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
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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
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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
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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
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8. Appendix 118

8. Appendix
Table S1: A lis o p ime s used in his s udy along wi h hei s a and s op posi ions and es ic ion enzymes ha ecognize hem.
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