The CLU-files: disentanglement of a mystery.

BioMol Concep s 2016; 7(1): 1–15 *Co esponding au ho : Philipp Rohne, Ins i u e o Pha macy and Biochemis y, The apeu ical Li e Sciences – Ins i u e o Biochemis y, Johann-Joachim-Beche Weg 30, D-55128 Mainz, Ge many, e-mail: philipp[email p o ec ed] Hans P ochnow: Ins i u e o Pha macy and Biochemis y, The apeu ical Li e Sciences – Ins i u e o Biochemis y, Johann-Joachim-Beche Weg 30, D-55128 Mainz, Ge many; and Depa men o Chemical Biology, Helmhol z Cen e o In ec ion Resea ch GmbH, D- B aunschweig, Ge many Claudia Koch-B and : Ins i u e o Pha macy and Biochemis y, The apeu ical Li e Sciences – Ins i u e o Biochemis y, Johann-Joachim-Beche Weg 30, D-55128 Mainz, Ge many Re iew Open Access Philipp Rohne*, Hans P ochnow and Claudia Koch-B and The CLU- iles: disen anglemen o a mys e y DOI 10.1515/bmc-2015-0026 Recei ed Oc obe 1, 2015; accep ed No embe 5, 2015 Abs ac : The mul i ace ed p o ein clus e in (CLU) has been challenging esea che s o mo e han 35 yea s. The cha ac e iza ion o CLU as a molecula chape one was one o he majo b eak h oughs in CLU esea ch. Today, sec e o y clus e in (sCLU), also known as apolipop o ein J (apoJ), is conside ed one o he mos impo an ex a- cellula chape ones e e ound. I is in ol ed in a b oad ange o physiological and pa hophysiological unc ions, whe e i exe s a cy op o ec i e ole. Desc ip ions o a i- ous o ms o in acellula CLU ha e led o u he and e en con adic o y unc ions. To un angle he cu en s a e o knowledge o CLU, his e iew will combine old iews in he ield, wi h new disco e ies o highligh he na u e and unc ion o his ascina ing p o ein(s). In his e iew, we u he desc ibe he exp ession and subcellula loca- ion o a ious CLU o ms. Mo eo e , we discuss ecen insigh s in o he s uc u e o CLU and assess how s uc- u al p ope ies as well as he edox en i onmen de e - mine he chape one ac i i y o CLU. E en ually, he e iew connec s he biochemis y and molecula cell biology o CLU wi h medical aspec s, o o mula e a hypo hesis o a CLU unc ion in heal h and disease. Keywo ds: Alzheime ’s disease; apolipop o ein J; clus- e in; LDL- ecep o s; molecula chape ones. In oduc ion: clus e in (CLU), i s his o y and he challenge o esea che s In he yea 1979, a p o ein was i s disco e ed, whose complexi y, abundance and in ol emen was ini ially unknown (1). La e , his sul a ed glycop o ein was iden i- ied by i ue o i s blood cell agg ega ing abili y in i o, and was he e o e named clus e in (CLU) (2). Mos s ik- ingly, i su aced in a b oad spec um o issues and is o e exp essed in he ace o pa hological p ocesses, such as a he oscle osis, cance and Alzheime ’s disease (3–6). Mo eo e , CLU was ound in he bodily luids o almos all e eb a es om zeb a ish o humans (7, 8). In acco d- ance wi h hese indings, an ex ensi e epe oi e o names eme ged anging om complemen lysis inhibi o (CLI) and es os e one ep essed p os a e message-2 (TRPM-2) o apolipop o ein J (apoJ). Ne e heless, in 1992 he name CLU was he gene ally accep ed e m o all disco e ed p o eins (9). Thus a , CLU has been demons a ed o be a highly glycosyla ed glycop o ein o 80 kDa, consis ing o wo polypep ide chains connec ed by ou o i e disul ide bonds (10). The p o ein is one o he mos p ominen ex acellula chape ones. The chape one ac i i y o CLU has been in ensi ely s udied by Ma k Wilson and his col- leagues (11–14). In connec ion wi h i s chape one ac i i y, CLU is desc ibed as a p o ein ha allows o he clea ing o cellula deb is and mis olded p o eins, as well as he clea ance o Aβ ia he blood-b ain ba ie (BBB) (15–17). The conce ed ac ion o chape one ac i i y, sca enging- and clea ance- unc ion, may be one basis o he cy o- and issue p o ec i e ole o he p o ein (5, 15, 18, 19). P e iously, i was u he shown ha he p o ein ac s as a signaling molecule, inducing cellula p osu i al and p oli e a o y pa hways, which may con ey ano he mech- anism o i s cy op o ec i e unc ion (20–22). This unde s anding o CLU (as p e iously desc ibed) became complica ed when in acellula CLU o ms we e desc ibed in damaged cells, in addi ion o he p edomi- nan sec e ed o m, and a ibu ed o a di e se and e en opposing ole (23–26). This e iew will summa ize he ©2015, Philipp Rohne e al., published by De G uy e . This wo k is licensed unde he C ea i e Commons A ibu ion-NonComme cial-NoDe i a i es 3.0 License. B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM 2      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y biosyn hesis o CLU, i s s uc u al ea u es and chap- e one ac i i y as well as ocus on aspec s o in acellu- la CLU o ms o assess hei cellula and physiological unc ion. Finally, he in ol emen o CLU in he modu- la ion o a ious signal ansduc ion pa hways, i s ole in he immune sys em and i s in ol emen unde pa ho- logical condi ions, such as ischemia and nec osis will be discussed. The goal is o summa ize cu en iews abou CLU o enable u u e s udies o inally sol e he ‘CLU mys e y’. Biogenesis o clus e in (CLU) The biosyn hesis o human sec e o y CLU (sCLU) ollows he canonical pa hway o sec e o y p o eins (Figu e 1). T ansla ion o CLU s a s om a s a codon loca ed on exon 2 o he CLU-mRNA, esul ing in a p e-p op o ein composed o 449 amino acids. The i s 22 amino acids ep esen a signal sequence ensu ing co- ansla ional ansloca ion in o he endoplasmic e iculum (ER). Subse- quen ly, he signal sequence is clea ed o and ou o i e disul ide bonds a e o med (10). He ea e , N-glycosyla ion a six Asn- esidues (Asn86, 103, 145, 291, 354, 374) akes place con e ing he p op o ein o a high-mannose ER-p ecu so (p e-sec e o y CLU, psCLU) o 60kDa (27, 28). A e ans- loca ion o he golgi-appa a us, complex ca bohyd a e moie ies a e a ached o he ma u a ing psCLU, composed o galac ose, ucose, mannose, N-ace ylglucosamine and N-ace ylneu aminic acid (29–31). The esul ing 80 kDa p o ein is u he clea ed by a u in-like p op o ein con- e ase (FC) (amino acid ecogni ion mo i : RIVR) be ween A g227 and Se 228 o p oduce an N- e minal α-chain and a C- e minal β-chain which a e in e linked by disul ide bonds (29–31). Finally, ma u e sCLU is sec e ed as a he - e odime ic complex o wo 40-45kDa subuni s (28, 29). Unde cellula s ess addi ional CLU o ms eme ge by di e sion om he canonical sec e o y pa hway (Figu e1). They encompass co e-glycosyla ed o ms, p esumably de i ed om e o ansloca ed CLU ou o he ER (32, 33) o in acellula o ms which ailed o be seg ega ed in o he ER and hus a e no ca ying any suga esidues o disul ide bonds, espec i ely (34). In addi ion, in acel- lula o ms may a ise om al e na i e splicing e en s o om non-canonical/al e na i e ansla ion-ini ia ion s a si es downs eam o he ER-leade pep ide (Figu e 1) (34). All men ioned in acellula CLU o ms a e single-chain p o eins, since hey a e no p o eoly ically p ocessed. FC clea age si e Al e na i e Exon 1 (P ochnow e al. 34) Exon 2–9 ER-Leade Diso de ed/mol en globule-like egion O de ed egion Disul ide bonds Amphipa hic helices O he in amolecula in e ac ions Ca bohyd a es Sec e ion Co e-glycosyla ed CLU o m Co ansla ional ansloca ion Disul ide bonds Complex glycosyla ion FC- clea age sCLU High mannose co e-glycosyla ion Endoplasmic Re iculum psCLU Cy osolic CLU o ms R e o a n s l o c a i o n Figu e 1: Illus a ion o CLU biosyn hesis. The canonical pa hway o sec e o y p o eins leads o he syn hesis o sec e o y CLU (sCLU). I unde goes p o eoly ic ma u a ion and ca ies complex ca bohyd a e moie ies as well as disul ide bonds. Upon cellula s ess, non-canonic CLU o ms eme ge, mainly de i ed om ailed ansloca ion, al e na i e splicing o ansla ion-ini ia ion e en s on exon 3, as well as om e o ansloca ion. All non-sec e ed CLU o ms a e incomple ely ma u a ed. Fo de ails please see ex . B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      3 S uc u e and unc ion o sec e o y clus e in (sCLU) S uc u e So a , one o he mos challenging ques ions pe ains o he s uc u e o sCLU. O e he pas h ee decades nume - ous s udies ha e been conduc ed, bu he s uc u al p op- e ies o sCLU a e s ill no ully unde s ood. One eason o his lack o knowledge lies in he agg ega ing na u e o sCLU. Besides in e ac ing wi h a ple ho a o ligands, i agg ega es wi h i sel , o ming di-, e a- and e en highe oligome s, depending on he pH- alue (2, 12–14, 35). In combina ion wi h dis inc ligands, such as ib inogen, sCLU can o m high molecula weigh complexes wi h a pu a i e molecula mass up o 40 000kDa and a diam- e e anging om 50 o 100nm (14). These cha ac e is- ics oge he wi h a he e ogeneous glycosyla ion pa e n, ende s i di icul o ob ain X- ay s uc u es o eliable NMR spec a om pu i ied sCLU-samples (13, 36). Addi- ionally, i is ha d o ob ain sui able p o ein samples in su icien quan i ies (30, 36). Ne e heless, a numbe o s udies ha e been conduc ed, which ocus on dis inc p ope ies o sCLU and shed ligh on i s s uc u al ele- men s (10, 13, 37–39). Beyond doub , he p ima y s uc u e o sCLU is highly conse ed be ween di e en species, wi h he highes homologies ound in he egions o disul ide bonding cys eins and he FC clea age si e poin - ing o hei signi icance ega ding sCLU- unc ion (8, 40). Al eady a e he i s success ul isola ion o sCLU, i s amphipa hic cha ac e was appa en (2). La e on, sec- onda y s uc u al elemen s we e in es iga ed by means o ci cula dich oism (CD)- and in a ed-spec oscopy (14, 30, 35, 38, 41, 42). In all o hese a p edominan α-helical con en o up o 60% was calcula ed. In silico analyses u he p edic ed i e amphipa hic α-helices (37). On he e ia y s uc u e le el, sCLU is belie ed o belong o he amily o in insically diso de ed p o eins, meaning ha i pa ially lacks a de ined e ia y s uc u e, hus expos- ing hyd ophobic egions, so-called mol en globule-like domains, owa ds he ex e nal space (43). This in u n allows o binding o o he molecules ia hyd ophobic in e ac ions (Figu e 2A). Rema kably, sCLU sha es his FC clea age si e Disul ide bonds Clien p o ein O he in amolecula in e ac ions Amphipa hic helices O de ed egions 100 Clea ed sCLU AC B Unclea ed sCLU 80 60 40 20 Rela i e chape oneac i i y (%) Non- ed. C l Time a e DTT emo al C l Time a e DTT emo al ed. 0 h 48 h Non- ed. ed. 0 h 48 h Di hio h ei ol Ca bohyd a es Diso de ed/mol en globule-like egions Figu e 2: Schema ic depic ion o sCLU chape one ac i i y and hypo he ical in luence o s uc u al elemen s. Fully ma u a ed sCLU is s abilized by disul ide bonds and in amolecula in e ac ions, such as hyd ophobic in e ac ions in he egion o amphipa hic helices (A). In con as , a i icial unclea ed sCLU elies on i s disul ide bonds and he e o e displays a high sensi i i y owa ds educing condi ions (B). P elimina y da a show ha unclea ed sCLU pa ly egains i s chape one ac i i y a e emo al o DTT wi hin 48h (C). The p ocedu e o sCLU pu i ica ion, DTT ea men and chape one ac i i y assay a e in acco dance wi h Rohne e al. (30). A e 12h o incuba ion wi h o wi hou 40 mm DTT a 37°C upon gen le agi a ion, sCLU was subsequen ly used o chape one ac i i y assays (C l educ- ing and non- educing) o DTT was subs i u ed by PBS using a Vi aspin 4 5000 MWCO PES (Sa o ius) a 4°C and used o chape one ac i i y assays subsequen ly o a e 48h a 4°C. All chape one ac i i y assays we e pe o med in he p esence o 5 mm DTT wi h Ca alase being he clien p o ein. Addi ionally, BSA was mixed wi h Ca alase and se ed as a nega i e con ol o calcula e he ela i e chape one ac i i y o sCLU as desc ibed by Rohne e al. (30). Fo e alua ion, he da a o wo o h ee independen expe imen s, each wi h he mean o wo o h ee measu emen s we e used (unpai ed - es *p < 0.05). The e o ba s co espond o he mean±s anda d e o s. B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM 4      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y ea u e wi h o he in insically diso de ed p o eins, mos p ominen ly hea shock p o ein (Hsp) 27 o α-c ys alline (11, 44). As poin ed ou in he ollowing, sCLU indeed pos- sesses a chape one ac i i y simila o ha o Hsp27 and o he hea shock p o eins. Chape one and sca enging unc ion Chape ones a e pa o he basic molecula de ense mech- anism o a cell o e coming s ess si ua ions induced by UV ligh , ionic i adia ion, hea , oxidan s, hea y me als, hype oxia o ce ain d ugs. They can be di ided in o di e - en classes: chape onins, Hsp100, Hsp90, Hsp70, Hsp60 and small Hsps (sHsps) (45). These in acellula chap- e ones egula e he co ec olding o ma u ing p o eins o p e en he agg ega ion o dena u ed p o eins hus adop ing a cy op o ec i e unc ion. Some e en ini ia e he e olding o mis olded p o eins in an ATP-dependen manne (Hsp70, Hsp90, chape onins). Fo chape ones, na i ely diso de ed egions a e equi ed o binding wi h a ple ho a o s uc u ally di e se clien p o eins (46). Simila o he abo e men ioned Hsps, CLU-mRNA is up- egula ed upon cellula s ess due o a hea shock elemen -like mo i p esen in he CLU p omo o (19, 34, 47, 48). The chape one unc ion o sCLU was e ealed 20yea s a e he p o ein’s disco e y (11). Thus i became he i s molecula chape one known o ac ou side o he li ing cell. Simila o sHsps inside he cell, i is assumed ha sCLU binds o clien p o eins h ough i s mol en globule-like egions ia hyd ophobic in e ac- ions (13, 14, 37, 49). In pa icula , p o eins ha a e on he o - olding pa hway, e.g. as induced by hea o educ- ing condi ions, se e as clien s o sCLU (12, 30). The binding occu s in an ATP-independen manne , leading o o ma ion o soluble high molecula weigh complexes wi h di e en mola chape one-clien a io be ween 1:5 and 1:0.33 (14, 50, 51). As a consequence, sCLU p e en s he uncon olled agg ega ion o he dena u ing p o eins. In e es ingly, binding o sCLU does no e ain po en- ial enzyma ic ac i i ies o dena u ing clien p o eins. Howe e , in coope a ion wi h ATP-dependen Hsps, such as Hsc70, he enzyma ic ac i i y o some p o eins can be es o ed (11, 50). In cell cul u e expe imen s i was obse ed, ha sCLU is able o acili a e he up ake o he bound clien complexes in o su ounding issue cells o allow hei emo al ia lysosomal diges ion (Figu e 3) (15, 52). Recep- o s esponsible o he binding o sCLU and/o sCLU-p o- ein complexes, such as megalin (15, 42, 53), LRP1 (15) and ApoER2, VLDLR (22, 54) a e membe s o he LDL- ecep o gene amily. In a ecen s udy, sCLU-clien complexes we e in a enously injec ed in o mice, con i ming he sCLU-chape one/sca enging ac i i y (17). sCLU-ligand complexes we e ound o be en iched in he mice li e and kidneys, which a e in ol ed in deg ada ion and sub- sequen exc e ion o cellula oxins. The inding ha he en ichmen in he li e can be blocked by adminis a- ion o ucoidan (a po en inhibi o o sca enge ecep o class A, E and F) indica es ha sCLU is able o in e ac wi h sca enge ecep o s beside hose o he LDL- ecep- o gene amily. In addi ion, a dec ease in he emo al o glome ula p o ein deposi s was ound in CLU-K.O. mice (55). In humans, educed sCLU sec e ion is accompanied by a highe isk o Alzheime ’s disease (56). These ind- ings a e in line wi h obse a ions ha show ha sCLU p e en s he agg ega ion and oligome iza ion o Aβ and ans hy e in (57, 58). Conclusi ely, hese da a a gue o he p onounced ole o sCLU in p o ein homeos asis in he body. Disul ide bonds & p o eoly ic ma u a ion: C ucial componen s o dispensable ea u es? One o sCLU’s p ominen ea u es is i s ma u a ion in o an α- and a β-chain occu ing wi hin he golgi appa a us o e eb a e cells. The wo subuni s a e connec ed by 4–5 highly symme ical disul ide bonds. As sugges ed by Bon- Hong Min’s g oup, he o ma ion o disul ide bonds is a p e equisi e o sCLU ma u a ion/syn hesis (59). In e es ingly, a educ ion o hese disul ide bonds in ma u e sCLU does no inhibi i s ac i i y in chape - one ac i i y assays (11, 12, 50). This ole ance o sCLU o educing condi ions aises he ques ion whe he he α- and β-chain o sCLU ac independen ly as chape ones o whe he he disul ide bonds a e ex aneous o hei in a- molecula associa ion. I mus be no ed ha he p ope ma u a ion o sCLU is dependen on app op ia e disul ide bond o ma ion. T ea men o cells wi h di hio h ei ol (DTT), which p e en s disul ide bond o ma ion in he ER, abolishes he sec e ion o sCLU (60). Mo e ecen ly i was demons a ed ha mu a ions in he cys- ich egion o he CLU p o ein a e leading o educed sec e ion o sCLU in pa ien s wi h Alzheime ’s disease (56). Addi ionally, i was shown ha he inhibi ion o he p o eoly ic ma u a ion by in i o mu agenesis o he u in-like p op o ein con e ase (FC) clea age si e, which gene a es unclea ed sCLU, does no in e e e wi h sCLU-ma u a ion and i s chape one ac i i y bu a he ende s i highly sensi i e o educing condi ions (30). The lack o p o eoly ic ma u a ion may he e o e cause B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      5 an impai ed ‘ lexibili y’o sCLU (Figu e 2B). S udies o Bailey e al. e ealed ha he N- and he C- e minus o sCLU a e egions wi h diso de ed/mol en globule-like domains which a e likely in ol ed in clien binding (37). The e o e, he only egions le o s abilizing he sCLU p o ein a e p obably he disul ide bonds and he pu a i e amphipa hic helices nex o he FC clea age si e. As men- ioned abo e, ma u e sCLU is s ill su icien ly ac i e e en a e long- e m educ ion (11, 12, 30, 50). Howe e , he ac i i y o unclea ed sCLU a e long- e m educ ion is emendously impai ed (Figu e 2) (30). Thus, he amphi- pa hic domains o o he neighbo ing egions may be esponsible o sCLU p o ein s abiliza ion. In iguingly, p elimina y da a om ou lab indica e ha unclea ed educed sCLU pa ly egains i s chape one ac i i y when DTT is emo ed (Figu e 2C). In conclusion, we p opose Spe m p o eins Cellula p o eins dsRNA P dSe P dSe ecep o Endocy osis Endosome Ac i a ion o anc ip ion ac o s Dab1/AKT/Co ilin VLDLR/ ApoER2 ECM emodelling/mig a ion Pseudomonas ae uginosa Su i al o ca diomyocy es P oli e a ion o neu onal p ecu so s Modula ion o mi ochond ial in eg i y Rep esen a i e cell Lysosome Endoplasmic Re iculum Au ophagosome o ma ion AKT/GSK3β CLU-media ed complexa ion LDL- ecep o s Nec o ic cell Spe m cell Complemen inhibi ion MAC Immune modula ion B ain Blood Figu e 3: Role o CLU in cell biology, pa hology, immunobiology and beyond. Fo desc ip ion please see ex . B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM 6      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y ha he disul ide bonds a e on one hand impo an o co ec olding and ma u a ion bu on he o he hand a e no essen ial o he chape one unc ion o ma u e sCLU. Howe e , we canno exclude he possibili y, ha hey a e in ol ed in o he p ocesses. The la e is suppo ed by he no ion ha sCLU is able o es o e he unc ion o glu amine syn he ase by ac ing as a hiol speci ic- an ioxidan . Howe e , his ac i i y is impai ed when he disul ide bonds o sCLU a e alkyla ed o when he elec- on dono is no hiol-based (59). Ca bohyd a es: negligible o essen ial? Ano he p ominen ea u e o sCLU is i s high le el o glycosyla ion esul ing in ca bohyd a es, which com- p ise abou 30% o he molecula weigh (61). The gly- cosyla ion pa e n o human se um sCLU was elucida ed in 1997. Six N-linked ca bohyd a e a achmen si es a e ound wi hin he molecule, wi h h ee on he α- and h ee on he β-chain in humans (29). The ca bohyd a e compo- si ion is highly di e se and depends on he exp essing issue (4, 31). Se um sCLU mainly con ains ca bohy- d a e moie ies wi h sialic acid in a mono- and bian en- na y ashion (29, 31). Simila glycosyla ion pa e ns we e ound wi h ecombinan human sCLU (30). In con as , semen sCLU ca ies mainly ucose- ich glycans, enabling semen sCLU o bind o he C- ype lec in ecep o DC-SIGN ound on dend i ic cells. This binding was no obse ed wi h se um sCLU (31, 62). The e o e, he glycan moie- ies ei he change he spa ial s uc u e o sCLU so ha i s ecep o a ini y is al e ed, o he ecep o s and o he in e ac ing molecules ha e a ying a ini ies o dis inc glycan moie ies. In e es ingly, i was obse ed ha he glycosyla ion o sCLU is c ucial o co ec pola sec e ion in epi he- lial cells (28), bu no o chape one unc ion (41). sCLU ea ed wi h he endoglycosidase PNGase F only showed li le dec ease in chape one ac i i y (41). Indeed, i was ecen ly shown ha he emo al o he e minal suga s by exoglycosidases does nei he al e he chape one ac i i y no he seconda y s uc u e o sCLU. Howe e , i became ob ious ha plain PNGase F-diges ion esul s in esidual suga s s ill being a ached o he p o ein-co e. Fully degly- cosyla ed ecombinan human sCLU was ob ained by applica ion o a mo e elabo a ed p o ocol and was shown o possess a emendously dec eased chape one ac i i y. CD-spec oscopy u he e ealed ha upon ull degly- cosyla ion he amoun o α-helices lowe ed om 60% o 40%, whe eas he amoun o diso de ed egions inc eased signi ican ly om abou 20% up o 30%. A e emo al o all ca bohyd a es he ea angemen o he seconda y s uc u e indica es ha he olding o sCLU is co ela ed wi h i s glycosyla ion (30). In summa y, he co e-glyco- syla ion is a p e equisi e o he olding and chape one ac i i y o sCLU and e minal suga s migh be ins umen- al o o he mechanisms, such as ecep o binding and signal ansduc ion (21, 31, 62). In acellula clus e in: signi icance s. occu ence Nuclea CLU (nCLU) Since i s disco e y in he ea ly 1980s un il he mid-1990s, CLU was ega ded solely as a sec e ed p o ein (sCLU). I was no un il 1995 ha esea che s obse ed an associa- ion o CLU wi h he nucleus a e induc ion o cell dea h wi h an i-es ogens (26) o TGF-β (63). They specula ed ha i ansla ion would s a a a s a codon loca ed on exon 3 o he CLU-mRNA, an o he wise c yp ic nuclea localiza ion sequence (NLS) could be ac i e. Thus, Reddy e al. pos ula ed in e nal ansla ion ini ia ion as one pos- sible mechanism leading o N- e minally unca ed o ms o CLU. These unca ed o ms a e lacking he signal sequence o seg ega ion in o he ER, exposing an NLS and he e o e localizing CLU in he nuclea compa men (Figu e 1). This hypo hesis, howe e , is s ill lacking sup- po ing da a. A ound he u n o he millennium, he g oup o Da id Boo hman epo ed he exp ession o a nuclea o m o CLU (nCLU) in MCF-7 cells a e ea men wi h ionizing adia ion. They p oposed ha his nCLU o m would ac as a p o-dea h ac o by in e ac ing wi h he DNA- epai - associa ed p o ein Ku70. While i was ini ially assumed ha nCLU de i es om an ER-bo ne psCLU o m (64), he au ho s s a ed in la e publica ions ha ionizing adia- ion a o s in e nal ansla ion ini ia ion a a s a codon on exon 3, esul ing in he exp ession o nCLU (Figu e 1) (24, 65). Finally, in 2003, he same g oup epo ed ha an exon-skipping e en , esul ing in a ansc ip lacking exon 2 wi h i s signal sequence coding egion (he ea e e med a ian 1 [Δex2]), p ecedes he exp ession o nCLU (25). Su p isingly, o e exp ession-expe imen s wi h a ian 1 [Δex2] esul ed in s ong cy osolic localiza ion o he co esponding p o ein as de e mined by con ocal mic oscopy. Nuclea associa ions, howe e , could only be de ec ed by o e exp ession o a i icial cons uc s lacking dis inc po ions o a ian 1 [Δex2]. Despi e his B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      7 con o e sial obse a ion, he au ho s hypo hesized ha he p o ein ansla ed om he exon 2-lacking mRNA is he p e-nuclea o m o nCLU. I would hen be ans- o med in o ma u e nCLU o unknown pos - ansla ional modi ica ions induced by ionizing adia ion. Howe e , expe imen al p oo o his hypo hesis is s ill missing. Th oughou he i s decade o he 2nd millennium many esea ch g oups claimed a nuclea localiza ion o CLU in s essed cells, including cellula s ess induced by a ious agen s such as E oposide (66–68), In e leukin-6, Soma os a in (69), hea (70), Ca2+-deple ion (67, 71), se um-s a a ion (72), TNF-α- ea men (73), indocyanine- ea men wi h g een ea ex ac s (74), p o easome inhibi ion (75), ea men wi h 5′-Fluo ou acil and Fas ecep o -binding an ibodies (76) o anadium- ea men (77). Fu he mo e, nuclea localiza ion o CLU has been obse ed upon o e exp ession o c- os (77), In e leukin-24 (78) o pVHL (79), as well as spon aneously in un ea ed cells (69, 76, 80–82). Addi ional o he mechanisms dis- cussed abo e, i was a gued ha inhibi ion o sCLU sec e ion could lead o in acellula accumula ion and subsequen ly nuclea localiza ion (83). Howe e , dimini- shed sec e ion and he e o e in acellula accumula ion o CLU, as desc ibed by Be ens e al. (56), did no p o ide any e idence o nuclea localiza ion o CLU. Ano he poin o in ense deba e is he po en ial exon 2-skipping o CLU-mRNA (Figu e 1). The esul ing a ian 1 [Δex2], was commonly conside ed o explain he occu ence o nCLU in many s udies, al hough igo - ous mRNA-analyses ha e no been pe o med. This led o con o e sies, as some g oups could no alida e he exis ence o a ian 1 [Δex2] (84–86). I was specula ed ha i s exp ession migh be limi ed o MCF-7 cells o e en an expe imen al a i ac (75, 84–87). Howe e , P ochnow e al. con i med he exis ence o a ian 1 [Δex2] in se e al human cell lines using alida ed, a ian -speci ic p ime se s o RT-PCR (34). Ye , mRNA-quan i ica ion indica ed ha a ian 1 [Δex2] accoun s o  < 0.13% o o al CLU- mRNA, e en in s essed cells. The exon 2-con aining a ian 1, in con as , ep esen s he p e-dominan CLU- mRNA ( > 99% o o al CLU-mRNA). In his s udy i was also shown ha he p o ein encoded by a ian 1 [Δex2] local- izes solely in he cy oplasm o uns essed and s essed cells. In ac , e y ecen s udies suppo his obse a- ion, challenging he heo y o a nuclea localiza ion o CLU (32, 79, 80, 88–91). Only by inco po a ing an a i i- cial nuclea localiza ion sequence (NLS) a he 5′-end o a ian 1 [Δex2], a nuclea localiza ion o he ansla ed p o ein could be achie ed (92). This in u n ende s he exis ence o a unc ional NLS hidden in he CLU p e- p op o ein unlikely. CLU associa ed wi h mi ochond ia O e he cou se o he las ew yea s, an associa ion o CLU wi h mi ochond ia has equen ly been epo ed and in e ac ions wi h in insic apop ose- ela ed p o eins Bax and Bcl-xL ha e been discussed (32, 93–96). Thus, i was specula ed, ha ei he psCLU o sCLU migh ac an i- apop o ic by seques e ing Bax in i s inac i e s a e in he cy osol (95, 96). Howe e , in none o hese s udies i was in es iga ed, how ex acellula sCLU o ER/golgi- esiden psCLU can each he cy osol in o de o bind Bax (Figu e1). Li e al. (32) ecen ly showed ha a hypoglycosyla ed o m o sCLU can escape he sec e o y pa hway wi h he aid o he chape one GRP78 (BiP) and s abilize he mi ochon- d ial memb ane o a oid pacli axel-media ed apop osis (Figu es 1 and 3). An in e ac ion wi h Bax was, howe e , no in es iga ed in his s udy. O he epo s sugges ha CLU can ac p o-apop o ic by p e en ing Bcl-xL om binding o Bax (88, 97). The au ho s assumed ha non- sec e ed CLU o ms simila o nCLU, a e esponsible o his e ec . This was a ibu ed o a po en ial BH3-domain ound in silico wi hin he nCLU-sequence (97). Howe e , upon o e exp ession o dis inc in acellula CLU o ms, no p o-apop o ic p ope ies could be assigned o any case (34). Thus, he signi icance o CLU associa ion wi h mi o- chond ia s ill needs o be challenged. How signi ican a e dis inc in acellula CLU o ms? The di e se esul s and p oposals pu o wa d in he s udies on in acellula CLU o e he pas 20 yea s a e inconclusi e and e en con adic o y. One eason o his may be ha he desc ibed e ec s a e es ic ed o s essed/ damaged cells and ha he abundance o in acellula CLU o ms is negligible (34). Mos impo an ly, o ou knowledge, no dis inc in acellula CLU o ms ha e been desc ibed in o he o ganisms besides humans, ye . Addi- ionally, CLU o ms lacking ca bohyd a es and/o p o eo- ly ic ma u a ion a e no capable o main ain a chape one ac i i y in he cell (30). Fu he mo e, P ochnow e  al. ound no e idence o an apop osis-modula ing e ec o unglycosyla ed cy osolic CLU o ms. The e o e, we sugges ha in acellula CLU o ms may acciden ly e ol e in com- p omised cells and migh ha e no bene icial unc ion o he cell. One excep ion may be hypoglycosyla ed psCLU o ms e o ansloca ed om ER/golgi. These o ms ha e been desc ibed in he cou se o modula ing mi ochond ia in eg i y o au ophagy o p omo e cell su i al (Figu e 3) (32, 91). B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM 8      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y Cellula challenges: om nec osis o immunobiology and beyond sCLU and nec osis The exp ession o sCLU is highly co ela ed o issue degene a ion, nec osis and edox imbalances (98, 99). The s ess-associa ed up egula ion in esponse o UV ligh , hea -shock, oxidan s, hea y me als and in pa icula p o eo oxic s ess is media ed by he clu p o- mo o which con ains HSF1, NFκB and AP-1 binding si es (100). Cy o oxic s ess can also be induced by nec osis in neighbo ing cells (Figu e 3) (101). As a consequence, mis olded p o eins, ee adicals, lipids and o he com- ponen s accumula e in he ex acellula space causing an ac i a ion o he immune sys em and a ec neighbo - ing cells (101, 102). Rema kably, i was obse ed ha CLU up egula ion also occu s in cul u ed cells upon exposu e o nec o ic cell deb is (103). This up egula ion is d i en by componen s o he nec o ic cells, such as memb anous phospha idylse ine (P dSe ) (103) o by RNA eleased om he cy osol (Figu e 3) (104). P dSe , unde i al con- di ions, is es ic ed o he cy osolic side o he plasma memb ane and is exposed o he ex acellula en i on- men upon damage o apop osis (105). Ex acellula RNA is endocy osed by su ounding cells and up egula es CLU ia he TLR3-signaling pa hway (104). Once sCLU is sec e ed in o he ex acellula space, i media es he binding and endocy ic up ake o cellula deb is by ecep- o s o he LDL- ecep o gene amily, such as megalin o LRP1 hus ees ablishing he ex acellula p o eos asis (Figu e 3) (15, 30). sCLU and immunobiology Qui e ecen ly, da a sugges ha semen sCLU can bind s ess-damaged p o eins, acili a ing he up ake by den- d i ic cells ia DC-SIGN hus making a p iming o immune cells easible (Figu e 3) (31, 62). As a u he consequence, sCLU has been a ibu ed o media e ole ance o he emale geni al ac owa ds male an igens (Figu e 3) (62). This sugges s an immune egula o y ole o sCLU, which is e iden om mul iple in i o and in i o s udies. Ini- ially, sCLU was ound o be co-localized wi h complemen SC5b-9/MAC (memb ane a ack complex) in glome ulo- neph i i is bu no in heal hy indi iduals (106). Subse- quen ly, in associa ion wi h he SC5b-9 complex sCLU was cha ac e ized as an inhibi o o he complemen sys em (Figu e 3) (38, 49, 107). The complemen sys em causes cell damage unde enal pa hologies. Thus, sCLU may alle ia e enal inju ies by p e en ing an uncon olled MAC ac i i y (108). Howe e , in 1999 Hochg ebe e al. (109) showed ha complemen inhibi ion by sCLU is no possible a physi- ological concen a ions. Despi e hese ac s, he mos ecen da a sugges ha Pseudomonas ae uginosa can p o ec i sel om complemen a ack by ec ui ing sCLU (110). Simila sugges ions we e made o spe m cells in he emale geni al ac (Figu e 3) (49). Thus, an inc ease in he local sCLU concen a ion may achie e a complemen inhibi ing e ec . Addi ionally, o he da a suppo a ole o sCLU in immune modula ion: I was shown ha sCLU syne gize wi h IL-2 o enhance he p oli e a ion o na u al kille cells (111). Mo eo e , sCLU is desc ibed as a ansc ip ional a ge o he lympho oxin-β ecep o in ge minal cen e s du ing an immune esponse (112, 113). I was u he dem- ons a ed ha sCLU imp o ed he iabili y o B lympho- cy es (113). In addi ion, i is impo an o no e ha CLU was desc ibed as a necessa y ac o o cu a ion o au oim- mune myoca di is, which is accompanied by an inc eased le el o nec osis and in lamma ion (102, 114). These con- clusions we e u he suppo ed by obse a ions a e in a enously adminis a ion o sCLU in a s du ing myo- ca dial in a c ion. A e ea men wi h sCLU, he in a c size was signi ican ly educed and led o mac ophage in asion (115). This inding is suppo ed by an ele a ed chemo ac ic mig a ion o mac ophages due o inc eased ma ix me allop o einase-9 (MMP9) exp ession induced by sCLU ia he ERK1/2-, PI3K/AKT- and NFκB-signaling pa hway (Figu e 3) (21, 116). The abili y o sCLU o modu- la e mac ophage unc ion was u he con i med by an addi ional s udy which demons a ed ha he mig a o y e ec is ela ed o TNF-α sec e ion induced by sCLU (116). Howe e , an Dijk e al. (20) ha e ound no e idence o a di e ence in wound healing a e sCLU applica ion. Ra he , sCLU p o ec s ca diomyocy es om apop osis by inducing he PI3K/AKT-signaling pa hway (Figu e 3). I is impo an o no e ha nei he he p o ec i e e ec o sCLU on ca diomyocy es, he educ ion o in a c size no he in e ac ion wi h dend i ic cells a e ela ed o ecep o s o he LDL- ecep o gene amily (20, 62, 115). I is he e o e impo an o emphasize ha he cha ac e iza- ion o o he sCLU binding ecep o s is essen ial o he un a eling o sCLU unc ion. sCLU and issue epai The signi icance o sCLU in issue p o ec ion/ emodeling was ound o be impo an because o i s po en ial o B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      9 p e en o gans om damage unde ischemic condi ions, e.g. ischemia- epe usion inju y (IRI) a e ansplan- a ion (117). The ischemia- epe usion damage occu s when he bloods eam is ees ablished a e pe iods o limi ed nu ien and oxygen supply. The consequence o his mechanism leads o ini ial nec osis, ollowed by massi e apop osis o he su ounding issue (102). The su ounding apop osis is belie ed o be due o he elease o ee adicals, gene a ing lipid adicals in he cellula memb ane and he e o e causing emendous cellula damage (Figu e 3) (101). In ac , sCLU is up eg- ula ed a e oxida i e s ess caused by H2O2 o ciga e e smoke, p o ec ing he cells and main aining hei iabil- i y (19, 20, 59, 118). In he case o ansplan medicine, i was shown ha supplemen sCLU in Uni e si y o Wis- consin-solu ion (UW-solu ion) is easible o p olong he handling- ime be o e an o gan can be ansplan ed (119). Simila ly o his inding, i was obse ed ha a gene ic inac i a ion o he CLU gene leads o an inc eased o gan ejec ion as well as impai ed cell iabili y. These p o ec- i e ac ions a e p obably due o inc eased memb ane lu- idi y in he p esence o sCLU, minimizing cold-induced s i ening and shea s ess (117). Fu he mo e, sCLU is a candida e bioma ke o damage in he p oximal ubule o he kidney (120). Addi ionally, i was shown ha o e ime, he absence o sCLU leads o an inc eased accumula ion o componen s om he immune sys em wi hin he glome uli (55). Se e al in i o s udies sugges a cy op o ec i e unc ion o sCLU a e gen amicin- o H2O2-induced cy o oxici y in kidney cells (121, 122). When inducing IRI by clamping he enal pedicles o mice, sCLU is an essen ial ac o o enal egene a ion. In con as o mac ophages, howe e , sCLU has no p o- mig a o y e ec on enal ubula epi helial cells (TECs) bu p omo es p oli e a ion and cell iabili y o TECs. In con as o wild ype mice, CLU K.O.-animals exhibi a ema kably dec eased su i al a e o abou 90% o 30%, espec i ely (123). P e iously i was shown ha a e uni- la e al u e e al obs uc ion (UUO) he p esence o sCLU was a p e equisi e o supp ess TGF-β media ed up egu- la ion o plasminogen ac i a o inhibi o -1 (PAI-1). PAI-1 p e en s ea angemen o he ex acellula ma ix and hus a enua es plasmin ac i a ion leading o ib osis (124). Howe e , in con as o Nguan e al. (123) no in lu- ence o sCLU on p oli e a ion o apop osis on ubula epi helial cells was obse ed. Finally, i is impo an o men ion ha al hough Gi on e al. (121) ha e excluded an in ol emen o megalin, he pa icipa ing ecep o s media ing he cy op o ec i e e ec o sCLU (di ec ly o indi ec ly) a e s ill unknown. sCLU and he ne ous sys em Al hough i is well es ablished ha sCLU is cy op o ec- i e in he kidney and in he hea , he ole o sCLU in he ne ous sys em is s ill challenging. As men ioned ini- ially, sCLU was also desc ibed as an apolipop o ein (apoJ) which is p esen in se um ( ee and bound o high densi y lipop o ein (HDL) pa icles). sCLU(apoJ)-con aining HDLs accoun o app oxima ely 2% o plasma HDLs (125) wi h abou 20% o he ci cula ing sCLU being ac ually bound o HDLs (126). In con as o he b ain, sCLU(apoJ)-con ain- ing HDL pa icles in plasma a e mo e lipid- ich (38, 127). In he mammalian b ain sCLU (apoJ) and apoE a e he wo mos abundan apolipop o eins [ e iewed by Wang and Eckel (128)]. In iguingly, bo h apolipop o eins a e in ol ed in simila mechanisms, such as Alzheime ’s disease o cog- ni i e and memo y p ocesses (18, 128–130). I is impo an o no e ha e en hough sCLU is a cons i uen o HDLs, which a e he only lipop o ein subclass c ossing he BBB, only LRP1 and SR-B1 a e in ol ed in lipid homeos asis o he b ain (128). The emaining lipop o ein ecep o s a e in ol ed in a ple ho a o o he unc ions (128, 129). Inside he b ain, CLU is p ima ily exp essed and sec e ed by as ocy es bu also by o he ypes o cells, e.g. neu ons (127, 131, 132). Upon neu onal dea h, ischemia and wi h ad ancing age, howe e , a signi ican up egula- ion o CLU is obse ed (7, 131, 133, 134). P e iously, i was no clea whe he an ele a ed le el o CLU in he b ain was help ul o ha m ul. In 2001, wo g oups pe o med b ain- ischemia expe imen s and epo ed con adic o y esul s (133, 134). Whe eas Han e al. (133) ound ha he absence o CLU educes cell dea h in neona al hypoxia-ischemia, Weh li e al. (134) obse ed a neu op o ec i e e ec o CLU in pe manen ocal ce eb al ischemia. I is belie ed ha hese di e en indings a e due o di e en expe imen al se ings; mo e bene icial e ec s o CLU in i o and in i o we e obse ed elsewhe e. Fo ins ance, b ain ischemia induced by pe manen middle ce eb al a e y occlusion shows hea ily impai ed issue emodeling in CLU K.O.- animals. In wild ype animals, howe e , a signi ican CLU up egula ion by as ocy es and inc eased mic oglia/ mac ophage in asion was de ec ed (135). In e es ingly, ano he s udy has shown ha mic oglia ac i a ion by sCLU leads o ch onic in lamma ion and hus neu o oxic- i y due o obse ing an inc eased elease o ni i e oxide and TNF-α (136). Mo eo e , sCLU in as ocy e-condi ioned media om he hippocampus and midb ain had a con- ibu ing e ec on cell su i al and di e en ia ion bu no on p oli e a ion o neu al p ecu so cells (137). Howe e , B ough o you by | Helmhol z-Zen um ü In ek ions o schung Au hen ica ed Download Da e | 2/15/18 10:19 AM