Biochemical and structural characterization of Sirtuins from mammals and Thermotoga maritima

Biochemical and s uc u al cha ac e iza ion o Si uins om mammals and The mo oga ma i ima DISSERTATION To ob ain he deg ee Dok o de Na u wissenscha en (D . e . na .) Fakul ä ü Biologie, Chemie und Geowissenscha en, Uni e si ä Bay eu h Submi ed by Mahade an Lakshmina asimhan Bay eu h, 2012 I This doc o al hesis was p epa ed a he Depa men o Physiological Chemis y, Ruh - Uni e si y Bochum, Ge many and a he Depa men o Biochemis y, Uni e si y o Bay eu h, Ge many, in a ilia ions wi h In e na ional Max Planck Resea ch School in Chemical Biology (IMPRS-CB) Ph.D. p og am, Max Planck Ins i u e o Molecula Physiology, Do mund Ge many, and Eli e Ne wo k o Ba a ia, BioMedTech In e na ional g adua e School o Science (BIGSS) Ph.D. p og am, Uni e si y o Bay eu h, Ge many om Decembe 2008 un il Ma ch 2012 supe ised by P o . D . Clemens S eegbo n. This is a ull ep in o he disse a ion submi ed o a ain he academic deg ee o Doc o o Na u al Sciences (D . e . na .) and app o ed by he Facul y o Biology, Chemis y and Geosciences o he Uni e si y o Bay eu h. Ac ing dean: P o . D . Bea e Lohne Da e o submission: 7 h Ma ch, 2012 Da e o de ense: 11 h May, 2012 Doc o al Commi ee: P o D . Clemens S eegbo n 1s e iewe P o D . Wul Blanken eld 2nd e iewe P o . D . Paul Rösch Chai man P o . D . Raine Schobe II Dedica ed o…. Ginaandmypa en s III Acknowledgemen s The e is a amous saying: “Behind e e y success ul man is a g ea woman”, bu in my case he e a e wo (my mom and Gina) and I dedica e his hesis o hem and also o my dad. Wi hou hei suppo , help and encou agemen , I wouldn’ be whe e I’m now. They a e my cons an sou ce o ene gy and inspi a ion and ha e always helped me pu sue my d eams. I’m eally o una e o ha e such people in my li e. I’m also g a e ul o my amily and iends o hei cons an suppo and encou agemen . I hank my esea ch ad ise P o . D . Clemens S eegbo n, o p o iding me he oppo uni y o wo k in his labo a o y wi h exci ing esea ch opics, o his cons an suppo and excellen scien i ic discussions. I would also like o hank all he membe s o he biochemis y depa men , Uni e si y o Bay eu h, Ge many o all he suppo and a good wo king a mosphe e. I also hank P o . D . Wul Blanken eld (Depa men o Biochemis y, Uni e si y o Bay eu h, Ge many), P o . D . Ola S emmann (Depa men o gene ics, Uni e si y o Bay eu h, Ge many), P o . D .Mike Schu kowski (Uni e si y o Halle, Ge many), D . U e Cu h (Hanno e Medical school, Ge many), D . Di k Wol e s (Ruh Uni e si y, Bochum, Ge many) and D . S e an Raunse (Max Planck Ins i u e o Molecula Physiology, Do mund, Ge many) o ui ul discussions and collabo a ions. Thanks also o P o . D . Roge Goody, P o . D . Ma in Engelha d, D . Wal aud Ho mann-Goody and Ms. Ch is a Ho nemann om he Max Planck Ins i u e o Molecula Physiology, Do mund, Ge many o helping me wi h he IMPRS-CB (In e na ional Max Planck Resea ch School in Chemical Biology) PhD p og am and suppo ing my scien i ic endea o s. I also hank P o . D . Paul Rösch, PD. D . S ephan Schwa zinge and Ms. Violaine Zigan o helping me wi h he ENB (Eli e Ne wo k o Ba a ia)/BIGSS (BioMedTec In e na ional G adua e School o Science) Ph.D. p og am a he Uni e si y o Bay eu h, Ge many. IV I hank he IMPRS-CB Ph.D. p og am, MPI, Do mund, Ge many and ENB (Eli e Ne wo k o Ba a ia)/BIGSS (BioMedTec In e na ional G adua e School o Science) Ph.D. p og am, Uni e si y o Bay eu h, Ge many, o unding and inancial suppo o ca y ou my esea ch.  V Publica ions and p esen a ions pe aining o his wo k Pee e iewed publica ions: Ch is ine Schlicke , Gina Boanca, Mahade an Lakshmina asimhan, Clemens S eegbo n (2011) S uc u e-based De elopmen o No el Si uin Inhibi o s. Aging. 3: 852-872. Mahade an Lakshmina asimhan, Clemens S eegbo n (2010) Eme ging mi ochond ial signaling mechanisms in physiology, aging p ocesses, and as d ug a ge s. Exp. Ge on ol. 46: 174-177. Manusc ip s submi ed and in p epa a ion: F ank Fische , Melanie Ge z, Benjamin Suenkel, Mahade an Lakshmina asimhan, Mike Schu kowski, Clemens S eegbo n (2012) A e ined Mass spec ome y deace yla ion assay e eals Si 5 as Nico inamide insensi i e sub amily. Manusc ip submi ed o ACS Chemical Biology. Mahade an Lakshmina asimhan, Melanie Ge z, Giang Thi Tuye Nguyen, Michael Weyand, Clemens S eegbo n (2012) The molecula mechanism o Si uin inhibi ion by Ex-527. Manusc ip in p epa a ion. Mahade an Lakshmina asimhan, Melanie Ge z, F ank Fische , Da id Rauh, U e Cu h, Mike Schu kowski, Clemens S eegbo n (2012) Res e a ol is a sequence-speci ic ac i a o o Si 1-dependen pep ide and p o ein deace yla ion. Manusc ip in p epa a ion. Da id Rauh, F ank Fische , Mahade an Lakshmina asimhan, Melanie Ge z, Tim Be gb ede, Mike Schu kowski, Clemens S eegbo n (2012) Speci ici ies and subs a es o human Si uin iso o ms e ealed by p o iling agains an ace ylome pep ide a ay. Manusc ip in p epa a ion. Con e ences, cou ses and symposiums: O al and pos e p esen a ion a he Eli e Ne wo k o Ba a ia S uc u e Days symposium – Thu nau, Ge many July’11, i led “S uc u al and biochemical cha ac e iza ion o Si uins.” Pos e p esen a ion a he EMBO p ac ical cou se on exploi ing anomalous sca e ing in mac omolecula s uc u e de e mina ion, ESRF, G enoble, F ance June’11m i led “S uc u al and biochemical cha ac e iza ion o mammalian Si uins”. VI O al p esen a ion a he Max Planck Ins i u e o Molecula Physiology – Do mund, Ge many, Sep’10, i led “S uc u al and biochemical cha ac e iza ion o mammalian Si 1.” O al and pos e p esen a ion a he Eli e Ne wo k o Ba a ia S uc u e Days symposium – Thu nau, Ge many July’10, i led “S uc u al and biochemical cha ac e iza ion o mammalian Si 1.” VII Lis o o he publica ions and p esen a ions Publica ions: Mahade an Lakshmina asimhan*, Pe e Madzelan*, Ru h Nan, Nicole M. Milko ic, Ma k A. Wilson (2010) E olu ion o new enzyma ic unc ion by s uc u al modula ion o cys eine eac i i y in Pseudomonas luo escens isocyanide hyd a ase. Jou nal o Biological chemis y. 285: 29651-29661. Je Blackin on*, Mahade an Lakshmina asimhan*, Kelly J Thomas, Rili Ahmad, Ash a S. Raza, Ma k R. Cookson and Ma k A. Wilson (2009) Fo ma ion o A S abilized Cys eine Sul inic Acid is C i ical o he Mi ochond ial Func ion o he Pa kinsonism P o ein Dj-1. Jou nal o Biological chemis y. 284: 6476–6485. Fea u ed on he Co e o he Jou nal o Biological chemis y, issue Ma ch 6, 2009. Anna C. Wi , Mahade an Lakshmina asimhan, Benjamin C. Reming on, Saha Hasim, Edwin Pozha ski and Ma k A. Wilson (2008) Cys eine pKa Dep ession by a P o ona ed Glu amic Acid in Human DJ-1. Biochemis y. 47: 7430-7440. Mahade an Lakshmina asimhan*, Ma ien Maldonado*, Wenbo Zhou, An hony Fink and Ma k A. Wilson (2008) The Impac o Th ee Pa kinsonism-Associa ed Poin Mu a ions on he S uc u e and Redox-Regula ed S abili y o DJ-1. Biochemis y. 47: 1381-1392. Fea u ed on he ACS Publica ions web si e as a mos -accessed a icle o he 1s qua e o 2008. * = Equal con ibu ion Mahade an Lakshmina asimhan, Alajos Bé czi, Han Asa d (2006) Subs a e-dependen educ ion o a ecombinan ch oma in g anule Cy -b561 and i s R72A mu an . Ac a Biol Szeged. 50(1-2): 61-65. Alajos Bé czi, Dan Su, Mahade an Lakshmina asimhan, Amy Va gas and Han Asa d (2005) He e ologous exp ession and si e-di ec ed mu agenesis o an asco ba e- educible cy och ome b561. A chi es o Biochemis y and Biophysics.443: 82-92. VIII P esen a ions and pos e s: O al p esen a ion a he Max Planck Ins i u e o Molecula Physiology – Do mund, Ge many, Ap ’09, i led “Molecula basis o he cy op o ec i e unc ion o he Pa kinsonism p o ein DJ-1.” Pos e p esen a ion in Ame ican C ys allog aphic Associa ion Annual mee ing 2008 a Knox ille, Tennessee, USA, i led “S uc u al Impac o Th ee Pa kinsonism-Associa ed Missense Mu a ions o Human DJ-1.” Pos e p esen a ion in Redox Biology cen e -Uni e si y o Neb aska-Lincoln, symposium i led “The s uc u al de e minan s o egula o y cys eine oxida ion in he Pa kinsonism- associa ed p o ein DJ-1” Pos e p esen a ion in Redox Biology cen e -Uni e si y o Neb aska-Lincoln, symposium i led “Cha ac e iza ion and mu a ional s udies on a mouse Cy och ome b561” XV 1. In oduc ion .......................................................................................................................... 1 1.1 Calo ie es ic ion and li espan ex ension ........................................................................ 1 1.2 Molecula mechanism o li espan ex ension by CR ......................................................... 1 1.3 Si uins, CR and aging ...................................................................................................... 1 1.4 Mammalian Si uins – Func ion, classi ica ion and localiza ion ...................................... 2 1.4.1 Classi ica ion o mammalian Si uins ........................................................................ 3 1.4.2 Localiza ion o mammalian Si uins .......................................................................... 4 1.5 Subs a es, unc ion, s uc u e and enzyma ic mechanism o Si uins ............................. 5 1.5.1 Subs a es o Si uins ................................................................................................. 5 1.5.2 Role o Si 1 in cellula unc ions and disease s a es ................................................. 6 1.5.3 S uc u al ea u es o Si uins .................................................................................... 8 1.5.4 Ca aly ic mechanism o Si uins ................................................................................ 9 1.6 Regula ion o Si 1 .......................................................................................................... 11 1.6.1 P o eins and PTMs in ol ed in Si 1 egula ion ...................................................... 12 1.6.2 Regula ion o Si 1 by small molecules ................................................................... 14 1.6.2.1 Regula ion by physiological me aboli es .......................................................... 14 1.6.2.2 Regula ion by pha macological small molecules .............................................. 14 1.6.3 Mechanism o Si 1 modula ion by small molecules ............................................... 17 1.6.3.1 Mechanism o inhibi ion ................................................................................... 17 1.6.3.2 Mechanism o ac i a ion ................................................................................... 19 1.7 Objec i es ....................................................................................................................... 20 2. Ma e ials and Me hods ...................................................................................................... 21 2.1 Ma e ials ......................................................................................................................... 21 2.1.1 Chemicals, Enzymes and S anda ds ........................................................................ 21 2.1.2 Bac e ial s ains .................................................................................................. 21 2.1.3 Plasmids and cDNA cons uc s .......................................................................... 21 2.1.4 Oligonucleo ides ...................................................................................................... 22 2.1.5 Miscellaneous ma e ials ........................................................................................... 22 2.2 Mic obiology me hods .................................................................................................... 22 2.2.1 S e iliza ion .............................................................................................................. 22 XVI 2.2.2 Cul u ing o E. coli cells .......................................................................................... 22 2.2.3 Compe en cells ........................................................................................................ 23 2.2.4 T ans o ma ion o compe en cells .......................................................................... 23 2.2.5 He e ologous o e exp ession o ecombinan p o eins ............................................ 23 2.2.6 Cell lysis ................................................................................................................... 26 2.3 Molecula biology me hods ............................................................................................ 26 2.3.1 Aga ose gel elec opho esis ..................................................................................... 26 2.3.2 Gene cloning ............................................................................................................ 26 2.3.3 Si e di ec ed mu agenesis ......................................................................................... 28 2.3.4 DNA sequencing ...................................................................................................... 28 2.4 Biochemical me hods ..................................................................................................... 28 2.4.1 De e mina ion o p o ein concen a ion ................................................................... 28 2.4.2 SDS-Polyac ylamide gel elec opho esis (SDS-PAGE) .......................................... 29 2.4.3 Blue-na i e polyac ylamide gel elec opho esis (BN-PAGE) ................................. 29 2.4.4 Pu i ica ion o ecombinan p o eins ....................................................................... 30 2.4.4.1 A ini y ch oma og aphy ................................................................................... 30 2.4.4.2 Size exclusion ch oma og aphy ........................................................................ 31 2.4.4.3 Ion exchange ch oma og aphy .......................................................................... 31 2.4.5 Clea age o a ini y ags .......................................................................................... 32 2.4.6 Fluo escence based pep ide deace yla ion assay ...................................................... 35 2.4.7 Enzyme-linked immunoso ben assay (ELISA) ...................................................... 35 2.4.8 Con inuous assay o deace yla ion based on coupled enzymes .............................. 36 2.4.9 Limi ed p o eolysis o p o eins ................................................................................ 36 2.4.10 The mal dena u a ion shi assay ........................................................................... 37 2.4.11 Mic oscale he mopho esis .................................................................................... 37 2.4.12 Analy ical ul acen i uga ion ................................................................................ 38 2.5 Mass spec ome y .......................................................................................................... 38 2.5.1 Mass spec ome y based deace yla ion assay ......................................................... 38 2.5.2 Analysis o p o ein samples om polyac ylamide gels using yp ic diges ........... 39 2.6 C ys allog aphy and s uc u e de e mina ion ................................................................. 39 XVII 2.6.1 C ys alliza ion ials o human Si 1 ........................................................................ 39 2.6.2 C ys alliza ion o The mo oga ma i ima Si uin (Si 2Tm) ...................................... 40 2.6.3 Collec ion and analysis o X- ay di ac ion da a o Si 2Tm c ys als ..................... 40 2.6.4 Molecula eplacemen o Si 2Tm c ys al s uc u es ............................................... 40 2.6.5 Re inemen , model building and s uc u e alida ion o Si 2Tm complex s uc u es ........................................................................................................................................... 40 2.6.6 Homology modeling o human Si 1 ....................................................................... 41 3. Resul s ................................................................................................................................. 42 3.1 S udies on Si 1 ............................................................................................................... 42 3.1.1 Recombinan exp ession and pu i ica ion o Si 1 ................................................... 42 3.1.2 In es iga ing he ole o e mini in he oligome iza ion o Si 1 ............................. 47 3.1.3 The ca aly ic domain o Si 1 is su icien o ac i a ion by polyphenols ............... 50 3.1.4 Iden i ica ion o no el pep ide subs a es o Si 1 .................................................. 53 3.1.5 Si 1 modula ion by es e a ol is sequence speci ic ............................................... 55 3.1.6 E ec o polyphenol un ela ed small molecules on Si 1 ........................................ 56 3.1.7 Res e a ol can di ec ly bind o Si 1 ...................................................................... 59 3.1.8 C ys alliza ion ials o Si 1 .................................................................................... 59 3.1.9 S udies on AROS ..................................................................................................... 61 3.2 S udies on he ole o Zinc in Si 3 ................................................................................. 64 3.3 S udies on Si 5 ............................................................................................................... 66 3.3.1 In luence o NAD+ on he ac i i y o Si 5 .............................................................. 66 3.3.2 Si 5 appea s o be insensi i e o Nico inamide inhibi ion ...................................... 67 3.4 S udies on Si 7 ............................................................................................................... 70 3.4.1 Exp ession and pu i ica ion o Si 7 ........................................................................ 70 3.4.2 E ec o a ious bu e s and sal s on he s abili y o Si 7 ...................................... 71 3.4.3 Iden i ica ion o new Si 7 cons uc s o c ys alliza ion ......................................... 71 3.4.4 C ys alliza ion ials o Si 7 .................................................................................... 73 3.4.5 PTMs in luence he ac i i y o Si 7 ........................................................................ 73 3.4.6 In es iga ing he modula ion o Si 7 by es e a ol and nico inamide ................... 75 3.5 S udies on Si 2Tm .......................................................................................................... 76 XVIII 3.5.1 In es iga ing he oligome iza ion beha io o Si 2Tm ............................................ 76 3.5.2 Iden i ica ion o subs a e-modula o pai s o Si 2Tm ........................................... 77 3.5.3 Ex-527 is a po en inhibi o o Si 2Tm .................................................................... 79 3.5.4 Ex-527 appea s o equi e bo h he subs a es o bind Si 2Tm ................................ 80 3.5.5 Si 2Tm - C ys alliza ion and c yop o ec ion ........................................................... 81 3.5.6 Da a collec ion ......................................................................................................... 83 3.5.7 S uc u e solu ion, e inemen and modeling ........................................................... 84 3.5.8 Analysis o c ys al s uc u es o Si 2Tm ................................................................. 86 3.5.9 EX-527 appea s o bind o he “C-pocke ” o Si 2Tm ............................................. 91 4. Discussion ............................................................................................................................ 93 4.1 Pu i ica ion and domain a chi ec u e o Si 1 ................................................................. 93 4.2 Si 1 modula ion by es e a ol ...................................................................................... 94 4.3 C ys alliza ion o Si 1 .................................................................................................... 96 4.4 Zinc is essen ial o he s uc u al s abili y and ac i i y o Si uins ............................... 97 4.5 Si 5 equi es unusually high amoun o NAD+ o i s deace ylase ac i i y ................... 97 4.6 Si 5 is a NAM insensi i e deace ylase among he Si uin amily ................................. 98 4.7 Exp ession, pu i ica ion and c ys alliza ion o Si 7 ...................................................... 99 4.8 Si 7 Phospho yla ion a Th 224 inc eases i s ac i i y ................................................. 100 4.9 Si 2Tm as a model sys em o s udying mammalian Si 1 ........................................... 100 4.10 Mechanism o Si uin inhibi ion by Ex-527 ............................................................... 101 4.11 Selec i i y o Ex-527 owa ds Si uins ....................................................................... 102 4.12 Insigh s o d ug de elopmen .................................................................................... 104 4.13 Ou look ....................................................................................................................... 105 5. Abs ac ............................................................................................................................. 107 6. Zusammen assung ............................................................................................................ 108 7. Re e ences ......................................................................................................................... 110 8. Appendix ........................................................................................................................... 118 9. E klä ung .......................................................................................................................... 130 1. In oduc ion 1  1. In oduc ion 1.1 Calo ie es ic ion and li espan ex ension Calo ie es ic ion o calo ic es ic ion (CR) is a die a y egimen whe ein he amoun o calo ies consumed is educed up o 20-40% om he amoun consumed ad libi um. Li espan ex ension by CR was i s epo ed by McCay e . al (McCay e al., 1989) in 1930’s. Calo ie es ic ed male a s li ed nea ly wice as long as hei coun e pa s ha we e ed wi h an ad libi um die . This su p ising and in e es ing phenomenon has since been esea ched ex ensi ely in a ious species anging om he unicellula yeas o wo ms, lies, ish, mice e c. and con i med ha up o 50% inc ease in li espan can be achie ed by CR (Bo done and Gua en e, 2005). CR has been linked no only o li espan ex ension bu also o an o e all well being o he o ganism by educing he incidences o age ela ed ailmen s such as cance , diabe es and ca dio ascula diseases, indica ing ha main enance o a disease ee s a e due o CR may in u n lead o li espan ex ension (Hu s ing e al., 2001; Lane e al., 1999). 1.2 Molecula mechanism o li espan ex ension by CR Al hough CR has been linked o li espan ex ension o a long ime, he molecula mechanisms behind he phenomenon a e s ill in ensely s udied and a ious heo ies ha e been p oposed. Ini ial s udies on CR aimed a explaining he e ec based on a slowing o me abolism. These e en s a e coo dina ed by signaling molecules such as TOR ( a ge o apamycin), AMPK (5′ adenosine monophospha e-ac i a ed p o ein kinase) and Si uins. The mechanisms p oposed o con ibu e o CR media ed li espan ex ension a e educ ion o oxida i e damage, imp o emen o mi ochond ial bioene ge ics, ho mesis, ho monal egula ion and a y acid me abolism (Bo done and Gua en e, 2005; Koubo a and Gua en e, 2003). 1.3 Si uins, CR and aging Aging in bake ’s yeas Saccha omyces ce e isiae was ini ially linked o a se o genes called Si 1-4 (Si s ands o silen in o ma ion egula o ), which a e in ol ed in he silencing o ch oma in nea elome es. La e , Go lieb and Esposi o (Go lieb and Esposi o, 1989) demons a ed ha Si 2 is he only gene among he se equi ed o silencing o genes nea elome es and also esponsible o he silencing o DNA ( ibosomal DNA). I was also shown ha he egions nea he elome es ha unde wen silencing by Si 2 con ained his ones, hypoace yla ed a he ε-amino g oup o hei lysine side chains, p edominan ly a he a N- 1. In oduc ion 2  e minus (Michan and Sinclai , 2007) and Si 2 was he only gene among he se ound o be esponsible o his obse a ion. When a budding yeas mo he cell’s di ision was ollowed, se e al cha ac e is ic changes we e obse ed including he accumula ion o ERC (ex ach omosomal DNA ci cles), which was in e sely co ela ed o li espan (Sinclai and Gua en e, 1997). Lin e . al. showed ha when yeas we e g own on CR media, Si 2 media ed educ ion in numbe o ERC was obse ed and was co ela ed wi h li espan ex ension, hus connec ing Si 2 and CR media ed li espan ex ension (Lin e al., 2002). They also showed ha CR enhances he ac i i y o Si 2, mos likely by inc easing he a ailabili y o i s co-subs a e NAD+ (nico inamide adenine dinucleo ide oxidized o m) (see below) due o slowing down o glycolysis and inc ease in espi a ion ia he TCA ( i ca boxylic acid) cycle. Al hough CR media ed li espan ex ension in yeas can also occu independen o Si 2 (Kaebe lein e al., 2005b) and a silencing independen mechanism can exis o supp essing ERC (Riesen and Mo gan, 2009), Si 2 and hei homologs called Si uins (Si 2-ins) in highe o ganisms appea , o con ibu e o CR media ed li espan ex ension. Si 2 homologs a e p esen in all kingdoms o li e anging om bac e ia o mammals and sha e a conse ed ca aly ic domain and migh be in ol ed in mo e han gene silencing, o example in cell cycle p og ession (B achmann e al., 1995). Roy F ye (F ye, 1999, 2000) la e showed ha he e a e se en Si 2 homologs in mammals (Si 1-7) and g ouped hem in o di e en classes (see below) and also showed ha some Si uins (such as CobB om E. coli (Esche ichia coli) and human Si 2) unc ion as weak NAD+ dependen mono-ADP - ibosyl ans e ases. This ac i i y was also ound o yeas Si 2 and is essen ial o i s gene silencing ole in he ch oma in (Tanny e al., 1999). Howe e , se e al independen g oups epo ed ha Si 2 and i s mammalian homologs (mouse Si 1 and human Si 1) ha e much highe NAD+ dependen deace ylase ac i i y and can deace yla e bo h his ones and o he p o eins such as he ansc ip ion ac o p53 (Imai e al., 2000; Land y e al., 2000; Vazi i e al., 2001). The hallma k o Si uins ha dis inguishes hem om o he HDAC’s (his one deace ylases) is he absolu e equi emen o NAD+ o hei ac i i y. Since Si uins can deace yla e p o eins o he han his ones, a new p o ein e minology called PDACs (Lakshmina asimhan and S eegbo n, 2011) has been coined o desc ibe hem. 1.4 Mammalian Si uins – Func ion, classi ica ion and localiza ion Regula ion o cellula unc ions by amino acid PTMs (pos - ansla ional modi ica ions) is an e icien and elegan p ocess. In pa icula lysine, he mos equen ly modi ied amino acid is in ol ed in se e al p ocesses anging om ansc ip ion, signaling, 1. In oduc ion 3  p o ein u no e o disease s a es (Zhang e al., 2011). Se e al lysine modi ica ions a e known ill da e and mo e a e s ill being disco e ed (Du e al., 2011; Zhang e al., 2011). Examples o lysine modi ica ions include; ace yla ion, me hyla ion, bio inyla ion, ubiqui ina ion, sumoyla ion, p opionyla ion, bu y yla ion, succinyla ion, malonyla ion. Lysine ace yla ion one o he mos abundan PTM (nex only o phospho yla ion) (Khou y e al., 2011), expanded ou unde s anding o how p o ein modi ica ions play a dynamic ole in all ypes o cellula unc ions. E en hough Si uins we e ini ially hough o unc ion as mono-ADP- ibosyl ans e ases, i is widely accep ed ha hei p ima y unc ion is lysine deace yla ion. Because o hei unique NAD+ dependence, hey ha e been g ouped in a class o hei own, HDAC Class III. Recen esea ch (Du e al., 2011) has also shown ha some iso o ms can in ac desuccinyla e and demalonyla e lysine esidues in p o eins, opening up new exci ing a enues in his ield. Since Si uins NAD+ is me abolized du ing he eac ion, i is a co-subs a e and no a co- ac o . The p esence o wo subs a es makes he enzyme e y challenging o s udy in e ms o unde s anding i s mechanism and egula ion, and he equi emen o NAD+ also makes i a me abolic senso . 1.4.1 Classi ica ion o mammalian Si uins The e se en mammalian Si uins ha e di e en subs a e p e e ences and localiza ion. Si uins ca y a conse ed ca aly ic domain consis ing o abou 275 esidues. This ca aly ic domain is he mos conse ed pa among all he Si uins. Based on phylogene ic analysis mammalian Si uins a e g ouped in o ou di e en classes (I-IV) (Figu e 1.1) (F ye, 2000; Michan and Sinclai , 2007). Si 1, 2 and 3 a e placed unde Class I along wi h mos euka yo ic Si uins such as he ounding membe yeas Si 2, HST 1 and 2 om yeas and Si 2.1 om D osophila melanogas e . The Class I is u he di ided in o Ia (Si 1) and Ib (Si 2 and 3). Si 4 belongs o Class II along wi h Si uins om bac e ia, nema odes, ungus and p o ozoans. Si 5 is placed in a class o i s own (Class III), along wi h Si uins om achea and p o ozoa, implying an ea ly e olu ion. Si 6 and 7 belong o class IV, which is u he subdi ided in o IVa and IVb, espec i ely. O he ep esen a i e membe s om his amily include Si uins om me azoans and plan s. Si uins om g am posi i e bac e ia such as The mo oga ma i ima all in o a unique unde e mined class (Class U) and seem o ha e appea ed e y ea ly in e olu ion. 1. In oduc ion 4  Figu e 1.1: Classi ica ion o mammalian Si uins. A igu e depic ing di e en classes o mammalian Si uins is shown along wi h ep esen a i e membe s om o he kingdoms. C. alb, Candida albicans; C. ele, Caenoh abdi is elegans; D. mel, D osophila melanogas e ; P. al, Plasmodium alcipa um; T. ma , The mo oga ma i ima. Figu e adap ed om Michan and Sinclai Biochem. J. 2007 (Michan and Sinclai , 2007). 1.4.2 Localiza ion o mammalian Si uins Mammalian Si uins a e localized in di e en compa men o he cells and seem o ha e di e en subs a e speci ici y. Si 1 is p edominan ly in he nucleus, al hough i has been epo ed o be p esen in he cy oplasm and also in mi ochond ia (Aquilano e al., 2010; Michan and Sinclai , 2007). Si 2 is p edominan ly cy oplasmic (F ye, 1999) (No h e al., 2003). Si 3, 4 and 5 a e mi ochond ial and a e localized in di e en compa men s o he mi ochond ia (Ge z and S eegbo n, 2010). Si 3 and 4 a e p esen in he mi ochond ial ma ix and Si 5 can be ansloca ed ei he o he in e memb ane space (IMS) o o he ma ix (Schlicke e al., 2008). Si 6 is p esen in he he e och oma in o he nucleus (Tennen e al., 2010) and Si 7 is p esen in he nucleolus (Figu e 1.2). Figu e 1.2: Schema ic a chi ec u e o se en mammalian Si uins. The se en mammalian Si uins, which all unde di e en classes con ain a conse ed ca aly ic co e (shaded egions). The N and he C- e mini (ha ched egions) a e mo e unique o each Si uin. 1. In oduc ion 5  1.5 Subs a es, unc ion, s uc u e and enzyma ic mechanism o Si uins 1.5.1 Subs a es o Si uins To da e he e a e o e 6800 ace yla ion si es known in mammalian p o eins indica ing he impo ance o his modi ica ion (Choudha y e al., 2009; Kim e al., 2006). Se e al o hese ace yla ions si es a e ela i ely uncha ac e ized and Si uins migh se e as a deace ylase o se e al o hem. Si 1, he mos s udied and he la ges membe o he amily, has mo e han wo dozen physiologically ele an subs a es iden i ied (La u e al., 2008), which anges om his ones (H1, H4 e c.), ansc ip ion ac o s (p53, FOXO e c.), eNOS, (endo helial ni ic oxide syn hase), pGC1-α (pe oxisome p oli e a o -ac i a ed ecep o -g co- ac i a o 1α). Di e en Si uins can also ac on same subs a es ei he a he same lysine esidue (Si 1 and Si 7 can deace yla e Lys382 o p53 (Vakh ushe a e al., 2008)) o a di e en lysine esidue in he same p o ein (Si 1 can deace yla e Lys1020, 1024 and Si 2 can deace yla e Lys1542 and 1707 espec i ely in p300 (Black e al., 2008)). These common subs a es migh be ei he deace yla ed in di e en issues o cellula compa men s o a a di e en ime poin in he cell cycle. The mi ochond ial Si uins Si 3, 4 and 5 modi y p o eins mainly in ol ed in me abolism and ETC (elec on anspo chain). Mice lacking Si 3 exhibi hype ace yla ion o many mi ochond ial p o eins, and con i med subs a es a e a g owing lis o me abolic enzymes including GDH (glu ama e dehyd ogenase), ICDH (isoci a e dehyd ogenase) and LCAD (long-chain acyl Coenzyme A dehyd ogenase) (Hi schey e al., 2010; Schlicke e al., 2008) and p o eins in ol ed in he ETC such as Complex I (Ahn e al., 2008) and Complex II, V (Finley e al., 2011). Si 4 has e y ew subs a es iden i ied so a . Ini ial s udies showed ha Si 4 can ADP- ibosyla e and inhibi GDH ac i i y (Haigis e al., 2006; Lakshmina asimhan and S eegbo n, 2011), bu esul s om ou lab show Si 4 is also a subs a e speci ic deace ylase (unpublished). Si 6 and Si 7 a e localized in he nucleus and nucleolus, espec i ely, and e y li le is known abou hei subs a e p e e ences. Si 6 was ini ially shown o au o-ADP- ibosyla e i sel (Lisz e al., 2005), bu Pan e . al. (Pan e al., 2011b) showed Si 6 possesses weak deace ylaase ac i i y. Si 7 in e ac s wi h RNAPolyme ase I and posi i ely egula es i s ansc ip ion (Fo d e al., 2006). Si 7 can deace yla e FdL-1 pep ide subs a e and mice de icien in Si 7 gene show inc eased ace yla ion le els o p53 a Lys382 and de elop in lamma o y ca diomyopa hy (Vakh ushe a e al., 2008) indica ing a ole o Si 7 in ca diac 1. In oduc ion 6  unc ion ia p53 egula ion. Si 1 has been shown o unde go PTMs leading o inc ease in ac i i y (see below), simila ly PTMs could also in luence ac i i y o he weak deace ylase Si 4, 5, 6 and 7. A lis o di e en mammalians Si uins and hei subs a es/in e ac ing pa ne s a e lis ed in Table 1.1. Table 1.1: A lis o mammalian Si uins, hei unc ion, subs a es and in e ac o s. Tex modi ied om La u e . al (La u e al., 2008) wi h inpu s om he ollowing: a (Fo d e al., 2006), b (Liu e al., 2011), c (Black e al., 2008), d (Smi h e al., 2008) e (Jin e al., 2008), (Schlicke e al., 2008), g (Ahn e al., 2008), h (Finley e al., 2011), i (Hi schey e al., 2010), j (Nakagawa e al., 2009), k (Kawaha a e al., 2009), l (G ob e al., 2009). ACS, ace yl-CoA syn he ase; ANT, ADP/ATP ca ie p o ein; AR, and ogen ecep o ; AROS, ac i e egula o o SIRT1; ART, adp- ibosyl ans e ase; BCL11A, B-cell CLL/lymphoma 11A (zinc inge p o ein); CPS1, caobamoyl phospha e syn he ase 1; CTIP2, COUP-TF in e ac ing p o ein2 (also known as BCL11B); DAC, deace ylase; DMC, demalonylase; DSC, desuccinylase; DBC1, dele ed in b eas cance 1; E2F1, E2F ansc ip ion ac o 1; FOXO, o khead box p o ein O1; IDE, insulin-deg ading enzyme; IRS2, insulin ecep o subs a e 2; LXR, li e X ecep o ; MCF2, myocy e-speci ic enhance ac o 2; MyoD, myogenic di e en ia ion 1; NBS1, Nijmegen b eakage synd ome 1; NCOR, nuclea ecep o co- ep esso ; NF-kB, nuclea ac o -kB; RB, e inoblas oma p o ein; SUV39H1, supp esso o a iega ion 3-9 homolog 1; TAT, ans-ac i a o o ansc ip ion; TLE1, ansducin-like enhance o spli 1; TORC2, ansduce o egula ed cAMP esponse elemen binding p o ein 2; UBF, ups eam binding ac o ; WRN, We ne synd ome p o ein. 1.5.2 Role o Si 1 in cellula unc ions and disease s a es Among all he mammalian Si uins, human Si 1 is he bes cha ac e ized iso o m wi h espec o i s unc ion. Si 1 was ini ially implica ed in CR media ed li espan ex ension and ch oma in silencing, bu ecen da a alida e i s in ol emen in se e al p ocesses anging 1. In oduc ion 13  e minal kinase 1), DYRK1A, DYRK3 (dual speci ici y y osine phospho yla ion kinase), CK2 (casein kinase 2), cyclinB/CdK1 (cell cycle-dependen kinase B) (Fo d e al., 2008; Guo e al., 2010; Kang e al., 2009; Nas in e al., 2009; Sasaki e al., 2008; Zschoe nig and Mahlknech , 2009). Si 1 was shown o be me hyla ed in i o by SET7/9 a Lys 233, 235, 236 and 238 and was ound o ha e no in luence on i s ac i i y (Liu e al., 2011), bu his ype o in i o me hyla ion migh ha e a oused om non-speci ic ac i i y o SET7/9 and may no e lec he ue s a us in he cell. Table 1.2: PTMs o Si 1 and hei modi ying enzymes. S14, S16, S26, S27, S47, S173, S535, S538, S539, T554, S569, T719 and S747 we e ound o be phospho yla ed om p o eomic s udies (Beausoleil e al., 2004; Beausoleil e al., 2006; Dephou e e al., 2008; Gauci e al., 2009; Ge ha -Hines e al., 2011; Mayya e al., 2009; Olsen e al., 2010). 1. In oduc ion 14  1.6.2 Regula ion o Si 1 by small molecules 1.6.2.1 Regula ion by physiological me aboli es The wo physiologically ele an small molecula egula o s o Si 1 a e NAD+ and NAM, because o he o me ’s absolu e equi emen o Si 1’s ac i i y and he la e ’s abili y o inhibi he enzyme. The equi emen o NAD+ canno be eplaced by NADH o NADP+/NADPH. Al hough he o al le el o NAD+/NADH in he cell is in he milliMola ange, he ee NAD+ le el in he cell is ~ 300 - 400 µM (Yang e al., 2007a), because he majo i y o NAD+ in he cell is seques e ed by p o eins in ol ed in me abolic p ocesses such as glycolysis and TCA cycle in o de o ca y ou hei unc ion. Si 1’s a ini y owa ds NAD+ being a he low (~ 150-170 µM, (Smi h e al., 2009)) makes NAD+ an impo an egula o o Si 1 and he enzyme a me abolic senso /signaling agen , and he a ailabili y o NAD+ can in luence se e al cellula e en s by inc easing he ac i i y o Si 1. Fo example, he a ailabili y o NAD+ can help decide he cellula machine y i i is necessa y o ca y ou me abolic ac i i ies o egula e gene ansc ip ion (such as ac i a ing Si 1). NADH has been shown o inhibi he ac i i y o Si uins bu only in he high millimola ange (Schmid e al., 2004) which is beyond physiological le els. In spi e o his, i is s ill a ma e o deba e i i is he p oduc ion o NAD+ ( he genes in ol ed in NAD+ biosyn hesis) o he a io o NAD+/NADH ha egula es he ac i i y o Si 1 in e en s such as CR. The disco e y o NAM as a inhibi o o Si 1 gene a ed a lo o in e es in inding small egula o s o Si 1 ha mimic NAM (Bi e man e al., 2002). NAM is physiologically ele an inhibi o , because i inhibi s Si 1 wi h an IC50 o <50 µM and he cellula le els o NAD ange om 11-400 µM. Se e al s uc u al and biochemical s udies ha e elucida ed he mechanism behind NAM inhibi ion (A alos e al., 2005; Sau e and Sch amm, 2003). NAM inhibi s Si 1 in a non-compe i i e manne by binding in he conse ed C pocke a e o ma ion o he alkylimida e, leading o a base exchange eac ion e e sing in e media e o ma ion. 1.6.2.2 Regula ion by pha macological small molecules Since Si uins we e linked o CR and li espan ex ension, ex ensi e esea ch was ca ied ou o ind small molecules ha can egula e hem. Howi z e . al. sc eened a lib a y o NAD+ analogs and plan polyphenols o iden i y small molecules ha can modula e he ac i i y o Si 1 and i s homologs. Se e al inhibi o s we e iden i ied, bu mos no ably se e al plan de i ed polyphenols seemed o ac i a e Si 1 (Howi z e al., 2003). These Si uin 1. In oduc ion 15  ac i a ing compounds a e collec i ely called STACs (Si uin ac i a o s). Among polyphenols, he mos po en ac i a o was es e a ol (Figu e 1.6), a la onoid ha is syn hesized by se e al plan species including g apes (high amoun s can be ound in ed wine). Res e a ol’s s uc u e consis s o a s ilbene moie y wi h OH g oups on bo h o he benzyl ings, he mos s able isome being ans- es e a ol. E en be o e he iden i ica ion o es e a ol’s abili y o ac i a e Si 1, i was shown o con e ca diac and neu o-p o ec ion h ough i s an ioxdia i e p ope ies (Pe aiz and Holme, 2009). Yeas g own in media supplemen ed wi h es e a ol showed li espan ex ension simila o CR and his e ec was di ec ly dependen on es e a ol’s abili y o ac i a e yeas Si 2 (Howi z e al., 2003). Res e a ol has also been shown o mimic an i-aging e ec s in lowe o ganisms such as Caeno habdi is elegans and D osophila melanogas e in a Si uin dependen manne (Wood e al., 2004). Fu he mo e, mice ed wi h es e a ol show highe mi ochond ial con en , p o ec ion agains me abolic diseases (Lagouge e al., 2006) and inc eased su i al in a Si 1 dependen manne (Bau e al., 2006). In ano he sc een, se e al small molecules ha do no sha e any s uc u al simila i y o polyphenols we e also iden i ied, and we e shown o be up o 1000 old mo e po en in ac i a ing Si 1 compa ed o es e a ol (SRTs (Si is) Figu e 1.6) (Milne e al., 2007). In die induced and gene ically obese mice, hese compounds we e claimed o inc ease insulin sensi i i y and lowe blood glucose le els, and a e cu en ly unde p eclinical ials o ea ype 2 diabe es. Figu e 1.6: Rep esen a i e small molecule ac i a o s o Si 1 (STACs). Figu es we e ei he c ea ed using Ma inSke ch o adap ed wi h pe mission om Blum e . al.(Blum e al., 2011) Copy igh 2011 Ame ican Chemical Socie y. 1. In oduc ion 16  Se e al small molecule inhibi o s o Si 1 ha e been iden i ied and cha ac e ized o e he pas se e al yea s ( e iewed by Blum e . al (Blum e al., 2011)), he majo i y o hem a e based on pep ide mimics and NAD+/NAM analogs (Figu e 1.7). Some o hese inhibi o s such as Ex-527 (IC50 0.098 µM) and Su amin (IC50 0.3 µM) inhibi Si 1 in he nanomola ange, whe eas o he s such as Si inol, Teno in and Cambinol and hei de i a i es inhibi in he mic omola ange. Se e al o hese so called lead compounds we e used as analogs o de elop mo e po en and bioa ailable compounds, bu a ely show speci ici y owa ds Si 1. Fo example, Sale mide was de eloped based on he sca old o Si inol and was shown o be mo e po en han i s pa en compound (IC50 o 43 µM s123 µM espec i ely) (Pasco e al., 2009). Sale mide was well ole a ed by mice up o 100 µM and caused umo speci ic cell dea h in a ious issues (La a e al., 2009), bu was also shown o equally inhibi Si 2 (IC50 25 µM). Simila ly, Teno in based de i a i es we e also de eloped wi h Teno in-6 showing he bes wa e solubili y and po ency (IC50 21 µM o Si 1) and dec eased umo g ow h by inc easing he ace yla ion le els o p53-Lys382. Kine ic s udies indica ed ha Teno in-6 inhibi s Si 1 in a non-compe i i e manne , bu i was also shown o inhibi Si 2 in he mic omola ange (IC50 10 µM), again leading o lack o selec i i y (Lain e al., 2008). Cambinol also inhibi s Si 1 and Si 2 equally and was shown o possess an i umo ac i i y in p eclinical models, bu i s analog con aining a B omide in he pa a posi ion o he phenyl ing imp o ed i s po ency and speci ici y owa ds Si 1 (IC50 o 13 µM s. >90 µM espec i ely) indica ing ha sub le changes in he small molecules can lead o iso o m speci ici y (Medda e al., 2009). A high h oughpu sc een using 50,000 compounds iden i ied ou sca olds wi h Si uin inhibi o y ac i i y, ou o which one had mode a e selec i i y o Si 1 wi h IC50 ~ 6 µM (Sande s e al., 2009). 1. In oduc ion 17  Figu e 1.7: Rep esen a i e small molecule inhibi o s o Si 1. Figu es we e ei he c ea ed using Ma inSke ch o adap ed wi h pe mission om Blum e . al.(Blum e al., 2011). Copy igh 2011 Ame ican Chemical Socie y. 1.6.3 Mechanism o Si 1 modula ion by small molecules 1.6.3.1 Mechanism o inhibi ion The lack o s uc u al in o ma ion o Si 1 has hampe ed mechanism based/s uc u e aided d ug de elopmen , bu se e al biochemical and CADD (compu e -aided d ug design) s udies ha e been pe o med o de elop no el compounds o inhibi Si 1. Mos Si 1 inhibi o s likely ei he bind o he ace yllysine binding si e (pep ide mimics) o o he NAD+/NAM binding si e o inhibi he enzyme. In he case o pep ide based inhibi o s, compe i i e inhibi o s can easily be made speci ic based on he Si uin’s subs a e p e e ence, bu such inhibi o s a e di icul o de elop in o sui able d ugs. E en a single amino acid analog o ace yllysine con aining a hioace yllysine (and aniline and benzyloxyca bonyl g oups a ached o he ca bonyl and amino g oups o he hioace yllysine) was shown o ha e an inhibi o y e ec agains Si 1 wi h an IC50 o 2.7 µM which had 8.5-37 old selec i i y o e Si 2 and Si 3 (Suzuki e al., 2009). The o ma ion o a s able hio-ace yl-ADP- ibose conjuga e (a ack o he hioace yllysine on he ibose ing o NAD+ which ca ies he NAM) was con i med by mass spec ome ic analysis. The mos likely mechanism o such a ype o inhibi ion, is he slow subs a e u no e due o he o ma ion o a s able hioimida e in e media e which blocks he ace yllysine binding cle and ende s he enzyme inac i e (Smi h and Denu, 2007). The mechanism behind NAD+/NAM based inhibi ion a ises ei he om compe i ion be ween he inhibi o and NAD+ o a mixed ype inhibi ion. Nappe e . al 1. In oduc ion 18  syn hesized se e al indole based de i a i es which ha e some simila i y o NAM and showed ha some o hese compounds inhibi ed Si 1 wi h a e y high po ency and speci ici y compa ed o Si 2, 3 and 5 (Nappe e al., 2005). Based on kine ic analysis hey p oposed a mixed ype inhibi ion and sugges ed ha he inhibi o binds o he C-si e a e he elease o NAM and p e en s u he elease o he p oduc s; 2OAADP and deace yla ed lysine. Howe e , binding si e and he inhibi ion mechanism emain unce ain. Till da e he only mechanis ically unde s ood Si uin inhibi o is su amin, because o he a ailabili y o i s c ys al s uc u e in complex wi h Si 5 (Schue z e al., 2007). The symme y o su amin allows i o bind wo molecules o Si 5 a he same ime, c ea ing a link be ween hem, which in u n leads o dime iza ion. Su amin makes con ac s wi h se e al esidues o he co-subs a e binding loop leading o i s s abiliza ion. The sul onyl g oups o su amin binds o he ca aly ic co e o Si 5 and media es majo i y o he in e ac ion. The sul onyl g oups mimic NAM and occupies he C-pocke , hus p e en ing NAD+ om binding. Compa ison o Si 2Tm complexed wi h p53 pep ide and NAM (pdb id: 1YC5) and he Si 5 s uc u e complexed wi h su amin, indica es ha Ss amin also occupies he ace yllysine binding si e and ex ends up o wo esidues u he o he C- e minus o he pep ide subs a e, hus p e en ing i s binding. Fu he mo e, he sul onyl g oups also occupy he ibose pa o NAD+ which is a ached o NAM. Taken oge he , his shows ha su amin inhibi s Si 5 by p e en ing he binding o bo h he subs a e and co-subs a e a he same ime. Due o hese easons, su amin by i sel may lack iso o m speci ici y, because he subs a e binding cle and NAD+ binding pocke a e e y simila among he Si uins. Ne e heless, se e al new compounds we e syn hesized and cha ac e ized using he sca old o su amin and we e ound o be iso o m speci ic based on he small modi ica ions on he sul onyl ings. Fo example, in oduc ion o aminoan h alic de i a i es o small u ea like g oups in he sul onyl head o su amin lead o Si 1 speci ic inhibi ion wi h IC50 as low as 93 nM (T app e al., 2007), indica ing ha sub le di e ences exis ing in he ca aly ic co e o Si uins can be exploi ed o disco e iso o m speci ic d ug a ge s. Kine ic s udies using inhibi o s iden i ied om high h oughpu sc een sugges ed non- compe i i e o mixed ype inhibi ion ela i e o bo h subs a e and co-subs a e, implying ha he inhibi o binds in he conse ed ca aly ic domain (Sande s e al., 2009) and he e o e may no show iso o m speci ici y. In o de o de elop Si 1 speci ic inhibi o s, u u e e o s could ocus on he e mini o Si 1, because o i s uniqueness among he iso o ms. 1. In oduc ion 19  1.6.3.2 Mechanism o ac i a ion The mechanism o Si uin ac i a ion by polyphenols and STACs is pe haps he mos in ensely deba ed opic among he Si uin esea ch communi y. P o ein/enzyme ac i a ion by a small molecule is less p e alen compa ed o inhibi ion. Examples o small molecules ac i a ing p o eins include; Si 1, GK (glucokinase), PDK1 (phosphoinosi ide-dependen kinase 1) and p300 (Zo n and Wells, 2010). Since Si 1 is in ol ed in a ious cellula e en s i would be bene icial o iden i y bo h inhibi o s and ac i a o s o Si 1, especially small molecules ha speci ically modula e Si 1 (among i s mammalian iso o ms) on a subs a e speci ic manne in o de o a ge a pa icula disease pa hway. Ini ial high- h oughpu sc eening assays employed luo escence based pep ides as subs a es o iden i y po en ial Si uin modula o s, which we e hen used in o he sc eens as well (Howi z e al., 2003; Milne e al., 2007). Based on kine ic s udies an ac i a ion mechanism was p oposed whe e es e a ol and STACs inc eased subs a e a ini y (by dec easing he Km o he luo escen pep ide) bu had no e ec on he Vmax. La e , i was ound ha es e a ol ac i a ion was dependen on he luo opho e (AMC (7-amino-4- me hylcouma in)) p esen on he subs a e pep ide and when unmodi ied pep ides (in i o) o ull leng h p o eins (in i o) we e used as subs a es he e was no es e a ol dependen ac i a ion o Si 1 (Behe e al., 2009; Bo a e al., 2005; Kaebe lein e al., 2005a). A es e a ol dependen ac i a ion mechanism was p oposed whe e he polyphenol binds o and induces a con o ma ional change in Si 1 nea he binding a ea o he couma in g oup o he luo opho e, leading o be e accommoda ion o he luo escen pep ide by he enzyme. Pacholec e al. (Pacholec e al., 2010) showed ha he SRT se ies o compounds (SRT1720, SRT2183 and SRT1460) also do no ac i a e Si 1 when unmodi ied pep ides o ull leng h p o eins we e used as subs a es and concluded ha hese compounds di ec ly in e ac wi h he luo opho e and o ma ion o a complex be ween he luo opho e and he compound may lead o Si 1 ac i a ion in an “indi ec ” manne . Bu in a ecen epo Dai e . al. (Dai e al., 2010) showed ha he ac i a ion o Si 1 by STACs depend on he s uc u al ea u es o he pep ide subs a e and p oposed an allos e ic mechanism o ac i a ion o Si 1 by STACs, whe e he speci ic ea u es o he subs a e pep ide plays a ole in he ac i a ion p ocess and he ac i a o may bind o he enzyme-subs a e complex. Indeed we ind in his hesis ha es e a ol can di ec ly bind o Si 1 and ha he sequence o deace yla ion si e decides whe he es e a ol modula es Si 1 (ac i a ion o inhibi ion) explaining he p e ious con adic o y epo s. 1. In oduc ion 20  1.7 Objec i es Si uins a e a conse ed amily o p o eins ound in all domains o li e. Si uins a e classi ied as class III His one deace ylases and con ain a special ea u e; whe ein hey me abolize NAD+ o deace yla e and o ADP- ibosyla e p o ein esidues, he eby ac ing as me abolic senso s. The mammalian Si uin (Si ) amily encompasses se en iso o ms (Si 1- 7), each wi h a conse ed ca aly ic co e consis ing o ~275 esidues. The N and C- e minal ex ensions and localiza ion in di e en cellula compa men s, di e en ia e mammalian Si uins among each o he . The human Si 1 is he la ges iso o m among all he mammalian Si uins and is in ol ed in se e al cellula p ocesses and disease s a es. Si 1 also pa icipa es in CR media ed li espan ex ension in lowe o ganisms and se e al small molecules can modula e i s ac i i y. The molecula mechanism o Si 1 modula ion by small molecules is s ill no ully unde s ood, p ima ily due o he una ailabili y o s uc u al in o ma ion. Unde s anding his mechanism would allow iso o m speci ic and subs a e speci ic modula ion o Si 1 leading o be e he apeu ic agen s o age ela ed diseases. The majo ocus o his s udy is s uc u al and biochemical cha ac e iza ion o Si 1, o gain insigh s in o he mechanism o modula ion by small molecules. In o de o achie e his, ull leng h and a ious cons uc s o human Si 1 we e o be cloned, exp essed and pu i ied o homogenei y. C ys alliza ion ials should be pu sued on hese pu e p o eins and hei subs a e/modula o complexes o unde s and he mechanism o modula ion om a s uc u al pe spec i e. Biophysical and biochemical s udies we e o be ca ied ou o analyze he ole o N and C- e minal domains o Si 1 owa ds i s ac i i y and modula ion by small molecules. O he iso o ms o Si 1 (Si 2, Si 3, Si 5 and Si 7) and he bac e ial homolog o Si 1 om Si 2Tm we e o be used as a models sys ems o unde s and and compa e he s uc u e, unc ion and modula ion mechanisms o Si uins. 2. Ma e ials and Me hods 21  2. Ma e ials and Me hods 2.1 Ma e ials 2.1.1 Chemicals, Enzymes and S anda ds All chemicals we e pu chased om Sigma, Applichem, Se a, Ro h o J.T. Bake unless s a ed di e en ly. Pep ides we e syn hesized by GL Biochem, Shanghai, China. Supplemen al able S4 lis s all he pep ides used in his s udy. DNA modi ying enzymes, DNA and p o ein s anda ds we e pu chased om Se a, Agilen Technologies, P omega, New England Biolabs, Bio-Rad and Fe men as. T ypsin, Chymo ypsin, Cy . c (cy och ome c), Glu ama e dehyd ogenase we e pu chased om Sigma. 2.1.2 Bac e ial s ains All bac e ial s ains used in his s udy we e de i ed om E. coli (Esche ichia coli) K- 12. The E. coli s ains XL-1 Blue (S a agene, USA) was used o cloning, plasmid p opaga ion and si e-di ec ed mu agenesis. The E. coli s ain BL21 S a (DE3) (In i ogen, USA) and Rose a (DE3) (Me ck, Ge many) we e mos ly used o o e exp ession o ecombinan p o eins. The s ains ha e he ollowing geno ypes: XL1-Blue: ecA1 endA1 gy A96 hi-1 hsdR17 supE44 elA1 lac [F´ p oAB lacIqZΔM15 Tn10 (Te )]. BL21 S a (DE3): F- ompT hsdSB ( B-mB-) gal dcm ne131 (DE3) BL21(DE3) pLysS: F – ompT hsdSB( B– mB–) gal dcm (DE3)pLysS (CamR) Rose a (DE3): F– ompT hsdSB( B – mB–) gal dcm lacY1(DE3) pRARE (CamR) C43 (DE3): F- ompT hsdSB ( B- mB-) gal dcm (DE3) (Kan ), ans o med wi h he F-ATPase subuni gene and cu ed and con ains a leas one uncha ac e ized mu a ion. BL21 (DE3) codonPlus RIL: a gU (AGA, AGG), ileY (AUA), leuW (CUA) (CamR). 2.1.3 Plasmids and cDNA cons uc s O e exp ession o ecombinan p o ein in E. coli cells was pe o med by cloning he genes o in e es in pET11a, pET15b, pET21a(+) (Me ck, Ge many), pASK-IBA17 (BioTAGnology, Ge many), pGEX-6P3 (GE Heal hca e, USA), pET151/D-TOPO (Li e 2. Ma e ials and Me hods 22  Technologies, USA) and modi ied pOPIN ec o s (Ox o d Uni e si y, UK). Mouse Si 1 was cloned using he mouse cDNA lib a y ob ained om DB Biosciences, USA. Full leng h human Si 1 in pCDNA3.1 ec o was a kind gi om D . Tony Kouza ides, The Gu don Ins i u e, Camb idge, UK. Human AROS in pCMV-SPORT6 ec o was pu chased om Sou ce BioScience, Ge many. All he genes o in e es a e unde he con ol o T7 p omo e . 2.1.4 Oligonucleo ides All oligonucleo ides used o cloning and si e-di ec ed mu agenesis we e pu chased ei he om Sigma, USA o Eu o ins MWG Ope on, Ge many. The oligonucleo ides we e HPLC (high pe o mance liquid ch oma og aphy) o HPSF (high pu i y sal ee) pu i ied. A comple e lis o oligonucleo ides used in his wo k is lis ed in supplemen al able S1. 2.1.5 Miscellaneous ma e ials O he ma e ials we e pu chased om Eppendo , Hamp on Resea ch, Molecula Dimensions, Jena Biosciences, Qiagen, Millipo e, S a Labs i no s a ed explici ly. 2.2 Mic obiology me hods 2.2.1 S e iliza ion Solu ions and ma e ials equi ed o s e ile bac e ial cul u e we e p epa ed by ei he au ocla ing a 121 C o 20 minu es in a Sys ec DX-150 au ocla e (Sys ec GmbH, Ge many) o s e ile il e ed using a 0.22 M memb ane il e (Millipo e, USA). 2.2.2 Cul u ing o E. coli cells Gene al p ocedu e E. coli cells we e g own ei he in LB (Lu ia-Be ani) media which consis s o 1% (w/ ) T yp one, 0.5% (w/ ) Yeas and 1% (w/ ) NaCl o in au oinduc ion media consis ing o 1.2% (w/ ) T yp one, 2.4% (w/ ) Yeas ex ac and 0.5% ( / ) Glyce ol au ocla ed and supplemen ed wi h 0.17 M KH2PO4, 0.72 M K2PO4, 0.05% (w/ ) glucose and 0.2% (w/ ) alpha-lac ose. LB pla es used o pla ing bac e ia also con ained 1.5% (w/ ) Aga ose. Be o e addi ion o E. coli o he media, app op ia e amoun o an ibio ics we e added. The cul u es we e ini ially g own a 37 ºC by shaking a a speed o 120 RPM ( e olu ions pe minu e) and hen shi ed o 22 ºC be o e induc ion o he p o ein o in e es . 2. Ma e ials and Me hods 29  In he second spec opho ome ic me hod, a UV scan om 350 – 200 nm was pe o med on he p o ein sample using a Ca y 50 UV-Vis spec opho ome e (Agilen Technologies, USA) a e pe o ming a blank wi h he bu e and he A280 was used o calcula e he concen a ion o p o ein acco ding o he Bee -Lambe law as ollows: P o ein concen a ion (mg/ml) = (A280/(ε280*pa h leng h))*dilu ion ac o The pa h leng h is 1 cm and he heo e ical ex inc ion coe icien (ε280) alue a 1 mg/ml o a pa icula p o ein is ob ained om he Expasy’s p o pa am se e (h p://web.expasy.o g/p o pa am/). Fo ull leng h human Si 1 wi h N- e minal 6x His- ag he ε280 a 1 mg/ml is 0.547 (45840 M-1 cm-1). 2.4.2 SDS-Polyac ylamide gel elec opho esis (SDS-PAGE) The pu i y and size o p o ein samples we e accessed using SDS-PAGE con aining he T is-glycine discon inuous bu e sys em adap ed om Laemmli (Laemmli, 1970). The pe cen age o ac ylamide in sepa a ing gel anged om 12 – 15 %. The s acking gel, unning gel, unning bu e and SDS loading bu e we e p epa ed acco ding o Samb ook, J., and Russell, D. W., (Molecula cloning: A Labo a o y manual, hi d edi ion). P o ein samples we e dena u ed by boiling o 5 minu es a 95 C be o e loading on he gel. The SDS loading bu e con ained ß-me cap o e hanol o ensu e a educing a mosphe e. The Mini-PROTEAN Te a Cell e ical elec opho esis sys em (Bio-Rad, USA) was used o elec opho esis, a a cons an ol age o 150 ol s. A e elec opho esis, he gel was insed in wa e , ollowed by ew seconds o soaking in ho Coomassiee blue solu ion (0.025 % (w/ ) Coomassiee-B ilian Blue R-250 (Applichem, Ge many), 50 % ( / ) me hanol, 10 % ( / ) ace ic acid). The gel was hen ans e ed o a des aining solu ion con aining 20 % ( / ) me hanol and 12 % ( / ) ace ic acid. 2.4.3 Blue-na i e polyac ylamide gel elec opho esis (BN-PAGE) To analyze he oligome ic s a e o Si uins, BN-PAGE (blue-na i e polyac ylamide gel elec opho esis) was pe o med acco ding o he p o ocol o Schaegge (Schaegge , 1991). BN- PAGE was pe o med using a Hoe e Migh y Small appa a us (GE Heal hca e, USA) a a cons an ol age o 150 V o he i s 15 minu es and hen a 250 V un il he end. The samples we e p epa ed by mixing hem wi h 10 % ( / ) glyce ol ollowed by cen i uga ion o 10 minu es a 13,200 RPM in a e ige a ed Eppendo mic o uge (Eppendo , Ge many). Molecula weigh ma ke s we e pu chased om Se a, Ge many. The gel consis ed o h ee 2. Ma e ials and Me hods 30  laye s wi h di e en pe cen age o ac ylamide in each o hem. The composi ion o he gels and he bu e s a e as ollows: Top laye : 50 mM Bis-T is/HCl, pH 7.0, 200 mM ε-aminocap oic acid, 0.1 % ( / ) TEMED, 1 % (w/ ) ammomium pesul a e, 5 % ac ylamide/bisac ylamide (37.5:1). Middle laye : 50 mM Bis-T is/HCl, pH 7.0, 200 mM ε-aminocap oic acid, 10 % ( / ) glyce ol, 0.5 % ( / ) TEMED, 0.5 % (w/ ) ammomium pesul a e, 10 % ac ylamide/bisac ylamide (37.5:1). Bo om laye : 50 mM Bis-T is/HCl, pH 7.0, 200 mM ε-aminocap oic acid, 20 % ( / ) glyce ol, 0.25 % ( / ) TEMED, 0.25 % (w/ ) ammomium pesul a e, 14 % ac ylamide/bisac ylamide (37.5:1). Anode bu e : 50 mM Bis-T is/HCl, pH 7.0, 50 mM T icine, 0.02 % (w/ ) Coomassiee B illian Blue G-250 (Se a, Ge many). Ca hode bu e : 50 mM Bis-T is/HCl, pH 7.0, 2.4.4 Pu i ica ion o ecombinan p o eins 2.4.4.1 A ini y ch oma og aphy Pu i ica ion o ecombinan p o eins con aining His- ag was pe o med using TALON esin (Clon ech, USA). The His- ag was used on majo i y o he p o eins including Si 1, Si 2, Si 3, Si 5, Si 7 and AROS. AROS wi h GST ag was pu i ied using Glu a hione sepha ose esin (GE Heal hca e, USA). P o eins wi h His-MBP usion ag we e pu i ied using TALON o Ni-NTA esin. Full leng h Si 1 (wild- ype and H363A mu an ) con aining S ep ag we e pu i ied using he S ep-Tac in sepha ose esin (IBA, Ge many). Fo e e y li e o E. coli cul u e, 1 ml bed olume o esin was used. P io o usage, he esin was washed wice wi h wa e , ollowed by equilib a ion in lysis bu e . The clea ed E. coli supe na an con aining he ecombinan p o ein in lysis bu e was incuba ed wi h he esin a 4 C o 1 h by s i ing o e icien binding o he p o ein o he esin. A e he incuba ion, he low- h ough was collec ed by g a i y low using a glass column (Bio-Rad, USA) and he esin was incuba ed o 30 minu es a 4 C wi h 20 g/ml (bed olume) o bo ine RNase A and 10 g/ml o bo ine DNase in 25 mM T is, pH 7.5, 20 mM NaCl, 5 mM MgCl2, 0.2 mM PMSF o emo e nucleic acids binding o he p o ein o in e es . A e incuba ion, he low- h ough was collec ed and he column washed wi h 20 bed olumes o wash bu e and hen elu ed 2. Ma e ials and Me hods 31  wi h elu ion bu e (bu e composi ions see below). The samples we e un on SDS-PAGE o access he size and pu i y be o e pe o ming addi ional pu i ica ion s eps. TALON esin: Lysis bu e : 50 mM T is, pH 7.5, 300 mM NaCl, 0.2 mM PMSF ( o AROS and Si 1 229 o516 10 mM Imidazole was added in he lysis bu e ). Wash bu e : 50 mM T is, pH 7.5, 300 mM NaCl, 0.2 mM PMSF, 10 mM Imidazole ( o AROS and Si 1 229 o516 15 mM Imidazole was added). Elu ion bu e : 50 mM T is, pH 7.5, 300 mM NaCl, 0.2 mM PMSF, 150 mM Imidazole GST esin: Lysis and wash bu e : 50 mM T is, pH 7.5, 300 mM NaCl, 0.2 mM PMSF. Elu ion bu e : 50 mM T is, pH 7.5, 300 mM NaCl, 0.2 mM PMSF, 10 mM Glu a hione. S ep-Tac in esin: Lysis and wash bu e s: 100 mM T is·Cl, 150 mM NaCl, 1 mM EDTA, pH 8, 0.2 mM PMSF. Elu ion bu e : 100 mM T is·Cl, 150 mM NaCl, 1 mM EDTA, 2.5 mM des hiobio in, pH 8, 0.2 mM PMSF. 2.4.4.2 Size exclusion ch oma og aphy Elu ion samples om he a ini y ch oma og aphy we e pooled, concen a ed using an Amicon cen i ugal concen a o (Millipo e, USA) o 5 ml and applied on o a p eequilib a ed Supe dex200 16/60 size exclusion column (GE Heal hca e, USA) and elu ed wi h 25 mM HEPES, pH 7.5, 100 mM KCl, 2 mM DTT (di hio h ei ol), 0.1 mM PMSF, 0.5 mM EDTA. Fo analy ical pu poses a Supe ose12 GL300 column was also used wi h he same bu e as men ioned abo e bu wi hou PMSF and EDTA. Following ch oma og aphy, he samples we e un on SDS-PAGE o access hei pu i y; app op ia e ac ions we e pooled and concen a ed. 2.4.4.3 Ion exchange ch oma og aphy Ion exchange ch oma og aphy was pe o med using he 1 ml HiT ap Q HP anion exchange column (GE Heal hca e, USA) ha was p eequlib a ed wi h bu e A (25 mM HEPES, pH 7.5, 100 mM KCl, 2 mM DTT, 0.1 mM PMSF, 0.5 mM EDTA). The samples 2. Ma e ials and Me hods 32  om SEC (size exclusion ch oma og aphy) we e concen a ed o 5 ml and applied on he ion exchange column a a e y low low a e o 0.05 ml/minu e. The column was washed wi h 2 column olumes o bu e A ollowed by elu ion o he bound p o ein using a linea g adien agains bu e B (bu e A con aining o al 500 mM KCl). App op ia e ac ions we e pooled a e accessing hei pu i y using SDS-PAGE, desal ed in s o age bu e (25 mM HEPES, pH 7.5, 100 mM KCl, 2 mM DTT), concen a ed, lash ozen in liquid Ni ogen and s o ed a -80 C un il equi ed. 2.4.5 Clea age o a ini y ags A ini y ags o usion p o eins we e clea ed using p o eases ha ecognize sequences p esen be ween he ags and he p o ein o in e es . The a ini y pu i ied p o eins we e dialyzed in 25 mM HEPES, pH 7.5, 100 mM KCl, 2 mM DTT a 4 C and concen a ion de e mined be o e adding p o eases. Th ombin p o ease (GE Heal hca e, USA) was added a 1 Uni /mg o ecombinan p o ein and incuba ed o e nigh a 4 C. TEV ( obacco e ch i us) p o ease and P eScission (human hino i us 3C p o ease) we e added a a a io o 1:20 mg o ecombinan p o ein and incuba ed o e nigh a 4 C. Following o e nigh incuba ion wi h he equi ed p o eases, he samples we e passed o e he same a ini y columns o e which hey we e ini ially pu i ied in o de o emo e he unclea ed p o ein and he p o ease (TEV and P eScission con ain His- ag and GST ag espec i ely). Th ombin p o ease was sepa a ed om he p o ein o in e es by passing i o e benzamidine sepha oase esin. P ocedu es de ia ing om he abo e p o ocol a e: Si 3 The cells con aining o e exp essed Si 3 we e esuspended in 50 mM HEPES, pH 7.5, 300 mM NaCl, 10 mM Imidazole, 1 mM TCEP ( is(2-ca boxye hyl)phosphine), lysed and a ini y pu i ied using TALON esin as men ioned abo e (excep o he addi ion o nucleases). The pu i ied p o ein was dialyzed in 25 mM HEPES, pH 7.5, 100 mM KCl, 2 mM DTT o 3 hou s (excep o he addi ion o nucleases) a 4 °C ollowed by addi ion o TEV p o ease (1 mg o TEV p o ease pe 20 mg o a ini y pu i ied Si 3) and s o ed a 4 °C o e nigh o clea age o he a ini y ag. The ollowing day, he sample was passed h ough His-Selec Nickel a ini y esin (Sigma, USA) o emo e unclea ed p o ein and he TEV p o ease (con ains His- ag a he C- e minus), concen a ed o 1 ml and applied on a Supe ose12 size exclusion column (GE Heal hca e, USA) equilib a ed in dialysis bu e , o 2. Ma e ials and Me hods 33  sepa a e Si 3 om agg ega es and o he con aminan s. Si 3 con aining samples we e pooled, concen a ed, snap ozen in liquid N2 and s o ed a -80 °C un il needed. Si 5 The cells con aining o e exp essed Si 5 we e hawed, lysed and a ini y pu i ied using TALON esin as men ioned abo e (excep o he addi ion o nucleases) wi h he ollowing wash and elu ion bu e s: wash bu e = 50 mM T is, pH 7.8, 200 mM NaCl, 20 mM Imidazole and elu ion bu e = 50 mM T is, pH 7.8, 200 mM NaCl, 150 mM Imidazole. The pu i ied p o ein was concen a ed and desal ed in 20 mM T is, pH 7.8, 200 mM NaCl, 1 mM DTT using a NAP column (GE Heal hca e, USA) ollowed by addi ion o TEV p o ease (1 mg o TEV p o ease pe 20 mg o a ini y pu i ied Si 5) and s o ed a 4 °C o e nigh o clea age o he a ini y ag. The ollowing day, he sample was passed h ough Nickel a ini y esin (Qiagen, USA) o emo e unclea ed p o ein and he TEV p o ease (con ains His- ag a he C- e minus), concen a ed o 1 ml and applied on a Supe ose12 size exclusion column (GE Heal hca e, USA) equilib a ed in desal ing bu e , o sepa a e Si 5 om agg ega es and o he con aminan s. Si 5 con aining samples we e pooled, concen a ed, snap ozen in liquid N2 and s o ed a -80 °C un il needed. Si 7 The cells con aining o e exp essed Si 7 we e esuspended in lysis bu e (50 mM HEPES, pH 8.0, 300 mM NaCl, 20 mM Imidazole, 1 mM TCEP), lysed and applied on o a 5 ml HisT ap FF esin (GE Heal hca e, USA) connec ed o an AKTAXP ess HPLC (Hi- Pe o mance Liquid Ch oma og aphy) sys em (GE Heal hca e, USA). A e washing he column wi h 20 column olumes o wash bu e (lysis bu e con aining 30 mM Imidazole), he column was equilib a ed wi h 5 column olumes o clea age bu e (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM TCEP), ollowed by addi ion o 0.7 column olumes o espec i e p o ease and incuba ion o 5 hou s. The ag-less p o ein was hen washed ou o he column using wash bu e . The la ges peak (based on a ea) was collec ed (maximum 13 ml) and passed o e 26/60 Supe dex size exclusion column (GE Heal hca e, USA) equilib a ed in 25 mM HEPES, pH 7.5, 40 mM NaCl, 1 mM TCEP, o sepa a e Si 7 om agg ega es and o he con aminan s. Si 7 con aining samples we e pooled, concen a ed, snap ozen in liquid N2 and s o ed a -80 °C un il needed. 2. Ma e ials and Me hods 34  Si 2Tm Pu i ica ion o S 2Tm was pe o med as desc ibed by Smi h e . al. (Smi h e al., 2002) wi h ew modi ica ions. The E. coli con aining o e exp essed Si 2Tm we e lysed (as men ioned abo e) in 50 mM T is pH 7.5, 100 mM NaCl, 1 mM EDTA, 5 mM DTT and he inclusion bodies con aining o e exp essed Si 2Tm we e isola ed by mul iple cen i uga ion and washing s eps as desc ibed by Smi h e . al. and solubilized in 50 mM T is, pH 8.0, 4M U ea, 100 mM NaCl, 5 mM DTT, 25 µM ZnCl2 by ocking o e nigh a 4 ºC. The solubilized p o ein was dilu ed o 1 mg/ml and dialyzed o 3 hou s a 4 ºC in 50 mM T is, pH 8.0, 100 mM NaCl, 5 mM DTT, 25 µM ZnCl2 in o de o e old i ollowed by ano he 3 hou dialysis in Bu e A (40 mM T is, pH 8.0, 5 mM DTT, 25 µM ZnCl2). The p o ein was bound o HiT apQ HP anion exchange column (GE Heal hca e, USA) in Bu e A and elu ed in a g adien o bu e A supplemen ed wi h 1M NaCl a a low a e o 0.3 ml/min. The elu ed ac ions we e analyzed by SDS-PAGE and ac ions con aining Si 2Tm we e pooled (~28.7 % B), concen a ed o 1 ml and applied o a Supe ose12 size exclusion column (GE Heal hca e, USA) equilib a ed in 20 mM HEPES, pH 8.0, 150 mM NaCl, 5 mM DTT in o de o emo e u he con amina ions and agg ega ed p o eins. Si 2Tm con aining samples we e pooled, concen a ed o 10 mg/ml, snap ozen in liquid N2 and s o ed a -80 °C un il needed. AROS AROS con aining His- ag and His-MBP- ag was pu i ied simila o Si 1. AROS con aining GST- ag was a ini y pu i ied using Glu a hione esin simila o o he AROS cons uc s wi h he ollowing lysis, wash and elu ion bu e s: lysis bu e = 50 mM T is, pH 7.5, 150 mM NaCl, 0.2 mM PMSF, wash bu e = 50 mM T is, pH 7.5, 150 mM NaCl, 5 mM DTT and elu ion bu e = 50 mM T is, pH 8.0, 1500 mM NaCl, 5 mM DTT, 20 mM educed L-Glu a hione. Soulbiliza ion and pu i ica ion o AROS om E. coli pelle s we e p e o med as ollows: he lysa es esul ing om sonica ion we e cen i uged a 18,000 RPM o 45 minu es a 4 º C. The esul ing pelle was solubilized in 50 mM T is, pH 7.5, 300 mM NaCl, 10 mM Imidazole and 6M Guanidin hyd ochlo ide by s i ing a oom empe a u e o ~ 30 minu es. The sample was hen cen i uged a 18,000 RPM o 45 minu es a 20 ºC; supe na an was emo ed and incuba ed wi h TALON esin o 1 hou a oom empe a u e. The mix u e was hen applied o a column and he low h ough was collec ed. The p o ein was e olded in a s epwise manne by washing wi h 5 column olumes each o bu e 1 (50 mM T is, pH 7.5, 300 mM NaCl, 20 2. Ma e ials and Me hods 35  mM Imidazole, 8 M U ea), bu e 2 (bu e 1 con aining 4 M U ea), bu e 3 (bu e 1 con aining 2 M U ea), bu e 4 (bu e 1 con aining 1 M U ea) and inally wi h bu e 5 (bu e 1 wi hou U ea). This was ollowed by elu ion bu e (50 mM T is, pH 7.5, 300 mM NaCl, 150 mM Imidazole). The elu ed p o ein was concen a ed o 1 ml and applied on o a Supe ose12 column which was equilib a ed in 25 mM HEPES, pH 7.5, 100 mM KCl, 2 mM DTT and ac ions co esponding o AROS we e collec ed. 2.4.6 Fluo escence based pep ide deace yla ion assay A luo escen ly labeled pep ide called Fluo de Lys1 (FdL-1) based on he sequence o p53 wi h ace yla ion a Lys382 (RHKK[ac]-couma in) (Enzo Li e Sciences, USA) was used as a subs a e in deace yla ion assays. The p inciple behind he assay is he abili y o he Si uin o deace yla e he luo escen pep ide in he p esence o he co-subs a e NAD+, ollowing which he addi ion o T ypsin clea es he luo escen ag (couma in) leading o an inc ease in he luo escence. The assay was pe o med by addi ion o 1 g o Si uin, 100 M FdL-1, 1 mM NAD+ in he assay bu e (50 mM T is, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/ml BSA (bo ine se um albumin) ollowed by incuba ion o 30 minu es a 37 C. A e he incuba ion, a de elope mix u e con aining 2 mM NAM , 10 mg/ml T ypsin was added o he eac ion mix u e and incuba ed o 45 minu es a oom empe a u e, a e which he luo escence was measu ed on a FluoDiaT70 mic opla e eade (Pho al O suka Elec onics, Japan) using an exci a ion wa eleng h o 360 nm and an emission wa eleng h o 460 nm. A eac ion mix u e con aining all he componen s o he assay sans he enzyme was used as a blank and sub ac ed om he samples con aining he enzyme. While es ing o po en ial Si uin ac i a o s, he concen a ion o he subs a e (FdL-1) and he co-subs a e (NAD+) was kep a 25 M and 25/50 M espec i ely. Fo Si 3, he subs a e used was Fluo de Lys2 (FdL-2) which is based on he sequence o p53 pep ide 317-320 (QPKK[ac]-couma in) (Enzo Li e Sciences, USA), because he deace yla ion e iciency was highe when compa ed o he FdL-1 pep ide subs a e. 2.4.7 Enzyme-linked immunoso ben assay (ELISA) Deace yla ion o Glu ama e dehyd ogenase (GDH) by Si 3 and Cy . c by Si 5 was ca ied ou using ELISA as desc ibed in Schlicke e . al., (Schlicke e al., 2008). 10 g o Cy . c o mi ochond ial GDH (bo h om Sigma, USA) was dissol ed in 100 l o TBS (T is bu e ed saline; 50 mM T is, pH 7.4, 150 mM NaCl) and incuba ed a 4 C o e nigh in a 96 well mic o i e pla e (Beck on and Dickenson, USA). The pla es we e washed wi h TBST 2. Ma e ials and Me hods 36  (TBS bu e con aining 0.1 % Tween-20) and incuba ed wi h blocking bu e (TBST con aining 3 % skimmed milk) o 2 h s a oom empe a u e. The pla es we e again washed wi h TBST, ollowed by TBS. 10 g o Si 5 o Si 3 was added o he pla es wi h a ying concen a ions o NAD+ and incuba ed a 37 C o 30 minu es ollowed by washing wi h TBST and TBS. An i ace yl lysine an ibody (S essgen, USA) a a a io o 1:1000 we e hen added o he pla es and incuba ed o 2 h s a oom empe a u e and washed wi h TBST. A seconda y an ibody (Sigma, USA) aised agains abbi IgG conjuga ed wi h ho se adish pe oxidase was added a a dilu ion o 1:2000 and incuba ed o 2 h s a oom empe a u e and washed ho oughly wi h TBST. The pe oxidase subs a e TMB (3,3’-5,5’- e ame hylbenzidin) (Sigma, USA) was added a a concen a ion o 1.5 mg/ml o he pla es and incuba ed o 3 minu es be o e s opping he eac ion wi h 1M H2SO4. The eac ion was quan i ied calo ime ically a a wa eleng h o 450 nm using an EL800 Mic opla e eade (BioTek, USA). 2.4.8 Con inuous assay o deace yla ion based on coupled enzymes A con inuous deace yla ion assay o Si uins ecen ly desc ibed by Smi h e . al., (Smi h e al., 2009) based on he p inciple o a coupled assay was also used o moni o he deace yla ion ac i i y o Si uins. In his assay, one o he eac ion p oduc NAM, se es as a subs a e o a downs eam enzyme Nico inamidase, which con e s i o NH3, which in u n is used by GDH in he p esence o NADPH and -ke oglu a a e o p oduce L-glu ama e and NADP+. The dec ease in abso bance a 340 nm due o he consump ion o NADPH (340 = 6.22 mM-1 cm-1) is moni o ed con inuously o e ime using a spec opho ome e (Ca y 50, Agilen echnologies, USA). A ypical eac ion mix u e consis ed o 1 M Si u in, 1 mM pep ide subs a e, 1 mM NAD+, 1 mM DTT, 3.3 mM -ke oglu a a e, 2 M ag clea ed Nico inamidase (Salmonella en e ica), 2 uni s o bo ine GDH o 0.3 uni s o p o eus GDH and 0.2 mM NADPH in 20 mM Na-PO4, pH 7.5 in a o al olume o 100 l. The eac ion was pe o med a 25 C using a Pel ie empe a u e con olle de ice. All componen s bu o he Si uin was added ini ially and he eac ion was moni o ed o 5 minu es a e blanking o es ablish a base line ollowed by he addi ion o Si uin. 2.4.9 Limi ed p o eolysis o p o eins In o de o iden i y po en ial s able cons uc s o Si 1 o c ys alliza ion, a limi ed p o eolysis app oach was pe o med. Roughly 10 g o p o ein was incuba ed wi h 0.1 g o p o ease (T ypsin, Chymo ypsin o Sub ilisin) o 30 minu es a 4 C o a oom empe a u e, 2. Ma e ials and Me hods 37  ollowed by quenching wi h ho SDS loading bu e (main ained a 80 C) and un on SDS- PAGE. The Coomassiee s ained and de-s ained gel was ans e ed o a PVDF (poly inylidene luo ide) memb ane, he mos p ominen band was cu and sen o N- e minal sequencing. (Toplab, Ge many). 2.4.10 The mal dena u a ion shi assay P o ein he mal dena u a ion was ollowed by exploi ing he change in he luo escence o he dye SYPRO O ange (Li e Technologies, USA), which upon binding o hyd ophobic (un olded) pa s o he p o ein inc eases i s in insic luo escence. 3 g o o al p o ein was aken in a 96 well mic o i e pla e (Bio-Rad, USA), 1 l o 1 o10 dilu ed SYPRO dye was added o i and made up o 50 l using di e en bu e s ollowed by 15 l o mine al oil. The bu e s used we e 50 mM each o sodium ci a e (pH 4.0 and 5.0), MES (2-(N- Mo pholino)E haneSul onic acid) (pH 6.0), HEPES (pH 7.0), T is (pH 8.0) and CAPSO (N- cyclohexyl-3-aminop opanesul onic acid) (pH 9.0) and he sal concen a ions we e 0, 50, 200 and 500 mM KCl. The empe a u e was g adually inc eased om 25 C o 73 C in 2 C in e als. The change in luo escence was ollowed using a FluoDiaT70 mic opla e eade (Pho al O suka Elec onics, Japan) using an exci a ion wa eleng h o 465 nm and an emission wa eleng h o 580 nm. 2.4.11 Mic oscale he mopho esis Binding measu emen s we e pe o med using he p inciple o mic oscale he mopho esis on a NanoTempe Monoli h NT.115 ins umen (NanoTempe Technologies GmbH, Ge many). 20 µM p o ein was dialyzed agains 100 mM ammonium ca bona e ((NH4)2CO3), pH 8.3 o 3 hou s a 4 °C and mixed wi h NT-647 luo escen dye a a a io o 1:1 in a o al olume o 250 µl and incuba ed a oom empe a u e o 30 minu es in he da k. The un eac ed dye was emo ed om he p o ein by applying he sample on a gel il a ion column (NAP column, GE Heal hca e, USA) ha was p e-equilib a ed in 25 mM HEPES, pH 7.5, 100 mM KCl, 2 mM DTT. The labeling e iciency was moni o ed spec opho ome ically by calcula ing he concen a ion o p o ein and dye using hei espec i e ex inc ion coe icien s ( he ex inc ion coe icien o NT-647 dye is 250000 M-1 cm-1 a 650 nm and he ex inc ion coe icien o human Si 1 a 280 nM is 45840 M-1 cm-1). An al e na i e label called FITC ( luo escein iso hiocyana e) was also used wi h he same p ocedu e, excep o changes in he pH o he bu e (100 mM (Na)2CO3, pH 9.3), he a io o p o ein o dye (1:2) and incuba ion ime (1 h a oom empe a u e in he da k). The ex inc ion coe icien o FITC 2. Ma e ials and Me hods 38  dye is 73,000 M-1 cm-1 a 495 nM in pH 9.3 bu e and he ex ension coe icien o Si 2Tm is 13410 M-1 cm-1 a 280 nM. The elu ion bu e o Si 2Tm was 20 mM HEPES, pH 7.5, 150 mM NaCl. Typical a ios be ween p o ein and dye we e ~ 1:1 (NT-647) and ~ 1:1.66 (FITC). 200 nM o he labeled p o ein o 1 µM o he label ee p o ein we e mixed wi h di e en concen a ions o he ligands o be i a ed and loaded on hin glass capilla ies o analysis. Mic oscale he mopho esis o he p o ein sample was ollowed a e applying a de ini e amoun o hea on he sample using an in a ed lase and ollowing he luo escence simul aneously, since i is coupled o he lase pa h using an in a ed dich oic mi o and ocused on he sample h ough he same objec i e. Analysis o he esul s we e pe o med using G aFi (E i hacus So wa e Limi ed, UK). 2.4.12 Analy ical ul acen i uga ion AUC (analy ical ul acen i uga ion) expe imen s we e pe o med a Hanno e medical school wi h he help o D . U e Cu h. B ie ly, di e en concen a ion o Si 1 samples we e p epa ed in 25 mM HEPES, pH 7.5, 100 mM KCl, 0.5 mM DTT bu e and cen i uged a 40,000 RPM on a Beckman Op ima XL-A ul acen i uge i ed wi h An-50 Ti o o s ha was main ained a 20 ºC. Concen a ion p o iles we e measu ed wi h he UV- abso p ion scanning op ics a a wa eleng h o 280 nm. The measu ed concen a ion p o iles we e e alua ed using he p og am SEDFIT (Pe e , 2000) which ans o med hem in o di usion co ec ed sedimen a ion coe icien c(s) dis ibu ions. 2.5 Mass spec ome y 2.5.1 Mass spec ome y based deace yla ion assay A no el MS (mass spec ome y) based assay was de eloped o assay Si uins. 100 o 250 nM Si uin was incuba ed wi h 5 o 50 M subs a e pep ide and 50 M NAD+ in 25 mM HEPES, pH 7.5, a 37 C. A e he equi ed ime was eached, an aliquo was wi hd awn and quenched wi h equal olume o 0.1 % ( / ) FA ( o mic acid), cen i uged using a 10 kDa cu o cen i ugal concen a o (Pall Li e Sciences, USA) and 5 o 10 l o he il a e applied o a LC-ESI-MS (LTQ XL mass spec ome e , The mo Scien i ic, USA) coupled o an HPLC (Shimadzu, USA). The samples we e loaded o e a K omasil 100 C18 5 µm p e-column (Higgins Analy ical, USA) ollowed by a e e se phase C18 column a a low a e o 300 nl/min. The ace yla ed and deace yla ed pep ides we e sepa a ed using a linea g adien om 0 % o 45 % bu e B wi hin 30 min (bu e A: 0.1 % TFA ( i luo oace ic acid), 0.02 % HFBA (hep a luo obu y ic acid); bu e B: 70 % ACN (ace oni ile), 0.1 % TFA, 0.02 % 3. Resul s 45  Figu e 3.2: Pu i ica ion and seconda y s uc u e analysis o Si 1. A) 10 % (w/ ) SDS polyac ylamide gel showing he pu i y o ull leng h Si 1 a e AC (A ini y Ch oma og aphy), SEC (Size Exclusion Ch oma og aphy) and AEC (Anion Exchange Ch oma og aphy). Al hough Si 1’s molecula weigh is ~82 kDa, i uns a ~ 110 kDa in SDS-PAGE, due o he high amoun o posi i e cha ge. B) CD spec a o 9.5 µM ull leng h Si 1 collec ed a 15 ºC, pH = 7.5. Mean esidue mola ellip ici y [ϴ] is plo ed agains wa eleng h in nm. Si 1 has p ope seconda y s uc u e wi h ~ 10.5 % α-helix and ~ 22.5 % β- s and con en as de e mined using he K2D3 p og am. Spec opho ome ic analysis o a ini y pu i ied Si 1 e ealed an unusually high abso bance a 260 nm compa ed o 280 nm (high A260/A280 a io) (Figu e 3.3a), an indica ion o nucleic acid con amina ion, because SEC and AEC pu i ied Si 1 beha es as expec ed. Fu he analysis o he ac ions using aga ose gel elec opho esis e ealed high amoun s o nucleic acids in he elu e ac ions (Figu e 3.3b). Incuba ion o hese ac ions wi h DNase lead o a mode a e dec ease in he amoun o nucleic acids, whe eas incuba ion wi h RNase lead o almos comple e loss o nucleic acids. These esul s indica e ha Si 1 co-pu i ies wi h nucleic acids, in pa icula RNA (Figu e 3.3b). Inclusion o ei he DNase o RNase o bo h du ing he pu i ica ion p ocess esul ed a dec ease in abso bance a 260 nm only when RNase was p esen (Figu e 3.3a). To a oid nucleo ide con amina ion, u he Si 1 a ini y pu i ica ion s eps included DNase and RNase ea men . 3. Resul s 46  Figu e 3.3: Si 1 appea s o co-pu i y wi h nucleo ides. A) Spec oscopic analysis o di e en ac ions o a ini y pu i ied Si 1 indica es high A260/A280 a io o con ol sample, whe eas samples ea ed wi h nucleases shows a dec ease in he a io. (A260/A280 a ios a e con ol: ~1.08, wi h DNase: ~1.06, wi h RNase: ~0.68, wi h DNase+RNase: ~ 0.70). B) 1 % (w/ ) aga ose gel elec opho esis o Si 1 in he p esence o nucleases indica es a dec ease in nucleic acid con en when Si 1 was incuba ed wi h bo ine DNase (mode a e dec ease) o bo ine RNase A (comple e loss). Si 1 was incuba ed wi h Sub ilisin o in es iga e i loss o Si 1 leads o a shi in he mig a ion o nucleic acids. ~ 6.25 µg Si 1 was incuba ed wi h 0.625 ng Sub ilisin (10000 old less), 0.25 µg DNase o 0.5 µg RNase o 15 minu es a 37 ºC and loaded on he gel. To u he in es iga e i Si 1 binds o nucleo ides, he p o ein was incuba ed wi h ei he DNA o RNA and esol ed on an aga ose gel. Figu e 3.4a shows a ep esen a i e gel whe e he mobili y o dsDNA (double s anded DNA) was no a ec ed by he p esence o Si 1, possibly indica ing a lack o di ec in e ac ion be ween he wo. Simila expe imen s o iden i y in e ac ions be ween Si 1 and RNA p o ed unsuccess ul. We also es ed he in luence o nucleic acids on he ac i i y o Si 1, by pe o ming ac i i y assays in he p esence and absence o nucleic acids (Figu e 3.4b). The assays e ealed ha Si 1’s ac i i y is no in luenced in he p esence o ei he DNA o RNA. I emains o be cla i ied why Si 1 seems o s ongly associa e wi h RNA when exp essed in E. coli. 3. Resul s 47  Figu e 3.4: Nucleo ides nei he in e ac wi h Si 1 no in luence i s ac i i y. A) Mobili y shi assay pe o med on 1 % (w/ ) aga ose gel shows no shi o dsDNA (296 base pai s) in he p esence o Si 1. 1X con ol = 45 ng DNA. The a io indica es he amoun o excess Si 1 added o e DNA. 1 mM NAD+ was added sepa a ely o access i s in luence on Si 1. The lack o shi in he mig a ion o dsDNA indica es no binding wi h Si 1. B) The ac i i y o 1 µg Si 1 wi h 100 µM FdL-1 pep ide and 1 mM NAD+ was measu ed wi h PCR ampli ied dsDNA (1467 base pai s, 1:0.3 a io o Si 1:DNA) o RNA o E. coli (~ 75 base pai s, 1:10 a io o Si 1:RNA). 3.1.2 In es iga ing he ole o e mini in he oligome iza ion o Si 1 Mammalian Si uins (Si 2-Si 7) and o he Si uins ha e so a been epo ed as monome s (Finnin e al., 2001; Schlicke e al., 2011; Schlicke e al., 2008; Schue z e al., 2007) excep o he yeas homolog Hs 2, which was epo ed as ime in bo h solu ion and c ys al s uc u e and he N- e minus was shown o be in ol ed in ime o ma ion (Zhao e al., 2003). SEC p o ile o lag- agged Si 1 o e exp essed and pu i ied om HeLa cells sugges ed ime iza ion wi h an appa en molecula weigh o ~ 350 kDa (Vaque o e al., 2004). We pe o med gel il a ion analysis on he ull leng h and dele ion cons uc s o Si 1 o analyze which domain(s) migh be esponsible o oligome iza ion. SEC p o iles o ull leng h, N- e minal and C- e minal dele ion cons uc s indica ed a highe o de oligome like beha io (simila o HeLa cells pu i ied Si 1), whe eas cons uc s lacking bo h e mini beha ed as lowe o de oligome s (Figu e 3.5). The appa en sizes seen on SEC could be a esul o oligome iza ion o an ex ended shape o ull leng h Si 1 and i s cons uc s. BN- PAGE on a ious Si 1 cons uc s showed esul s simila o size exclusion p o iles o ull leng h and 214 o747 cons uc s bu he 1 o664 and 214 o664 cons uc s we e seen as a smea on he gel despi e epea ed a emp s, leading o a lack o clea unde s anding on he na u e o 3. Resul s 48  oligome iza ion (Figu e 3.6). I ull leng h Si 1 indeed is a ime o highe o de oligome , i should ha e a molecula weigh o a leas ~ 240 kDa o abo e and he e o e migh be a good candida e o EM (Elec on Mic oscopy) s udies. C yo EM s udies on ull leng h Si 1 (Figu e 3.7a) indica es i o be a monome , howe e his could ha e also esul ed due o dilu ion du ing sample p epa a ion. We he e o e used AUC o analyze he oligome iza ion o Si 1, because AUC is insensi i e o molecula shape. Figu e 3.7b shows he esul s o sedimen a ion eloci y AUC uns pe o med on di e en Si 1 cons uc s. Unde he condi ions es ed, all Si 1 samples seem o beha e as monome s in AUC expe imen s. Figu e 3.5: SEC indica es oligome iza ion o Si 1. A) SEC elu ion p o iles o di e en Si 1 cons uc s: no malized abso bance (a 280 nm) is plo ed agains elu ion olume. B) A plo o Loga i hm o SEC s anda ds agains elu ion olume. Compa ing he elu ion olume o Si 1 cons uc s o ha o he s anda ds indica e oligome ic beha io o all Si 1 excep o he ca aly ic co e cons uc . FL = ull leng h. Appa en molecula weigh s based on SEC s anda ds: FL – 230 kDa ( ime ), 1 o664 – 158.7 kDa (dime ), 214 o747 – 95.3 kDa, 214 o664 – 90.9 kDa, 225 o664 – 77.2 kDa ( he molecula weigh s o he la e h ee cons uc s co esponds in be ween monome and dime ) and 229 o516 – 33.4 kDa (monome ). 3. Resul s 49  Figu e 3.6: BN-PAGE Analysis o Si 1 indica es oligome iza ion. Le panel: A ca oon ep esen a ion o Si 1 cons uc s used in p obing he domain in ol emen in oligome iza ion o Si 1. Righ panel: BN-PAGE analysis o di e en Si 1 cons uc s along wi h p o ein ma ke s indica e an oligome iza ion p o ile o Si 1 cons uc s simila o SEC. 10 µg o each ma ke p o eins we e loaded in he gel along wi h 27 µg FL ( ull leng h), 30 µg 1 o664, 27 µg 214 o747, 32 µg 214 o664 Si 1 cons uc s espec i ely Figu e 3.7: EM and AUC analysis indica es Si 1 o be monome ic. A) Si 1 a a concen a ion o ~10 µg/ml was nega i ely s ained wi h U anyl o ma e, adso bed on g ids and obse ed unde a JOEL-1400 elec on mic oscope a a magni ica ion o 50,000. Full leng h Si 1 can be isualized as small iny pa icles which a e globula and homogenous indica ing monome ic beha io . EM image analysis was pe o med a MPI-Do mund in he lab o D . S e an Raunse . B) A ep esen a i e plo o con inuous c(s) dis ibu ion agains sedimen a ion coe icien (co ec ed o wa e a 20 ºC) om sedimen a ion eloci y AUC uns o Si 1 cons uc s indica e a clus e ing a a ound sedimen a ion coe icien alue o ~ 3.1-3.9 S implying a monome ic beha io o all he cons uc s. All uns we e pe o med a 20 ºC and 40,000 RPM and he p o ein concen a ion was moni o ed a 280 nm. 3. Resul s 50  3.1.3 The ca aly ic domain o Si 1 is su icien o ac i a ion by polyphenols Res e a ol was one o he i s small molecules iden i ied ha can ac i a e Si 1. The unique N- e minus o Si 1 was epo ed o be essen ial o he ac i a ing e ec o es e a ol (Milne e al., 2007), bu o he s udies ha e shown ha es e a ol can also ac i a e yeas Si 2, whose N- e minal domain is un ela ed o i s human coun e pa (Howi z e al., 2003). To u he in es iga e he egions esponsible o Si 1 ac i a ion by es e a ol, we assessed he ac i i y o ull leng h Si 1 and he dele ion cons uc s in he p esence and absence o es e a ol using he luo ogenic subs a e pep ide FdL-1 employing bo h he luo escence as well as MS based assays (Figu e 3.8). The assay e ealed a ious basal ac i i ies o di e en cons uc s (Figu e 3.9), bu s imula ion by es e a ol was obse ed in each cons uc including he sho es one comp ised o jus he ca aly ic domain. To e i y i his ac i a ion can also be achie ed by picea annol (ano he na u ally occu ing polyphenol e y simila o es e a ol in s uc u e) we pe o med he same assay in he p esence o picea annol. Indeed as expec ed ac i a ion by picea annol also equi ed only he ca aly ic domain (Figu e 3.10). Compa ison o ac i a ion ela i e o basal le el in bo h es e a ol and picea annol indica es ha all cons uc s can be s imula ed in a simila manne , e en hough hei basal ac i i y di e s. To es i he di e ence in basal ac i i y is in pa due o he s abili y o he cons uc s, we pe o med he mal shi dena u a ion assays. In a he mal shi assay, he p o ein is dilu ed in a a ie y o bu e and sal condi ions and a luo escen dye is added o i . P o ein un olding is moni o ed by eco ding he inc ease in luo escence ( he dye binds o hyd ophobic pa s o he p o ein) wi h g adual aise in empe a u e, which is hen i ed o an equa ion, o ob ain he hal - ansi ion poin , Tm (Tempe a u e o Mel ing). Figu e 3.11 shows ep esen a i e he mal shi cu es o di e en Si 1 cons uc s. Wi h he excep ion o ull leng h Si 1, mel ing cu es wi h mo e han one ansi ion we e ob ained, so ha no Tm alues could be ob ained wi h he s anda d wo-s a e model, Howe e , i appea s ha all he cons uc s ha e simila s abili y, sugges ing a ole o he e mini in modula ing he ac i i y o Si 1 (see discussion sec ion). 3. Resul s 51  Figu e 3.8: Ca aly ic domain is su icien o ac i a ion by es e a ol. A) Full leng h (FL) Si 1 along wi h di e en unca ion cons uc s we e used in he luo escence assay con aining FdL-1 pep ide subs a e, NAD+ and 100 µM Res e a ol (R) . B) The same assay was pe o med wi h FL ( ull leng h) and he ca aly ic cons uc 225 o664 and analyzed by MS. In bo h he cases ac i a ion by es e a ol can be obse ed e en o he sho es cons uc which con ains only he ca aly ic domain. Con ol samples con ained 1 % ( / ) DMSO and he assay was pe o med a 37 ºC. Figu e 3.9: The ac i i y o Si 1 appea s o be egula ed by i s e mini. ~ 200 nM each Si 1 cons uc was used in he FdL-1 subs a e based luo escence assay. The basal ac i i y o Si 1 appea s o be dependen on he e mini, as he cons uc con aining only he ca aly ic co e domain has lowe ac i i y compa ed o he ull leng h p o ein (FL = ull leng h). 3. Resul s 52  Figu e 3.10: Picea annol can also ac i a e he ca aly ic co e o Si 1. Simila o es e a ol 100 µM Picea annol (P) is also able o ac i a e Si 1 comp ising only he ca aly ic domain. The FdL-1 subs a e was used in he luo escence based assay. (FL = ull leng h). Figu e 3.11: The mal dena u a ion shi assay on di e en Si 1 cons uc s. Rep esen a i e he mal shi cu es o ou di e en Si 1 cons uc s comp ising ei he FL ( ull leng h), lacking N o C- e minus o bo h indica e an unusual ansi ion s a e o he p o eins. Ne e heless, all he p o ein samples appea o ha e simila s abili ies. Fi ing he cu e o FL using a wo s a e ansi ion e eals a T m close o ~ 47 ºC. 3. Resul s 53  3.1.4 Iden i ica ion o no el pep ide subs a es o Si 1 O e 6800 ace yla ion si es ha e been iden i ied in mammalian p o eome, p esen ing an excellen oppo uni y o pe o m subs a e p o iling on mammalian Si uins o iden i y no el iso o m speci ic subs a es as well as hei subs a e sequence p e e ences. In collabo a ion wi h D . Mike Schu kowski’s labo a o y a Uni e si y o Halle, Ge many, we es ablished a chip-based a ay sys em ha can be used o cha ac e ize iso o m speci ic subs a e p e e ences o di e en mammalian Si uins (Rauh e . al. manusc ip in p epa a ion). Roughly 6800 ace yla ed pep ides comp ising six esidues N and C- e minal om he ace yla ion si es we e syn hesized and spo ed on a chip o c ea e an a ay (he ein e e ed o as ace ylome chip) simila o he one desc ibed by Schu kowski e . al. (Schu kowski e al., 2004). To iden i y no el subs a e pep ides o Si 1, he p o ein was mixed wi h NAD+ and incuba ed on he ace ylome chip. As a con ol, we incuba ed a chip wi h only bu e and NAD+ o Si 1 ca aly ic mu an H363A and NAD+. The deace yla ion e iciency o Si 1 was analyzed by employing an ELISA ype me hod as shown in igu e 3.12. Se e al new po en ial subs a es we e iden i ied o Si 1 such as Lys12 o HMG-B1 (High Mobili y G oup B1), Lys628 o TFIID (T ansc ip ion ini ia ion ac o TFIID subuni 3) o name a ew. Figu e 3.12. Schema ic ep esen a ion o he p inciple behind he ace ylome-chip assay. The chip con aining se e al ace yla ed pep ides is i s incuba ed wi h Si 1, NAD+ and modula o s. This is ollowed by incuba ion wi h an i-ace yllysine an ibody and a luo opho e labeled seconda y an ibody. E icien deace yla ion by Si 1 will lead o a dec ease in luo escence, which is compa ed wi h a bu e con ol. Figu e kindly p o ided by D . Da id Rauh, Uni e si y o Halle, Ge many. To ensu e ha subs a e pep ides iden i ied using he a ay a e also e icien ly deace yla ed in solu ion, we es ed se e al pep ides using he con inuous assay (Figu e 3.13). As expec ed, pep ides we e deace yla ed by Si 1 in solu ion wi h a ying deg ee. Kine ic cha ac e iza ion o H3-Lys116 using he con inuous assay esul ed in simila kine ic alues 3. Resul s 54  compa ed o p53-Lys382 pep ide, a widely used subs a e pep ide o Si 1 (Figu e 3.14). I Lys116 and Lys12 o ull leng h H3 and HMG-B1 p o eins, espec i ely, a e accessible o Si 1 hey will be deace yla ed, indica ing ha deace yla ed lysine esidues om a ay expe imen s se e as excellen candida es o es i hey se e as, physiological Si 1 subs a es. Figu e 3.13. No el subs a es o human Si 1. A con inuous assay was pe o med using 1 µM Si 1, 0.64 mM espec i e pep ides and 1 mM NAD+. The dec ease in abso bance a 340 nM was ollowed o e ime in a 96 well pla e eade . The abso bance o a con ol (wi hou pep ide subs a e) was sub ac ed om each pep ide alue and plo ed o e ime. The known Si 1 subs a e si e p53-K382 pep ide was used as a posi i e con ol and AATase-K159 (Aspa a e amino ans e ase 2) which was no deace yla ed on he a ay as a nega i e con ol. Figu e 3.14: Michaelis-Men en plo o Si 1 kine ics. A hype bolic ep esen a ion o he kine ics o ca alysis o Si 1 using p53-Lys382 pep ide (A) and H3-Lys116 pep ide (B) shows ha he kine ic p ope ies o H3 pep ide iden i ied om he ace ylome-a ay is simila o he commonly used p53 pep ide. 3. Resul s 61  Fgi e 3.21: C ys alliza ion o Si 1. A ep esen a i e pic u e o po en ial Si 1 c ys als s ained wi h a ed dye. The concen a ion o dye in he c ys al could indica e po en ial p o ein c ys al. The c ys alliza ion condi ions a e: hSi 1 225-664 SER mu an a a concen a ion o 10 mg/ml, 300 mM ci ic acid and 40 % ( / ) MPD ((±)-2-Me hyl-2,4-pen anediol). Figu e 3.22: Phospho yla ion mimic o Si 1 may egula e i s ac i i y. ~ 200 nM ull leng h WT (wild ype) and Th 530Asp Si 1 we e incuba ed wi h 100 µM FdL-1 subs a e and 1 mM NAD+. A wo old highe ac i i y can be obse ed o he mu an compa ed o wild ype Si 1. 3.1.9 S udies on AROS Human AROS is a small nuclea p o ein (15.4 kDa), shown o ac i a e human Si 1 by in e ac ing wi h i s N- e minus ( esidues 114 o217) (Kim e al., 2007). To da e he e a e no biochemical o s uc u al s udies ha cha ac e ize he Si 1 in e ac ion wi h AROS. In o de o unde s and how AROS egula es Si 1 unc ion, we a emp ed o e exp ession and pu i ica ion o AROS in E. coli. AROS con aining an N- e minal hexa his idine ag was o e exp essed in high yield, bu was mos ly insoluble (Figu e 3.23A). Se e al a emp s we e made o inc ease he solubili y o AROS, wi hou success. A emp s o solubilize he p o ein om inclusion bodies using guanidine hyd ochlo ide ollowed by on-column e olding and a ini y pu i ica ion we e success ul and yielded pu e p o ein which elu es as a single peak in SEC 3. Resul s 62  wi h an appa en molecula weigh co esponding o ha o a monome (Figu e 3.23 B) and a CD spec um indica es ha i possesses seconda y s uc u e (Figu e 3.23C). When FdL-1 based luo escence assay was pe o med using AROS and Si 1 a e y mild dec ease in Si 1 ac i i y was obse ed (Figu e 3.24A). To pu i y AROS in i s na i e o m, we c ea ed cons uc s ha bo ing GST and MBP on he N- e minus o AROS espec i ely. Al hough GST- agged AROS was mo e soluble han His- agged AROS, pu i ica ion was no possible as he GST ag did no bind o Glu a hione esin. MBP- agged AROS was soluble and easily pu i ied wi h oughly 60 % pu i y (Figu e 3.23A), bu e o s o clea e he MBP ag o u he pu i ica ion o he p o ein esul ed in almos comple e p ecipi a ion o AROS. Ne e heless MBP- agged AROS was used in FdL-1 assay o in es iga e he modula ion o Si 1. Al hough no s a is ically signi ican , he esul s show a mild inhibi ion o Si 1 (Figu e 3.24B). Figu e 3.23: Pu i ica ion and seconda y s uc u e analysis o AROS. A) 15 % SDS polyac ylamide gel showing he exp ession and pu i y o His- agged AROS (indica ed wi h a s a ) and a 12 % SDS polyac ylamide gel showing he pu i y o MBP-AROS (hexagon) a e a ini y ch oma og aphy. B) The SEC p o ile o e olded AROS indica es an appa en monome ic beha io . The inse shows he pu i y o e olded AROS on a 15 % SDS polyac ylamide gel. C) The CD spec um o e olded AROS indica es p esence o seconda y s uc u e. 3. Resul s 63  Figu e 3.24: In luence o AROS on he ac i i y o Si 1. The in luence o e olded AROS p o ein on he ac i i y o ~ 200 nM Si 1 was analyzed using 100 µM FdL-1 pep ide, 1000 µM NAD+ and 1:1 o 1:10 mola excess o AROS. B) The same assay pe o med in he p esence o 20 old mola excess o MBP agged AROS wi h 50 µM FdL-1 subs a e, 1000 µM NAD+ and ~ 200 nM Si 1. Unde he condi ions es ed, AROS seems o weakly inhibi Si 1, bu u he in es iga ion is equi ed o con i m his esul . 3. Resul s 64  3.2 S udies on he ole o Zinc in Si 3 The human Si 3 p o ein cloning, exp ession and pu i ica ion p ocedu es we e pe o med as men ioned in sec ions 2.2.5, 2.2.6 and 2.4.4 espec i ely. Sligh modi ica ions adop ed o Si 3 a e de ailed explici ly. The pu i y o he p o ein was ≥ 95 % and he yield was ~ 3 mg/li e o E. coli cul u e. Figu e 3.25 shows he pu i y o Si 3 isualized on an SDS-PAGE. The enzyme showed ac i i y in assays (see below) indica ing a well olded and ac i e p o ein.  Figu e 3.25: Pu i ica ion o Si 3. 15 % (w/ ) SDS polyac ylamide gel showing he pu i y o ag clea ed Si 3 (114-399) a e size exclusion ch oma og aphy. To unde s and he ole o Zinc in he s uc u e and unc ion o mammalian Si uins, Si 3 was used as a model because o i s ease o pu i ica ion, ac i i y, and a ailabili y o c ys al s uc u ed. Si 3, like all o he mammalian Si uins s udies so a con ains ou conse ed Cys eines (256, 259, 280 and 283) (supplemen al igu e S2), which coo dina e a Zinc ion. To p obe he ole o Zinc, si e di ec ed mu agenesis s udies we e pu sued by mu a ing he Cys eines ha coo dina e he Zinc ion (C259A o C259/280A). The mu an p o eins we e o e exp essed in E. coli Rose a 2 (DE3) cells simila o he wild ype p o ein. P o ein o e exp ession was obse ed, bu a emp s o pu i y ei he he single o he double mu an p o ed u ile, because he p o eins we e ound in he insoluble ac ion (Figu e 3.26). Inclusion o ZnCl2 (10 µM) ei he in he pu i ica ion bu e o while o e exp essing he p o ein also did no yield soluble p o ein. These exp ession s udies indica e ha he mu an p o eins appea no o old p ope ly o o be uns able. 3. Resul s 65  Figu e 3.26: Pu i ica ion o Si 3 Cys eine mu an s. A ep esen a i e 12 % (w/ ) SDS polyac ylamide gel showing he p esence o Si 3 Cys eine mu an (C259A) in he pelle .  Since mu a ional s udies p o ed unsuccess ul in ob aining soluble p o ein, he wild ype p o ein was used o p obe he ole o Zinc. Si 3 was incuba ed wi h EDTA a 4 ºC o 30 minu es, ollowed by cen i uga ion o 5 minu es a 14,000 RPM a 4 ºC and assayed using he comme cially a ailable luo escence based assay ki as men ioned in sec ion 2.4.6. As shown in Figu e 3.27a, he ea men o EDTA did no a ec he ac i i y o Si 3, indica ing ei he a s ong a ini y be ween Zinc and Si 3 o a lack o unc ion o Zinc. On he o he hand, incuba ion o Si 3 wi h 200 µM 1,10-phenan h oline immedia ely p ecipi a ed he p o ein leading o a comple e loss o ac i i y as shown in Figu e 3.27b. Thus, Zinc is essen ial o s abili y o Si 3. Figu e 3.27: Zinc is essen ial o he s abili y and ac i i y o Si 3. The luo escence assay was used o measu e he ac i i y o Si 3, using 100 µM FdL-2 pep ide subs a e (QPKK[ac]- couma in) and 1 mM NAD+. A) 1 µg o Si 3 was incuba ed wi h ei he bu e o 2 mM EDTA o 30 minu es on ice and cen i uged be o e pe o ming he assay. B) Si 3 was incuba ed wi h bu e o wi h 200 µM O-phenan h oline and cen i uged be o e pe o ming he assay. 3. Resul s 66  3.3 S udies on Si 5 The human Si 5 p o ein cloning, exp ession and pu i ica ion p ocedu es we e pe o med as men ioned in sec ions 2.2.5, 2.2.6 and 2.4.4 espec i ely. Sligh modi ica ions adop ed o Si 5 a e de ailed explici ly. The pu i y o he p o ein was ≥ 95 % and he yield was ~ 15 mg/li e o E. coli cul u e. Figu e 3.28 shows he pu i y o Si 5 isualized on an SDS-PAGE. The enzyme showed ac i i y in se e al assays (see below) indica ing a well olded and ac i e p o ein. Figu e 3.28: Pu i ica ion o Si 5. 15 % (w/ ) SDS polyac ylamide gel showing he pu i y o ag clea ed Si 5 (34-302) a e size exclusion ch oma og aphy. 3.3.1 In luence o NAD+ on he ac i i y o Si 5 The human Si 5, which is localized in he mi ochond ia, is poo ly cha ac e ized in e ms o subs a es and egula o s compa ed o i s iso o ms and o da e only one in i o subs a e (CPS1) is known. Ou lab had p e iously iden i ied ha Cy c, can be deace yla ed in i o by Si 5 in an ELISA (Schlicke e al., 2008). In gene al, Si 5 seems o beha e as a weak deace ylase compa ed o o he Si uins when luo ogenic pep ides o Cy . c a e used as subs a es (Schlicke e al., 2008). In o de o iden i y i Si 5’s ac i i y is dependen on i s co- subs a e NAD+ an ELISA was pe o med in he p esence o inc easing amoun s o NAD+ and 10 µg o Cy c. As shown in Figu e 3.29, he deace ylase ac i i y o Si 5 inc eased d ama ically wi h inc easing concen a ion o NAD+ un il 10 mM, leading o a ~ 200 % inc ease in ac i i y. 3. Resul s 67  Figu e 3.29: Si 5 appea s o equi e highe amoun s o NAD+ o i s deace ylase ac i i y. An ELISA was pe o med wi h 10 µg Cy . c as subs a e and 10 µg Si 5 and inc easing amoun s o NAD+. A dec ease in abso bance indica es an inc ease in he abili y o Si 5 o deace yla e he subs a e Cy . c. The ac i i y o Si 5 inc eased linea ly un il 10 mM NAD+, beyond which no inc ease was seen. 3.3.2 Si 5 appea s o be insensi i e o Nico inamide inhibi ion NAM, one o he p oduc s o Si uin ca alyzed deace yla ion is a po en physiological inhibi o (IC50 in he mic o mola ange) o se e al Si uins including Si 1 and Si 3. In o de o iden i y i NAM can also inhibi Si 5 an ELISA was pe o med using 10 µg Cy . c and 5 mM NAD+ (Figu e 3.30). E en in he p esence o 10 mM NAM, he ac i i y o Si 5 was no a ec ed, indica ing ha Si 5 migh possess unique ea u es in he NAM binding pocke (“C pocke ”) ha ende s i insensi i e o inhibi ion by NAM (A alos e al., 2005; Sau e and Sch amm, 2003). 3. Resul s 68  Figu e 3.30: Si 5 appea s o be insensi i e o NAM. An ELISA was pe o med wi h 10 µg o Si 5 and 10 µg Cy . c, 5 mM NAD+ as subs a es. Inc easing amoun s o NAM we e added o es i s abili y o inhibi Si 5. E en a 10 mM concen a ion, NAM did no inhibi Si 5. In o de o iden i y he eason behind Si 5’s insensi i i y owa ds NAM, a poin mu an (Th 69Asp) was gene a ed based on sequence alignmen o Si uin homologs (supplemen al igu e S2). All mammalian Si uins excep Si 5 con ain Asp in he lexible co- subs a e binding loop, which akes pa in NAM binding (A alos e al., 2005). The Th 69Asp mu an p o ein showed less ac i i y compa ed o he wild ype and did no sensi ize Si 5 o NAM (Figu e 3.31), indica ing ha o he molecula di e ences should be esponsible o he unique NAM insensi i i y o human Si 5. 3. Resul s 69  Figu e 3.31: Th 69Asp mu a ion impai s he ac i i y o Si 5. An ELISA was pe o med wi h 10 µg o Si 5T69D and 10 µg Cy . c, 5 mM NAD+ as subs a es. The ac i i y o he mu an enzyme was impai ed when compa ed o he wild ype (Figu e 3.30) and addi ion o NAM had no e ec on i s ac i i y. 3. Resul s 70  3.4 S udies on Si 7 3.4.1 Exp ession and pu i ica ion o Si 7 Cloning, exp ession and pu i ica ion p ocedu es o human Si 7 cons uc s (Table 3.2) we e pe o med as men ioned in sec ions 2.2.5, 2.2.6 and 2.4.4 espec i ely. Sligh modi ica ions adop ed o Si 7 a e de ailed explici ly. The di e en p o eases used o clea e a ini y ags a e lis ed in able 3.2. The pu i y o he elu ed samples we e ≥ 90 % as judged by capilla y elec opho esis (Calipe Li e Sciences, USA) o SDS-PAGE A ep esen a i e gel showing di e en cons uc s o Si 7 is shown in Figu e 3.32. Typical yields o Si 7 cons uc s o e exp essed in E. coli we e ~1 -3 mg/li e media. Table 3.2: Table ep esen ing he cons uc s o Si 7 used in his s udy. The cloning, exp ession and ini ial pu i ica ion s eps we e pe o med a he DPF.  Figu e 3.32: Pu i ica ion o Si 7. 12 % (w/ ) SDS polyac ylamide gel showing he pu i y o a ious cons uc s o Si 7 (indica ed on op) a e size exclusion ch oma og aphy. 3. Resul s 77  Figu e 3.40: Si 2Tm exhibi s oligome like beha io in PAGE. A) 10 µg o ull leng h Si 2Tm con aining di e en amoun s o educa an s esol ed on a 14 % SDS-gel. B) BN- PAGE analysis o di e en amoun s o ull leng h Si 2Tm indica es he p o ein o ha e a molecula weigh o ~ 45 KDa. 3.5.2 Iden i ica ion o subs a e-modula o pai s o Si 2Tm Unde s anding he mechanism o Si 1 modula ion by small molecules has been hampe ed by he lack o i s c ys al s uc u e. Si 2Tm sha es ~57 % sequence simila i y wi h he ca aly ic domain o Si 1 (Figu e S1), can be modula ed simila ly o Si 1 (see Ex-527 sec ion below) and is mo e amenable o c ys alliza ion. Due o hese easons, Si 2Tm was chosen as a model sys em o s uc u ally cha ac e ize he modula ion o Si 1 by small molecules. Con inuous assay was used o iden i y he subs a e p e e ence o Si 2Tm using well known pep ide subs a es o Si 1. As shown in igu e 3.41, Si 2Tm has a sligh p e e ence among he di e en subs a es es ed, wi h p53lg and HMG-B1 showing he highes and lowes ac i i y espec i ely. HMG-B1 is s ill an in e es ing candida e o sc eening expe imen s, because i can e eal po en ial small molecule ac i a o s o Si 2Tm. 3. Resul s 78  Figu e 3.41: Si 2Tm p e e en ially deace yla es subs a e pep ides. A con inuous assay was pe o med using 1 µM Si 2Tm, 1mM o each pep ide subs a e and 2 mM NAD+. The dec ease in abso bance a 340 nM was ollowed o e ime in a 96 well pla e eade . The abso bance o a con ol (wi hou pep ide subs a e) was sub ac ed om each pep ide alue, no malized and plo ed o e ime. (p53lg = p53 long), (p53sh = p53 sho ). When Si 2Tm was assayed in he p esence o es e a ol using he FdL-1 subs a e pep ide, no ac i a ion was obse ed (Figu e 3.42a), whe eas he same subs a e-modula o pai was able o ac i a e Si 1 (Figu e 3.8, sec ion 3.1.3). MS based assays, using polyda in (glycosyla ed o m o es e a ol wi h inc eased solubili y in aqueous solu ions) and unmodi ied pep ide subs a es, p53sh (p53 sho ), p53lg (p53 long) and H3, also showed no change in he ac i i y o Si 2Tm (Figu e 3.42b). These esul s seem o indica e ha ac i a ion by es e a ol and o he polyphenols migh ha e di e en subs a e equi emen s. 3. Resul s 79  Figu e 3.42: Si 2Tm modula ion by polyphenols. A) The ac i i y o 1 µg Si 2Tm wi h 100 µM FdL-1 pep ide and 200 µM NAD+ was measu ed wi h 100 µM es e a ol. B) 3.5 µg Si 2Tm was incuba ed wi h 500 µM espec i e pep ides, 1 mM NAD+ and 100 µM PD (PolyDa in) o 15 minu es a 37 ºC and quenched wi h 0.05 % ( / ) FA and analyzed by MS. 2 % ( / ) DMSO was p esen in all he samples. p53sh pep ide was analyzed only once. Ex-527 is a po en iso o m speci ic inhibi o o Si 1, wi h IC50 in he nM ange ((Nappe e al., 2005), see below). When Ex-527 was es ed agains Si 2Tm using he FdL-1 subs a e, inhibi on was obse ed (Figu e 3.43a). In o de o a oid luo ogenic pep ide subs a es which can o en gi e ise o a i ac s, we also pe o med he same assay using unmodi ied H3 pep ide. As shown in Figu e 3.43b, Si 2Tm was comple ely inhibi ed by 100 µM Ex-527, iden i ying a good subs a e-modula o pai o u he cha ac e iza ion. Figu e 3.43: Ex-527 inhibi s Si 2Tm using unmodi ied pep ide subs a es. A) 1 µg Si 2Tm was mixed wi h 25 µM FdL-1 subs a e and 200 µM NAD+ and incuba ed wi h a ious amoun s o Ex-527 o 30 minu es a 37 ºC. B) 3.5 µg o Si 2Tm was mixed wi h 500 µM H3 pep ide, 1 mM NAD+ and 100 µM Ex-527 and incuba ed a 37 ºC o a ious ime poin s and quenched wi h 0.05 % ( / ) FA ollowed by MS analysis. In he con ol samples a ime dependen deace yla ion can be seen, whe eas he p o ein sample con aining Ex-527 we e inac i e. Con ol samples con ained 1 % ( / ) DMSO. 3.5.3 Ex-527 is a po en inhibi o o Si 2Tm To cha ac e ize Si 2Tm and Si 1 inhibi ion by Ex-527, a dose esponse s udy was pe o med using unmodi ied pep ide subs a es. Since Ex-527 does no abso b a 340 nM, con inuous assay was used o de e mine he IC50 alues. As shown in igu e 3.44, Ex-527 inhibi s bo h Si 2Tm and Si 1 in he nanomola ange. The IC50 alues a e 390 nM ± 170 nM and 470 ± 20 nM o Si 2Tm and Si 1 espec i ely. The IC50 alue o Si 1 is highe in compa ison o Nappe e . al. who epo ed a alue o 100 nM (Nappe e al., 2005), he 3. Resul s 80  di e ence may a ise due o he pep ide subs a es used in he assay, becuase Nappe e . al used he a i icial FdL-1 subs a e, whe eas we used an unmodi ied subs a e pep ide based on he C- e minus o p53 con aining ace yla ed Lys382. Figu e 3.44: Ex-527 is a po en inhibi o o Si 2Tm and Si 1. A) 15 µg Si 2Tm was mixed wi h 500 µM H3 pep ide, 1 mM NAD+ and a ious amoun s o Ex-527 and ac i i y was measu ed using he con inuous assay. B) 12.6 µg human Si 1 was mixed wi h 500 µM p53sh pep ide, 1 mM NAD+ and ac i i y was measu ed using he con inuous assay.The IC50 alues a e 390 nM ± 170 nM and 470 ± 20 nM o Si 2Tm and Si 1 espec i ely. 3.5.4 Ex-527 appea s o equi e bo h he subs a es o bind Si 2Tm The exac mechanism o Si uin inhibi on by Ex-527 is s ill unclea . Kine ic s udies on Si 1 showed ha Ex-527 mos likely inhibi s in a non-compe i i e manne , which includes bo h subs a es. To in es iga e he binding be ween Si 2Tm and Ex-527 and unde s and how he p esence o subs a es a ec s binding, mic oscale he mopho esis measu emen s we e pe o med. No binding was obse ed when Ex-527 was i a ed agains he apoenzyme, in he p esence o NAD+, ace yla ed pep ide o ADP plus ace yla ed pep ide (Figu e 3.45). This clea ly indica es ha Ex-527 does no bind o Si 2Tm when only one o he subs a e is p esen o e en in he p esence o subs a e pep ide and a p oduc mimic. Nex we ied binding measu emen s a e incuba ing he enzyme wi h bo h subs a es (ace yla ed H3 pep ide and NAD+). This ime binding was seen, wi h an a ini y in he mic omola ange (Kd = 24.5 ± 6.3 µM). Since his s a egy can lead o a complex mix u e o e en s, as he eac ion can p oceed be o e he enzyme-subs a e mix u e encoun e s he inhibi o , we pe o med he same expe imen using H3 pep ide con aining a hioace yllysine. Thioace yllysine (whe e he ca bonyl oxygen o he ace yl g oup is eplaced by a sul u ) has been shown o be a po en inhibi o o Si uins, because he s-alkylimida e in e media e o med a e he elease o NAM s alls he enzyma ic eac ion due o slow u no e and was in ac apped in he c ys als o 3. Resul s 81  Si uins (Hawse e al., 2008; Jin e al., 2009; Smi h and Denu, 2007). Binding o Ex-527 o Si 2Tm-subs a e mix u e was also seen in he p esence o hioace yllysine pep ide wi h a Kd o 11.5 ± 2.5 µM, showing u he ha bo h he subs a es o he p oduc (s) a e mos likely equi ed o e icien binding o Ex-527 o Si 2Tm. Figu e 3.45: Ex-527 binds o Si 2Tm only in he p esence o bo h subs a es. 200 nM FITC labeled Si 2Tm was mixed wi h a ious compounds and i a ed agains Ex-527. The binding was analyzed using mic oscale he mopho esis. 500 µM pep ide, 1 mM NAD+, 1 mM ADP we e used in he eac ion. The eac ion con aining Si 2Tm, pep ide and NAD+ was incuba ed o 10 minu es a oom empe a u e be o e adding co esponding amoun s o Ex- 527. The he mopho esis ob ained wi h 17 % LASER powe was used o analysis om all he measu emen s. 3.5.5 Si 2Tm - C ys alliza ion and c yop o ec ion To s uc u ally elucida e he binding and mechanism o Si 2Tm inhibi ion by Ex-527, se e al c ys alliza ion sc eens we e pu sued as men ioned in sec ion 2.6.2. C ys als we e o med in many condi ions as soon as 24 hou s a e se up and we e con inuously moni o ed o g ow h. The d ops wi h Si 2Tm, pep ide and Ex-527 we e he mos p omising and ou o se e al condi ions which yielded c ys als; ou bes condi ions we e selec ed and g id sc eens we e se up. Figu e 3.46 shows ep esen a i e condi ions and he appea ances o he c ys als 3. Resul s 82  which we e used o u he op imiza ion. I is in e es ing o no e ha Si 2Tm did no c ys allize in he apoenzyme o m and a emp s o ep oduce Si 2Tm c ys als using published condi ions p o ed u ile. Figu e 3.46: C ys alliza ion o Si 2Tm. Rep esen a i e c ys als and condi ions o Si 2Tm in he p esence o Ex-527 and pep ide subs a es. A) 1M i-sodium ci a e, 0.1 M CHES (N- Cyclohexyl-2-aminoe hanesul onic acid), pH 9.5. B) 20 % (w/ ) PEG (PolyE hylene Glycol) 6000, 0.1 M BICINE (2-(Bis(2-hyd oxye hyl)amino)ace ic acid), pH 8.5. C) 20 % (w/ ) PEG 3350, 0.2 M di-sodium hyd ogen phospha e. D) 25 % (w/ ) PEG 4000, 0.1 M T is, pH 8.5, 50 mM Li2SO4. Among he ou condi ions ha we e used in op imiza ion p ocess, he bes looking c ys als g ew in 25 % (w/ ) PEG 4000, 100 mM T is, pH 8.5, 50 mM Li2SO4. The c ys al ays we e se up using he si ing d op apo di usion me hod by mixing 1.5 µl sample and 1.5 µl ese oi and incuba ed a 18 ºC. The sample mix u e con ained ~9.6 mg/ml Si 2Tm, 1 mM H3 ace yla ed/ hioace yla ed pep ide and 1.5 mM Ex-527. The pep ide subs a es used in he c ys alliza ion p ocess was changed om p53 o H3-Lys116/ hioace yla ed H3-Lys116 pep ide ( hio-H3) in mos se ups, as H3-Lys116 pep ide was used in o he biophysical and kine ic s udies. Long od shaped c ys als o Si 2Tm complex c ys als appea ed o e nigh and g ew o a size o a leas 200 µm in one o he dimension. Figu e 3.46 shows c ys al pic u es o Si 2Tm g own in he p esence o hioace yllysine con aining H3-Lys116 pep ide and Ex- 527. C ys als o Si 2Tm we e c yop o ec ed by supplemen ing he ese oi wi h 25 % ( / ) 3. Resul s 83  e hylene glycol, 1 mM co esponding H3-Lys116 pep ide and 1.5 mM Ex-527. A e sol ing he c ys al s uc u e, we obse ed densi y only o he pep ide (see below), he e o e a c yo apping s a egy was employed whe e he c ys als we e ans e ed o he c yop o ec an ollowed by addi ion o 1 mM ß-NAD+ o he solu ion con aining he c ys al and incuba ed o wo minu es be o e lash eezing in liquid N2. In he case o hioace yllysine condi ion, he soaking was ca ied on o 5 minu es. As soon as he NAD+ was added, he c ys als con aining ace yla ed H3-Lys pep ide s a ed disin eg a ing o small needles and we e ully dissol ed a ound ou minu es, whe eas o he hioace yllysine-H3-Lys116 pep ide he c ys als s a ed dissol ing only a e oughly 3 o 4 minu es and we e ully dissol ed a ound 9 o 10 minu es, pe haps due o he s alling o he enzyma ic eac ion by he hioalkylimida e in e media e. The e o e he c ys als we e ozen a 1, 2, 3 and 4 minu e ime in e als o ace yllysine pep ide and o 5, 8 and 9 minu es o he hioace yllysine pep ide. Since he alkylimida e/ hioalkylimida e in e media e is o med be ween he pep ide and NAD+, he s uc u al ea angemen s happening du ing he eac ion and especially du ing he elease o p oduc s may dis up he c ys al as ime p og esses, leading o hei de e io a ion. 3.5.6 Da a collec ion Di ac ion da a collec ion was pe o med as men ioned in sec ion 2.6.3. All he c ys als we e o a ed a an oscilla ion ange o 1º and was exposed o X- ays a di e en ime in e als: 4 sec exposu e and 120 images we e collec ed wi h s a ing angle o 80 o Si 2Tm/H3-Lys116, 6 sec exposu e and 110 images we e collec ed wi h s a ing angle o 150 o Si 2Tm/ligand mix + Ex-527 and 5 sec exposu e and 100 images we e collec ed wi h s a ing angle o 90 o Si 2Tm/ hio-H3-Lys116. The uni cell cons an s, da a collec ion and p ocessing s a is ics a e lis ed in Table 3.3. The c ys als di ac ed a 1.72 (Si 2Tm + H3-Lys116), 1.90 (Si 2Tm/ligand mix + Ex-527) and 2.8 (Si 2Tm + Thio-H3-Lys116) Å espec i ely. The uni cell cons an s (Table 3.3) di e ed sligh ly be ween he wo s uc u es, which could ha e a isen due o changes in he c ys al packing du ing c yo apping. 3. Resul s 84  C ys al Si 2Tm/H3-Lys116 Si 2Tm/ p53 ligand mix + Ex-527 Si 2Tm/H3- hio- Lys116 Space g oup P212121 P212121 P212121 Uni cell cons an s a = 58.1 Å, b = 61.3 Å, c = 75.7 Å a = 46.8 Å, b = 59.9 Å, c = 109.0 Å a = 45.3 Å, b = 58.2 Å, c = 105.8 Å Resolu ion (Å) 47.6 - 1.72 46.8 - 1.9 19.6 – 2.8 Unique e lec ions 29180 24821 7264 <I>/<σ(I)> (a) 18.0 (2.8) 20.7 (3.5) 10.8/(2.3) Comple eness (a) (%) 99.3 (99.5) 99.8 (100) 99.3 (99.9) Rme ge (%) (a)(b) 4.9 (57.3) 5.3 (41.4) 13.1 (68.3) Rmeas (%) (a)(c) 5.5 (64.2) 6 (46.9) 15 (78.5) (a) Numbe s in pa en heses a e o he ou e mos shell. (b) Rme ge = Σ (I - <I>)/ ΣI; I is he in ensi y o an indi idual measu emen and <I> he co esponding mean alue. (c) Rmeas = Σ[N/(N-1)]1/2 (I - <I>)/ ΣI; I is he in ensi y o an indi idual measu emen and <I> he co esponding mean alue. N indica es mul iplici y and [N/(N-1)]1/2 he co ec ion ac o o mul iplici y. Table 3.3: Da a collec ion and p ocessing s a is ics o c ys als o Si 2Tm 3.5.7 S uc u e solu ion, e inemen and modeling S uc u es we e sol ed h ough Pa e son sea ches as men ioned in sec ion 2.6.4. The Pa e son sea ch yielded a unique solu ion wi h an RFZ (Ro a ion Func ion Z-sco e) o 22.2 and TFZ (T ansla ion Func ion Z-sco e) o 30.4 o Si 2Tm/H3-Lys116 s uc u e, RFZ o 26.6 and TFZ o 37.7 o Si 2Tm/ligand mix + Ex-527 and an RFZ o 20.4 and 29.3 o Si 2Tm /H3- Thio-Lys116 espec i ely. The high RFZ and TFZ indica e an unambiguous co ec solu ion o all he s uc u es. The o e all e inemen o all he s uc u es seem o p oceed sa is ac o ily and he Rc ys and R ee alues con e ged well du ing e inemen and s ays a a easonable pe cen age app op ia e o he esolu ion (Table 3.4). In he case o Si 2Tm/H3- hio-Lys116, he di e ence be ween Rc ys and R ee seems o be mo e han ha obse ed o he o he wo s uc u es, his could be due o he mo emen o he p o ein molecule in he p esence o subs a es o in e media e. Figu es 3.47 shows a ep esen a i e Ramachand an plo (φ and ψ backbone dihed al angles) o one o he sol ed s uc u es. The e we e 99.2 %, 98.9 % and 3. Resul s 85  98.4 % o esidues in he a o ed egion, 0.8 %, 1.1 % and 1.6 % in he allowed egion and none in he o bidden egion o he H3-Lys116, ligand mix+Ex-527 and H3- hio-Lys116 bound s uc u es espec i ely, indica ing an excellen ag eemen wi h s e eochemical pa ame e s obse ed o s uc u es wi h simila esolu ion. The esidues 35-40 had no elec on densi y o he H3-Lys116, 37-43 o ligand mix + Ex-527 and 34-44 o he H3- hio-Lys116 bound s uc u es espec i ely and he e o e we e no modeled. This is e y much in ag eemen wi h majo i y o he c ys al s uc u es o Si 2Tm a ailable in he PDB (p o ein da a bank) and indica es ha hese esidues, which a e pa o he co-subs a e binding loop a e lexible and ac i ely pa icipa e in ca alysis. C ys al Si 2Tm/H3-Lys116 Si 2Tm/ ligand mix + Ex-527 Si 2Tm/H3- hio-Lys116 Re inemen esolu ion (Å) 30.6 - 1.7 40.3 - 1.9 19.6 – 2.8 To al e lec ions used 29177 24820 6896 P o ein a oms 1893 1872 1845 Sol en a oms modeled 141 206 63 R.m.s.d. bond leng hs (Å) 0.01 0.02 0.01 R.m.s.d. Bond angles (°) 1.3 2.3 1.6 A e age B- ac o (Å2) 26.5 25.7 35.5 Final Rc ys / R ee (%) (a)(b) 19.49/22.69 18.00/22.71 18.67/27.08 (a) R- ac o = Σ||Fobs| - |Fcalc|/ Σ|Fobs|, |Fobs| is he obse ed and |Fcalc| he calcula ed s uc u e ac o ampli ude. (b) R ee was calcula ed om 5 % o measu ed e lec ions omi ed om e inemen . Table 3.4: Re inemen s a is ics o s uc u es o Si 2Tm 3. Resul s 86  Figu e 3.47: Rep esen a i e Ramachand an plo o Si 2Tm c ys al s uc u es. Ramachand an plo o Si 2Tm + H3-Lys116 pep ide s uc u e. The e a e 99.8 % and 0.2 % esidues in he a o ed and allowed egions espec i ely. 3.5.8 Analysis o c ys al s uc u es o Si 2Tm Ini ial a emp s o co-c ys allize Ex-527 and Si 2Tm in he p esence o pep ide subs a e esul ed in a s uc u e con aining only he pep ide subs a e (Figu e 3.48), indica ing ha Ex-527 binding o Si 2Tm equi es ei he bo h he subs a es o p oduc s, simila o ou solu ion s udies. 4. Discussion 93  4. Discussion Re e sible lysine (de)ace yla ion, i s desc ibed o his one p o eins, is now known o be a widesp ead pos - ansla ional modi ica ion, wi h o e 6800 known mammalian ace yla ion si es in di e en p o eins and s ill coun ing making he p o eins in ol ed in hese modi ica ions all he mo e impo an o cha ac e ize. Al hough His one deace ylases we e he i s se o enzymes disco e ed o deace yla e lysine esidues, he iden i ica ion o Si uins as PDACs c ea ed a pa adigm shi , because Si uins equi e NAD+ as a co-subs a e o deace yla e lysine esidues and his coupling has been p oposed o link changes in cellula ene gy le els o deace yla ion ac i i y o Si uins, which would indica e hem as me abolic senso s (Schlicke e al., 2008). 4.1 Pu i ica ion and domain a chi ec u e o Si 1 To analyze he ole o N and C- e minal domains o Si 1 owa ds i s ac i i y and s uc u e, we exp essed and pu i ied se e al cons uc s o Si 1 con aining di e en egions. Human Si 1 can be o e exp essed abundan ly in a s able and ac i e o m in a p oka yo ic sys em such as E.coli wi hou majo p oblems such as hos oxici y, insolubili y o a need o PTMs, al hough i is in e es ing o no e ha mouse Si 1 can only be exp essed in Rose a 2 (DE3) E. coli s ain which con ains RNAs o a e codons, implying codon bias speci ically in he mouse Si 1 gene. The pu i ica ion o Si 1 u ns ou o be di icul when compa ed o o he Si uins and equi ed se e al pu i ica ion and op imiza ion s a egies. Concen a ion o Si 1 be o e comple e pu i ica ion ( ha is p io o SEC) leads o agg ega ion wi h a dec ease in ac i i y. This p oblem was o e come by employing a s a egy whe e he a ini y pu i ied p o ein was concen a ed minimally and loaded o an SEC column o emo e E. coli con aminan s and agg ega es. I is possible ha concen a ion o Si 1 p io o SEC leads o agg ega ion due o he exposu e o hyd ophobic pa s o he p o ein ha can in e ac wi h E. coli p o eins. CD analysis on ull leng h Si 1 indica es ha he p o ein possesses seconda y s uc u e, bu he amoun o epe i i e seconda y elemen s (α-helices and β-s ands) is highe han wha is no mally obse ed, example o o he Si uins. The p esence o uns uc u ed/ lexible pa s mos likely s ems om he N and C- e minus o Si 1 as indica ed by diso de p edic ion p og ams (X alP ed and PHYRE), which a e shown o be in ol ed in binding o pa ne p o eins and egula e i s ac i i y (Kang e al., 2011; Kim e al., 2007; Kim e al., 2008; Pan e al., 2011a). The p esence o such lexible egions is likely he eason o he appa en oligome ic beha io o Si 1 in SEC, as uns uc u ed egions can o en lead o ex ended shape o p o eins and in luence hei mig a ion in SEC. This is also suppo ed by 4. Discussion 94  ou sedimen a ion eloci y cen i uga ion and EM analysis whe e Si 1 seems o beha e as a monome in he physiologically ele an concen a ions. Mo eo e , he cons uc comp ising only o he ca aly ic co e (229 o516) beha es as a monome in SEC (Figu e 3.5 abo e), p o iding u he e idence ha uns uc u ed egions o Si 1 a he N and C- e minus a e esponsible o appa en di e ences seen be ween SEC and AUC expe imen s. Simila o ou indings Pan e . al. (Pan e al., 2011a) ecen ly epo ed ha a Si 1 cons uc con aining he ca aly ic co e (160-665) beha es as a monome . We hus conclude ha Si 1 is mos likely a monome in solu ion. The obse a ion ha nucleic acids co-pu i y wi h Si 1 du ing he a ini y pu i ica ion s ep and he ac ha Si 1 is p edominan ly localized o he nucleus make i emp ing o specula e on an in e ac ion be ween Si 1 and nucleic acids. P og ams ha p edic binding in e ac ions be ween p o ein and RNA/DNA such as BindN (h p://bioin o.ggc.o g/bindn/) and DP-Bind (h p://lcg. i .albany.edu/dp-bind/) in ac sugges po en ial binding a he N- e minus, especially o RNA wi h esidues Lys233, A g234 and Lys237 o Si 1. ChIP (ch oma in immunop ecipi a ion) assays show Si 1 o be co-localized a he p omo e egion o se e al genes, indica ing he possibili y o in e ac ion be ween Si 1 and DNA, al hough his localiza ion can also be due o p o ein/p o ein in e ac ions. Si 1, o example is known o deace yla e se e al ansc ip ion ac o s and his ones. Ou in i o s udies con i m ha RNA is dominan in he co-pu i ied nucleic acid mix u e. Since we we e no able o obse e any di ec binding when pu e Si 1 was incuba ed wi h ei he RNA o DNA, and no change in ac i i y o Si 1 in he p esence o nucleic acids was obse ed, i is possible ha he co-pu i ica ion was ei he an a i ac o o he E. coli con aminan s p esen along wi h Si 1 bind o nucleic acids. Howe e , we migh ha e also no es ed he igh ype o RNA, and u he ho ough s udies wi h he co-pu i ied nucleic acids will ha e o e eal whe he Si 1 binds RNA. 4.2 Si 1 modula ion by es e a ol Using he di e en Si 1 cons uc s, we we e also able o show ha he ca aly ic domain o Si 1 is su icien o i s ac i a ion by polyphenols such as es e a ol and picea annol, in con as o a p e ious epo claiming in ol emen o he N- e minus (Milne e al., 2007). One possible explana ion o hese wo di e en esul s could be due o he o e all ac i i y and s abili y o he di e en p o ein samples used. Ou assays sugges ha he ac i i y o Si 1 is highe in p esence o he e mini, simila o a ecen epo by Pan e . al (Pan e al., 2011a) and Milne e . al. epo (Milne e al., 2007). The basal ac i i y seems o co ela e, howe e , wi h he s abili y o hese cons uc s (Milne e . al. and ou da a) indica ing ha he 4. Discussion 95  e mini a e p ima ily impo an o Si 1 s abili y. Ne e heless, all he cons uc s we e equally ac i a able by es e a ol implying ha he ca aly ic domain o Si 1 is su icien o i s ac i a ion and he N- e minus migh indi ec ly suppo es e a ol ac i a ion by s abilizing Si 1. Ac i a ion o p o ein o enzyma ic unc ion due o small molecules is uncommon compa ed o inhibi ion. When Si 1 was ound o be s imula ed by es e a ol, i opened up an exci ing oppo uni y o he apy. The abili y o es e a ol o ac i a e Si 1 has been well documen ed (Howi z e al., 2003; Wood e al., 2004), bu he mechanism and ele ance has been ques ioned due o con lic ing epo s. Si 1 ac i a ion by es e a ol was shown o equi e a luo opho e modi ica ion o he subs a e on he C- e minus and no ac i a ion was obse ed when a na i e pep ide subs a e was used (Behe e al., 2009; Bo a e al., 2005; Kaebe lein e al., 2005a; Pacholec e al., 2010). We we e also able o show ha es e a ol binds di ec ly o Si 1, which sugges s ha he mechanism o es e a ol modula ion should also in ol e he p o ein. Ou indings show ha he modula ing e ec s o es e a ol is indeed subs a e speci ic; i does no equi e, howe e , a luo opho e modi ica ion bu a he depends on he local subs a e sequence. This also explains da a no only om p e ious ac i i y assays (Kaebe lein e al., 2005a) bu also he obse a ion ha es e a ol impa s posi i e e ec s in Ceano habdi is elegans in a Si 2 dependen manne , which is o e lapping bu no iden ical o yeas Si 2 e ec s (Viswana han e al., 2005). This leads o he conclusion ha in o de o analyze es e a ol speci ic modula ion o Si 1 i is impo an o conside he e ec s based on he subs a e used, i.e. he e ec depends on he subs a e-modula o pai . This conclusion can be gene alized o any small molecule employing he same binding si e/modula ion mechanism and we indeed ind a simila end in he case o he es e a ol un ela ed small molecule SRT1720, which was also shown o bo h ac i a e, inhibi o no a ec Si 1 (Dai e al., 2010; Milne e al., 2007; Pacholec e al., 2010). I is in e es ing o no e ha a ay esul s based on SRT1720 yielded subs a e-modula o pai s ha we e di e en om he es e a ol esul s, clea ly showing ha each compound has i s own se o compa ible subs a e sequence o ac i a ion. Ou esul s o m a basis o u he s udies media ed owa ds cla i ying which o he iden i ied es e a ol sensi i e si es con ibu e o i s in i o e ec . Based on ou obse a ion ha he ca aly ic co e o Si 1 is su icien o i s modula ion by es e a ol, i is emp ing o specula e ha es e a ol migh in luence o he Si uin iso o ms as well, which could again depend on he subs a e-modula o pai . Human Si 1 is 4. Discussion 96  implica ed in se e al diseases such as cance , diabe es, neu onal diso de s (Haigis and Sinclai , 2010), making i an a ac i e d ug a ge . Small molecules modula o s o Si 1 a e in clinical ials o ea ype 2 diabe es (Milne e al., 2007), e en hough hei exac mechanism o ac ion is s ill deba ed. We we e able o show ha es e a ol modula es he ac i i y o Si 1 in a subs a e sequence speci ic manne . Al hough es e a ol and Si 1 in e ac ion has been ex ensi ely s udied bo h in i o and in i o, i is a poo choice as a d ug, because o i s low solubili y and bioa ailabili y. The a ay expe imen s desc ibed he e will allow us in es ing and iden i ying di e en compounds ha a e no only mo e po en and speci ic o Si 1 bu also possess be e pha macological p ope ies. This ype o subs a e-modula o pai could se e as excellen ools o Si 1 speci ic d ug de elopmen ha p e e en ially a ge s pa icula Si 1 modula ed sys em such as his ones and ch oma in o a disease s a e which in ol es Si 1. 4.3 C ys alliza ion o Si 1 S uc u e aided d ug disco e y e o s, which ha e been used success ully o iden i ying lead compounds o se e al p o eins including Si 2 (Schlicke e al., 2011), a e hampe ed in he case o Si 1 by he una ailabili y o s uc u al in o ma ion. In spi e o se e al c ys alliza ion a emp s, di ac ion quali y c ys als o Si 1 we e no ob ained. Due o high lexibili y in he N and C e minal domain egions o Si 1, e o s o sol e he c ys al s uc u e o Si 1 should ocus u he on he ca aly ic co e wi h a iable N and C- e minal ex ensions which migh be mo e amenable o c ys alliza ion, an app oach s a ed in his hesis. Al hough s uc u al in o ma ion ob ained om his ype o cons uc would no highligh he unique ea u es o Si 1, i could s ill be used o gain aluable insigh s on how small molecules bind o i s ca aly ic co e and modula e i s ac i i y. Fu he e o s o ob ain he c ys al s uc u e o ull leng h Si 1 could ocus on o e exp ession and pu i ica ion om insec /mammalian cells, because some PTMs occu only in euka yo ic cells and his may make he p o ein mo e amenable o c ys alliza ion. Also, physiological pa ne p o eins o Si 1 binding o he e mini and s abilizing hem could be used o co-c ys allize Si 1, as ied he e wi h AROS. Co-c ys alliza ion a emp s in he p esence o non-physiological pa ne p o eins, such as DARPins (Designed Anky in Repea P o eins) (Hube e al., 2007) o complexes wi h subs a e-modula o pai s can also be a emp ed, which may “lock” he p o ein in a s able con o ma ion leading o c ys als. Ne e heless, ou homology model o Si 1 can a leas be used o analyze he esidues in ol ed in subs a e binding ( ia elec os a ic su ace po en ial) o unde s and i s subs a e p e e ences. Mo eo e , o he Si 1 4. Discussion 97  homologs such as Si 2Tm, which a e easy o pu i y and c ys allize can se e as Si 1 models and aid in s uc u e based d ug disco e y e o s as demons a ed by ou Ex-527 s udy. 4.4 Zinc is essen ial o he s uc u al s abili y and ac i i y o Si uins Si uins con ain a Zn2+ ion coo dina ed ypically by ou Cys eine esidues. The Zinc binding domain o Si uins is pa o he conse ed ca aly ic co e bu a ies among he Si uins in de ail, bo h a he s uc u al and sequence le el. Unlike o he HDACs, Si uins do no use Zinc o ca alysis. Zinc in Si uins ei he is equi ed o hei s uc u al s abili y o unc ion in an indi ec way. Ou s udies indica e ha Si 3 has an absolu e equi emen o Zinc o i s s abili y. The insolubili y o he Cys eine mu an s indica es ha he Zinc ion is mos likely equi ed du ing p o ein olding and plays an impo an ole in he s uc u e o Si 3. EDTA has a high a ini y owa ds Zinc (Kd ~ 10-16 M) and can easily s ip Zinc om Zinc binding mo i s in ansc ip ion ac o s (Kd o ~ 10-9-10-11 M) (Nybo g and Pee sen, 2004). In ou expe imen s, up o 2 mM EDTA was no able o a ec he unc ion (and he eby s abili y) o Si 3 whe eas 1,10-phenan h oline (a e en mo e po en Zinc chela o ) eadily p ecipi a ed he p o ein, indica ing an unusually high a ini y o Zinc among he Si uins in compa ison o o he Zinc binding p o eins. In a ecen s udy Chen e . al (Chen e al., 2010a) also epo ed ha Si 1 e ained ac i i y e en in he p esence o 10 mM EDTA, co obo a ing ou indings. I is in e es ing o no e ha he Cys eine o Se ine mu an s o Si 1 we e s able du ing exp ession and we e bound o Zinc e en a e pu i ica ion, bu we e inac i e (Chen e al., 2010a). Based on hese esul s i is emp ing o specula e ha Zinc binding migh be in luenced by he e mini o Si uins (highly a iable among Si uins) and he Zinc binding domain may also pa icipa e in e en s such as p o ein-p o ein in e ac ions he eby con ibu ing o iso o m speci ici y among Si uins. 4.5 Si 5 equi es unusually high amoun o NAD+ o i s deace ylase ac i i y Mammalian Si uins a e localized in di e en o ganelles o he cell and deace yla e a a ie y o p o eins. In spi e o his, he e a e e y ew subs a es iden i ied o he mi ochond ial iso o m Si 5. Si 5 knockou mice did no show a clea pheno ype compa ed o hei con ol li e ma es, whe eas Si 3 knockou mice showed ema kable hype ace yla ion o mi ochond ial p o eins (Lomba d e al., 2007). A plausible eason o his inding could be ha unde no mal condi ions, he basal ac i i y o Si 5 migh be much lowe compa ed o i s mi ochond ial homolog, Si 3 and he e o e a Si 5 knockou mouse may no show hype ace yla ion. Based on ou indings ha high amoun s o NAD+ is equi ed o decen 4. Discussion 98  Si 5 ac i i y, i is emp ing o specula e ha Si 5’s deace yla ion ac i i y may be coupled o he amoun o NAD+ p esen in he mi ochond ia a a gi en ime. No mal le els o NAD+ in he mi ochond ia has been es ima ed o be ~ 250 µM, a which ime he e is high le els o deace yla ion by Si 3, whe eas Si 5 dependen deace yla ion may be negligible in compa ison. In he case o s ess (CR, apop osis, e c.) he e is a chance o NAD+ le els o ise, hus leading o inc eased deace yla ion le els also due o Si 5. Al hough he physiological le els o NAD+ will ne e each he high le els epo ed he e, he appa en a ini y be ween NAD+ and Si uins in he p esence o subs a e p o ein is lowe in gene al, indica ing ha NAD+ could ac as a me abolic senso and lead o physiological changes in he mi ochond ia in a Si 5 dependen manne . An al e na i e explana ion o he high amoun s o NAD+ equi ed o Si 5’s ac i i y could be due o he echnical limi a ions o he assay i sel (ELISA and Fluo escence based assays), since a mo e e ined MS based assay (Fishe e . al. manusc ip submi ed o chemical biology) indica ed ha NAD+ in ac has be e binding a ini y o Si 5 (~ 0.2 mM) in he p esence o pep ide subs a e. I is also possible ha he appa en a ini y o NAD+ owa ds Si 5 may also be in luenced by he p o ein/pep ide subs a e used in he assay. Recen ly Du e . al. (Du e al., 2011) epo ed ha Si 5 is a desuccinylase and a demalonylase and exhibi s obus ac i i y owa ds succinyla ed and malonyla ed lysine esidues and Si 5 dele ion mice exhibi ed inc eased le els o succinyla ion in CPS1. I was concluded ha he a chi ec u e o Si 5 in he acyl pocke con e s his ype o ac i i y o he enzyme. I would s ill be in e es ing o know i du ing ex eme cellula condi ions, whe he NAD+ le els can ise o ce ain PTM e en s can occu ha can inc ease he a ini y be ween Si 5 and i s subs a es (ace yllysine and NAD+), hus leading o inc eased Si 5 dependen deace yla ion. 4.6 Si 5 is a NAM insensi i e deace ylase among he Si uin amily NAM is a known physiological inhibi o o Si uins. Ou esul s show ha Si 5 appea s o be insensi i e o NAM a physiological le els (now con i med in ou g oup wi h he mo e sensi i e MS assay (Fische e . al. manusc ip submi ed)), indica ing ha NAM can egula e Si uins in an iso o m speci ic manne , as di e en Si uins espond di e en ly o NAM. This iso o m speci ic egula ion by NAM could also be ela ed o he localiza ion o Si uins, as he nucleola Si 7 also seems o be insensi i ie o NAM, indica ing ha egula ion be ween di e en Si uin classes can occu na u ally, which could p o e bene icial a a ious imes du ing he cell cycle and could also be in luenced by ex e nal e en s such as s ess, pa hogen a ack e c. The highly conse ed Asp esidue among mammalian Si uins 4. Discussion 99  which is pa o he co-subs a e binding loop is eplaced by a Th in he case o Si 5. Mu a ing his Th o Asp (T69D) seems o dec ease he deace ylase ac i i y o Si 5, bu s ill con e insensi i i y owa ds NAM, indica ing ha he o e all NAD+ binding loop and he NAM binding pocke (o o ien a ion o NAM) a e in ol ed in he egula ion by NAM. In conclusion, his lack o inhibi ion by NAM could e y well be an in e es ing mechanism o mi ochond ial egula ion and can guide owa ds de eloping Si 3 speci ic small molecule he apeu ics owa ds ace yla ed p o ein subs a es ha does no a ec Si 5 dependen deace yla ion. 4.7 Exp ession, pu i ica ion and c ys alliza ion o Si 7 In o de o in es iga e he s uc u e and unc ion o Si 7, se e al cons uc s o Si 7 we e cloned, exp essed and pu i ied. The majo i y o he p ocess was pe o med a DPF in collabo a ion wi h D . Tim Be gb ede and colleagues using high- h oughpu echnologies in a semi-au oma ed ashion. The o e all pu i y and yield o di e en Si 7 cons uc s we e qui e sa is ac o y. In o de o iden i y he bes bu e and sal condi ions o u he s udies on Si 7, he mal dena u a ion shi assays we e pe o med using a ying bu e and sal condi ions. The assay e ealed ha Si 7 is mo e s able a a basic pH and wi h a sal concen a ion o a leas 50 mM. Due o he basic na u e o Si 7 (calcula ed pI ~ 8.9 o abo e), a ca ion exchange ch oma og aphy was used o u he pu i ica ion om E. coli p o eins, yielding Si 7 wi h highe pu i y ha was used in c ys alliza ion ials. An addi ional p o ein specie ob ained a e ion exchange ch oma og aphy was analyzed using MS and N- e minal sequencing and iden i ied as a deg ada ion/p o eolysis p oduc . The new N- e minus s a s jus be o e he α3 helix o he ypical seconda y s uc u e opology o Si uins (supplemen al igu e S2), which is al eady loca ed inside he conse ed ca aly ic co e. In o de o o e come s abili y issues, based on he sequence alignmen and bioin o ma ic p edic ions se e al new cons uc s we e planned which consis ed o esidues 98-338, 98-342 and 98-356. Al hough se e al c ys alliza ion ials we e pu sued o sol e he c ys al s uc u e o Si 7, no di ac ion quali y c ys als we e ob ained. Se e al new cons uc s and c ys alliza ion ials we e planned o c ys allize Si 7, bu so a wi hou success. This could indica e ha Si 7 equi es addi ional ac o s such as PTMs, in e ac ion p o ein pa ne s, subs a es/p oduc s o small molecule egula o s o make i mo e amenable o c ys alliza ion. 4. Discussion 100  4.8 Si 7 Phospho yla ion a Th 224 inc eases i s ac i i y Among all he mammalian Si uins, Si 7 is pe haps he leas cha ac e ized iso o m in e ms o s uc u e, subs a e p e e ence and egula ion. Si 7 o e exp essed in E. coli shows weak deace ylase ac i i y agains FdL subs a es when compa ed o Si 1 o example, whe eas when insec cell o e exp essed Si 7 was used, be e deace yla ion was obse ed wi h FdL subs a es, his could indica e ha Si 7 is di e en o o he human Si uins in a way ha exp ession o p ope ly olded, ac i e Si 7 equi es euka yo ic exp ession sys ems. Howe e , i migh also indica e ha PTMs migh play a ole in he ac i i y o Si 7. In ac i has been shown ha Si 7 is phospho yla ed by he CDK1-cyclin B pa hway, al hough he exac esidue phospho yla ed was no iden i ied (G ob e al., 2009). Ou MS esul s on he insec cell o e exp essed Si 7 shows ha Th 224 is indeed phospho yla ed, o ou knowledge his is he i s ime a phospho yla ion/PTM si e has been iden i ied o Si 7. Th eonine o Aspa a e mu a ion is an es ablished p ocedu e used o mimic phospho yla ion s a e due o s uc u al simila i ies be ween Aspa a e and phospho yla ed Th eonine. Ou a emp s o exp ess T224D mu an s in E. coli o in es iga e whe he he mu an s con e ed ac i i y o Si 7 esul ed in no p o ein exp ession. This could possibly indica e ha he phospho yla ion mimic impa s be e ac i i y o Si 7 esul ing in oxici y o E. coli and he e o e leading o p o ein deg ada ion immedia ely du ing exp ession. In conclusion, we ha e shown ha he ac i i y o Si 7 is in luenced by PTMs and new s a egies a e being planned o c ys allize Si 7. 4.9 Si 2Tm as a model sys em o s udying mammalian Si 1 Mammalian Si uins play c ucial oles in a ious p ocesses anging om cellula housekeeping o disease s a es (Haigis and Sinclai , 2010; La u e al., 2008). S uc u al elucida ion o all he se en mammalian Si uins would be e y use ul in inding d ugs a ge s ha can selec i ely modula e each Si uin, leading o a a ge ed app oach owa ds diseases caused by a pa icula iso o m. The lack o s uc u al in o ma ion on mammalian Si uins; Si 1, Si 4 and Si 7 p esen s a challenge on he a ional le el, owa ds iden i ying and cha ac e izing small molecules ha a ge only hese iso o ms and modula es hem agains speci ic subs a es. In spi e o his, he e a e se e al small molecule modula o s a ailable o Si 1 (Blum e al., 2011) wi h low speci ici y and po ency (ba ing a ew) compa ed o al eady ma ke ed he apeu ics, hus necessi a ing be e d ug a ge s. To o e come he lack o s uc u al in o ma ion on Si 1, we ha e used Si 2Tm as a model sys em o i s s uc u al cha ac e iza ion due o i s ease o pu i ica ion and c ys allizabili y. Nume ous c ys al 4. Discussion 101  s uc u es o Si 2Tm a e a ailable in he PDB and a e in ac used as model sys ems o s udying he ca aly ic mechanisms and egula ion o Si uins (A alos e al., 2005; A alos e al., 2004; Ho e al., 2006). Un il he s uc u e o Si 1 is a ailable, i would be bene icial o s uc u ally cha ac e ize Si 2Tm wi h small molecules ha can modula e bo h Si 1 and Si 2Tm equally. 4.10 Mechanism o Si uin inhibi ion by Ex-527 The c ys al s uc u es o Si 2Tm bound pa ially o Ex-527 and hioace yllysine o e s possible mechanis ic insigh s in o he inhibi ion o Si uins by Ex-527. The ca aly ic mechanism o Si uins in ol es he o ma ion o he alkylimida e in e media e and he elease o NAM. The o e all s uc u e o Si 2Tm bound o Ex-527/alkylimida e is simila o he s uc u e wi h bo h he subs a es bound (PDB id H4F (Ho e al., 2006), excep o he co- subs a e binding loop. The Phe33 which plays a ole in o ma ion o he alkylimida e in e media e and shields he in e media e om a ack by NAM, is p esen in he closed con o ma ion in bo h he s uc u es. Despi e his, he e is a s iking di e ence in he o ien a ion o he phenyl ing. The phenyl ing o Si 2Tm bound o Ex-527 is almos pe pendicula (~ 94 º) o he phenyl ing o 2H4F (Figu e 4.1) and is displaced by ~ 2.2 Å away om he phenyl ing o 2H4F s uc u e, owa ds he di ec ion o he C-pocke . This could be due o he ac ha he NAM moie y o NAD+ is no longe p esen nea he phenyl ing o o e s e ic hind ance. Since he cyclohexene ing o EX-527 is bu ied mo e deepe in o he C-pocke , i may p o ide mo e eedom o he phenyl ing o mo e in o such a posi ion. Thus he s abiliza ion o he inhibi o could in pa be due o he closu e o he co- subs a e binding loop and he mo emen o Phe33 leading o a apped inhibi o which p e en s p oduc elease he eby ende ing he enzyme inac i e. Al hough he s uc u e p esen ed he e does no ha e ull occupancy o he inhibi o , he c ys al s uc u e o Si 3 in complex wi h he inhibi o clea ly shows ha he inhibi o is bound o he same si e in bo h he p o eins, he eby allowing us o make easonable conclusions ega ding he inhibi o binding si e in Si 2Tm. Because he inhibi o is close o he subs a e o p oduc molecules (close p oximi y be ween he A and B ings o Ex-527 and me hyl g oup o ace yllysine and ibose ing) i is also possible ha he e is no enough oom in he ac i e si e o he elease o he p oduc s o een y o esh subs a es, making he enzyme inac i e. We ha e also shown ha he e exis ano he mechanism by which Ex-527 can inhibi Si uins whe e he IC50 is in he mic omola ange (example 60 µM o Si 3) using Si 3 as a model sys em. In his case, Ex-527 binds o he same exac ECP as in he case o Si 2Tm, bu he NAD+ is posi ioned in a 4. Discussion 102  di e en con o ma ion, whe e he NAM moie y o NAD+ is poin ing owa ds he ace yllysine binding cle and he eby p e en s he binding o he p o ein/pep ide subs a e. In he case o Si 3 also, he co-subs a e binding loop is in he close con o ma ion, which mos likely p e en s he exi o he inhibi o . Figu e 4.1: S uc u al compa ison o he ac i e si es o Si 2Tm in he p esence and absence o Ex-527. An o e lay o Si 2Tm s uc u es bound pa ially o Ex-527 and pep ide subs a e/NAD+ (PDB id: 2H4F) indica es ha he o e all a chi ec u e o Si 2Tm ac i e si e emains unchanged when Ex-527 is bound. In bo h he s uc u es he co-subs a e loops exis in he closed con o ma ion. The majo di e ence lies in phenyl ing o Phe33. In he p esence o Ex-527 he phenyl ing mo es 2.2 Å away and is almos pe pendicula o Phe33 wi hou Ex-527. The phenyl ing also con ac s Ex-527. The esidues co esponding o Ex-527 bound s uc u e a e ep esen ed as g ay cylinde s and esidues co esponding o 2H4F a e ep esen ed as g een cylinde s. Aly s ands o ace yllysine. 4.11 Selec i i y o Ex-527 owa ds Si uins The esidues in ol ed in binding o Ex-527 o Si 2Tm a e e y simila in Si 1 and o he mammalian iso o ms (Supplemen al igu e S1). In spi e o his, Ex-527 selec i ely inhibi s Si uin iso o ms. Si 1 and Si 2Tm a e e y well inhibi ed by Ex-527 (IC50 in he nanomola ange) whe eas Si 3 and Si 5 show mode a e and no inhibi ion espec i ely. The c ys al s uc u es o Ex-527 bound o Si 2Tm and Si 3 indica e ha he inhibi o binds o he same pocke o he p o ein ( he ECP) and is he icini y o simila esidues, wi h one excep ion being he Me 71 o Si 2Tm which con ac s he cyclohexene ing ha bea s he 6. Zusammen assung 109 527, einem po en en Si 1 Inhibi o , wu de gelös . In Ve bindung mi biophysikalischen Un e suchungen e gaben sich da aus E kenn nisse zu Bindung und Inhibi ionsmechanismus on Ex-527. Eine äumlich enge Nachba scha zwischen Ex-527 und eine Fo m on ADP- Ribose, mögliche weise dem P oduk O-ace yl-ADP-Ribose (noch nich olls ändig e i izie ) in de geschlossenen Enzymkon o ma ion e hinde die P oduk eise zung und s opp dadu ch die enzyma ische Reak ion. Diese E gebnisse bie en Einsich en in die Iso o m- spezi ische Hemmung on Si uinen du ch Ex-527 und lie e n In o ma ion ü die wei e e Ve besse ung on Si 1-spezi ischen Inhibi o en. 7. Re e ences 110  7. Re e ences Abdelmohsen, K., Pullmann J , R., Lal, A., Kim, H.H., Galban, S., Yang, X., Ble h ow, J.D., Walke , M., Shube , J., Gillespie, D.A., e al. (2007). Phospho yla ion o HuR by Chk2 Regula es SIRT1 Exp ession. Molecula Cell 25, 543-557. Adams, P.D., A onine, P.V., Bunkoczi, G., Chen, V.B., Da is, I.W., Echols, N., Headd, J.J., Hung, L.W., Kap al, G.J., G osse-Kuns le e, R.W., e al. 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Name Posi ion S op codon Res ic ion si e Sequence hsi 1_225_ndei_ 225 NA NdeI CGG CCG CGC CAT ATG ATT AAT ATC CTT TCA GAA CCA CC hsi 1_664_Nos op_xhoi_ 664 No XhoI GCA AGC GCG CTC GAG GTC ATC TTC AGA GTC TGA ATA TAC hsi 1_225_ndei_new_ 225 NA NdeI CAG CTG CGC CAT ATG ATT AAT ATC CTT TCA GAA CCA CC hsi 1_664_xhoi_new_ 664 No XhoI GCA ATC GCG CTC GAG GTC ATC TTC AGA GTC TGA ATA TAC hsi 1_225_quick_chng_ 225 NA NdeI GA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATGATT hsi 1_664_quick_chng_ 664 No XhoI CGG ATC TCA GTG GTG GTG GTG GTG GTG CTC GAG GTC ATC TTC AGA lsi 1_sdm_xhoi_ NA GAT GGT CCC GGC CTG GAG CGG AGC CCG lsi 1_sdm_xhoi_ NA CGG GCT CCG CTC CAG GCC GGG ACC ATC hsi 1_ 1_ndei_ 1 NA NdeI CAG ATC ATA CAT ATG GCG GAC GAG GCG hsi 1_ 1_xhoi_Nos op_ 747 No XhoI GCA ATC GCG CTC GAG TGA TTT GTT TGA TGG hsi 1_in 101 NA GGA GAC AAT GGG CCG GGC CTG CAG GGC hsi 1_ 1_xhoi_ 747 Yes XhoI GCA GTC GCG CTC GAG CTA TGA TTT GTT TGA TGG hsi 1_ 313a_ e e _ NA CCA TTC TTC AAG TTT GCA AAG GAA ATA TAT CC hsi 1_ 313a_ e e _ GG ATA TAT TTC CTT TGC AAA CTT GAA GAA TGG si 1_576-578_eek_aaa_ CA AAA GGT TGT ATG GCA GCA GCA CCA CAG GAA GTA C si 1_576-578_eek_aaa_ G TAC TTC CTG TGG TGC TGC TGC CAT ACA ACC TTT TG hsi 1_664_s op_xhoi_ 664 Yes XhoI GCA AGC GCT CTC GAG TCA GTC ATC TTC AGA GTC hsi 1_ l_ehei_ 1 NA EheI GTT AAA GGC GCC ATG GCG GAC GAG GCG hsi 1_214_lic_pET15b_ 214 NA NdeI CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC CAT hsi 1_225_lic_pET15b_ 225 NA NdeI CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG ATT AAT ATC hsi 1_214_lic_pET21a_ 214 NA NdeI GA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG GAG hsi 1_664_lic_pET15b_ 664 Yes XhoI CTT TCG GGC TTT GTT AGC AGC CGG ATC CTC GAG TTA GTC ATC TTC hsi 1_1_lic_pET15b_ 1 NA NdeI CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG GCG GAC GAG hsi 1_747_lic_pET15b_ 747 Yes XhoI CTT TCG GGC TTT GTT AGC AGC CGG ATC CTC GAG CTA TGA TTT GTT hsi 1_1_lic_pET21a_ 1 NA NdeI GA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG GCG 8. Appendix 125  225-664 H363A 5 JCSG (Molecula Dimensions) 18 1 Si ing d op/Manual Ca aly ic His idine mu an 225-664 10 Mic oly ic capilla y ay 18 New capilla y manual se ing Ca aly ic His idine mu an 225-664 H363A 5 PACT I (Molecula Dimensions) 18 1 Si ing d op/Manual Ca aly ic His idine mu an 225-664 H363A 5 PACT II (Molecula Dimensions) 18 1 Si ing d op/Manual Ca aly ic His idine mu an 225-664 H363A 5 PEG Ion (Hamp on) 18 1 Si ing d op/Manual Ca aly ic His idine mu an 225-664 SER 10 Pen ae hy i ol (Jena Biosciences) 18 1 Si ing d op/Manual SER= Su ace En opy Reduc ion mu an EEK-AAA (574-578) 225-664 SER 10 JCSG (Molecula Dimensions) 18 1 Si ing d op/Manual SER= Su ace En opy Reduc ion mu an EE K -AAA (574-578) 225-664 SER 5 JCSG Co e II (Qiagen) 18 1 Si ing d op/Manual SER= Su ace En opy Reduc ion mu an EE K -AAA (574-578) 225-664 10 JCSG Co e I (Qiagen) 20 0.1 Si ing d op/Robo T ypsinized p o ein 225-664 10 JCSG Co e I (Qiagen) 20 0.1 Si ing d op/Robo T ypsinized p o ein + 1 mM H3 + 1 mM ADP 225-664 10 JCSG Co e I (Qiagen) 20 0.1 Si ing d op/Robo T ypsinized p o ein + 1 mM H3 + 1 mM ADP + 200 µM Res e a ol 225-664 H363A 5 JCSG Co e II (Qiagen) 18 1 Si ing d op/Robo Ca aly ic His idine mu an 225-664 H363A 4.7 JCSG Co e II (Qiagen) 20 0.1 Si ing d op/Robo Ca aly ic His idine mu an +1 mM p53 + 2 mM ADP 225-664 H363A 4.7 JCSG Co e II (Qiagen) 20 0.1 Si ing d op/Robo Ca aly ic His idine mu an +1 mM p53 + 2 mM NAD 225-664 H363A 4.7 JCSG Co e II (Qiagen) 20 0.1 Si ing d op/Robo Ca aly ic His idine mu an +1 mM p53 + 2 mM NAD + 500 µM Res e a ol 225-664 H363A 4.7 JCSG Co e III (Qiagen) 20 0.1 Si ing d op/Robo Ca aly ic His idine mu an +1 mM p53 + 2 mM ADP 225-664 H363A 4.7 JCSG Co e III (Qiagen) 20 0.1 Si ing d op/Robo Ca aly ic His idine mu an +1 mM p53 + 2 mM NAD 225-664 H363A 4.7 JCSG Co e III (Qiagen) 20 0.1 Si ing d op/Robo Ca aly ic His idine mu an +1 mM p53 + 2 mM NAD + 500 µM Res e a ol 225-664 SER 5 JCSG Co e I (Qiagen) 20 0.1 Phoenix Robo SER= Su ace En opy Reduc ion mu an EE K -AAA (574-578) 8. Appendix 126  225-664 SER 5 JCSG Co e I (Qiagen) 20 0.1 Phoenix Robo SER= Su ace En opy Reduc ion mu an EE K -AAA (574-578) 225-664 SER 5 JCSG Co e III (Qiagen) 20 0.1 Phoenix Robo SER= Su ace En opy Reduc ion mu an EEK-AAA (574-578) 225-664 SER 5 JCSG Co e IV (Qiagen) 20 0.1 Phoenix Robo SER= Su ace En opy Reduc ion mu an EEK-AAA (574-578) 229-516 5 JCSG (Molecula Dimensions) 20 0.1 Si ing d op/Robo Tag clea ed 229-516 5 JCSG (Molecula Dimensions) 20 0.1 Si ing d op/Robo P o ein + 1mM p53 229-516 5 JCSG I Co e (Qiagen) 20 0.5 Si ing d op/Manual 229-516 5 JCSG II Co e (Qiagen) 20 0.5 Si ing d op/Manual 229-516 5 JCSG I Co e (Qiagen) 20 0.5 Si ing d op/Manual P o ein + 1.5 mM Ex527 + 0.5 mM p53 +2 mM NAD 229-516 5 JCSG II Co e (Qiagen) 20 0.5 Si ing d op/Manual P o ein+ 1.5 mM Ex527 + 0.5 mM p53 +2 mM NAD 229-516 5 JCSG III Co e (Qiagen) 20 0.5 Si ing d op/Manual P o ein 229-516 5 JCSG III Co e (Qiagen) 20 0.5 Si ing d op/Manual P o ein + 1.5 mM Ex527 + 0.5 mM p53 + 2 mM NAD 206-737 20 Index (Hamp on) 18 1 Si ing d op/Manual Mouse Si 1 206-737 20 S uc u e (Molecula Dimensions) 18 1 Si ing d op/Manual Mouse Si 1 206-737 10 S uc u e (Molecula Dimensions) 18 0.125 Si ing d op/Robo A) Mouse Si 1 +1 mM p53, B) P o ein + NAD, C) P o ein +ADP and p53 206-737 10 PEG Ion (Hamp on) 18 0.125 Si ing d op/Robo A) Mouse Si 1 + p53, P o ein + NAD, P o ein + ADP and + p53 8. Appendix 127  Table S3: C ys alliza ion ials o Si 7. The able shows a ious cons uc s o Si 7 used in he c ys alliza ion p ocess along wi h he condi ions. Si 7 Cons uc P o ein concen a ion (mg/ml) Sc een Tempe a u e (° C) Sample olume (µl) Me hod Commen s Full leng h 5 JCSG Co e 20 0.1 Si ing d op/Robo 14-367 9.2 JCSG Co e 20 0.1 Si ing d op/Robo 14-367 9.2 JCSG+ 18 0.7 Si ing d op/ Manual 14-367 9.2 JCSG Co e 20 0.1 Si ing d op/ Robo 14-367 9.2 JCSG Co e 20 0.1 Si ing d op/ Robo In si u p o eolysis wi h 1/10000 h T ypsin 14-367 9.2 JCSG Co e 20 0.1 Si ing d op/ Robo In si u p o eolysis wi h 1/10000 h Chymo ypsin 59-356 5 JCSG Co e 20 0.1 Si ing d op/Robo 81-356 4.7 Pac 1&2 18 1 Si ing d op/ Manual 81-356 4.7 JCSG Co e 18 1 Si ing d op/ Manual 81-356 6.8 JCSG Co e 18 1 Si ing d op/ Manual A e CEC 81-356 6.8 JCSG Co e 18 1 Si ing d op/ Manual In si u p o eolysis wi h 1/1000 h T ypsin 81-356 6.8 JCSG Co e 18 1 Si ing d op/ Manual In si u p o eolysis wi h 1/1000 h Chymo ypsin 81-356 6.8 JCSG Co e 18 1 Si ing d op/ Manual In si u p o eolysis wi h 1/10000 h Sub ilisin Table S4: Lis o pep ides and hei sequence used in his wo k. The Thio-H3 = hioace yllysine. Pep ide name P o ein name Sequence Ace yla ed p53sh (p53 sho ) p53 RHKK[Ac]LMFK Lys382 p53lg (p53 long) p53 STSRHKK[ac]LMFKTE Lys382 H3 His one 3 IHAK[ac]RVT Lys116 Thio-H3 His one 3 IHAK[ hio-ac]RVT Lys116 HMG-B1 High Mobili y G oup B1 KKPRGK[ac]MSSY Lys12 SF38A P e-mRNA-splicing ac o 38A PQYLVEK[Ac]IIRTRI Lys23 TFIID T ansc ip ion ini ia ion ac o TFIID subuni 3 DREKGKK[Ac]DKDKRE Lys628 Ku70 Ku70 TKRK[Ac]HDN Lys544 AATase Aspa a e amino ans e ase 2 VFLPK[ac]PTWG Lys159 8. Appendix 128  Figu e S1: Sequence alignmen o human Si 1 ca aly ic domain (214-497) (Hs_Si 1) and Si 2Tm (Tm_Si 2) wi h seconda y s uc u e elemen s om Si 2Tm. The alignmen was c ea ed using BioEdi (Hall, 1999). 8. Appendix 129  Figu e S2: Mul iple sequence alignmen o all he se en human Si uin iso o ms. The seconda y s uc u e elemen s co espond o he s uc u e o Si 2 (1J8F). The numbe ing co esponds o Si 1. The ca aly ic His idine (indica ed as g een s a ), Cys eines coo dina ing Zn2+ (g een iangles) and he Th 69 o Si 5 (g een hexagon) a e indica ed on op o he esidues. The alignmen was c ea ed using BioEdi (Hall, 1999). 9. E klä ung/Decla a ion 130  9. E klä ung Hie mi e klä e ich, dass ich die A bei selbs ändig e ass und keine ande en als die on mi angegebenen Quellen und Hil smi el benu z habe. Fe ne e klä e ich, dass ich ande wei ig mi ode ohne E olg nich e such habe, diese Disse a ion einzu eichen. Ich habe keine gleicha ige Dok o p ü ung an eine ande en Hochschule endgül ig nich bes anden. Bay eu h, Da e: 7 h Ma ch, 2012 Mahade an Lakshmina asimhan Decla a ion I he eby decla e ha his disse a ion is my own o iginal wo k and ha I ha e acknowledged all sou ces used. Fu he mo e I decla e ha I ha e no a emp ed, wi h o wi hou success, o submi his disse a ion o any o he academic ins i u ion and ha I ha e no ailed a simila doc o al exam a any o he academic ins i u ion. Bay eu h, Da e: 7 h Ma ch, 2012 Mahade an Lakshmina asimhan