Endolysosomal proteases

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i clea ed at the proximal e d of CLIP. The CLIPMHCcla II complex i the recog ized y the o -cla icalMHC cla II molec le H2DM a d H2DO i mice(HLA-DM a d HLA-DO i h ma ), hich are thechapero e that mediate tit tio of CLIP for a ti-ge ic peptide (FIG. 1).

The protea e that co ert Iip10 to CLIP are ellcharacterized: cathep i Li thymic epithelial cell a dcathep i s i b cell , macrophage a d de dritic cell(DC )2. Cathep i F a al o tho ght to clea e Iip10 imacrophage 3, t later a aly i of k ocko t mice di -co ted thi po i ility 4. M ltiple protea e ca clea ef ll le gth Ii a d Iip22, ho i g that there i protea ered da cy i the e t o reactio . The e of i hi i-tor of a paragi yl e dopeptida e (AEP; al o k o a

leg mai ) i dicated that thi e zyme might e i ol edi f ll le gth Ii proce i g, t ch a role i pro a ly red da t 5,6. It i orth oti g that Ii ha other f ctioi additio to co trolli g the peptide loadi g of MHCcla II molec le , i cl di g the reg latio of e do omalarchitect re 7,8, mod latio of DC migratio 9, recog itioof the pro-i flammatory cytoki e macrophage migratioi hi itory factor 10 a d clear factor-b (nF-b) ig al-li g11. Thi i rele a t he i g protea e i hi itor ork ocko t mice to a e the role of Ii-degradi g e zymei a tige pre e tatio a d T cell primi g eca e per-t r atio of imm e re po e co ld al o ari e i directly thro gh effect o a y of the e acti itie .

u like the clea age of Ii, the degradatio of e docy-to ed a tige i ot pecific or ordered; i tead, eachclea age poi t i determi ed mai ly y it acce i ility to the acti e ite of a protea e. Degradatio i a i ted

y the lo pH of e doly o ome a d the red ctio of di lphide o d , hich promote a tige foldi g toexpo e protea e- e iti e regio 2. A tige proce i gge erate a ra dom array of peptide , ome of hich

ill ha e the right eq e ce to fit i to the i di g iteof the partic lar et of MHC cla II allotype expre ed

y the APC. Ma y, if ot all, ch peptide fir t i dto MHC cla II molec le a lo ger polypeptide iththeir ami o a d car oxyl termi i protr di g o t of thepeptide- i di g groo e, to e eq e tly trimmed

til o ly a regio of the peptide protected y the MHC

cla II molec le remai 12. The e doly o omal protea epro a ly ha e a red da t a d, i ge eral, eco dary role i the electio of peptide that ill e e t ally epre e ted o the APC rface. ne erthele , there areexample of a tige that req ire a partic lar protea efor pre e tatio . For example, mo e a d h ma APClacki g AEP acti ity are impaired i the pre e tatio of teta toxi eca e AEP i req ired to i itiate proce -i g of thi a tige 13. There are al o example i hichthe a e ce of o e protea e ca i crea e the pre e ta-tio of a partic lar epitope: ome peptide from myeli

a ic protei (MbP) a d myoglo i are more efficie tly pre e ted y h ma APC that lack AEP a d y mo e

Box 1 | Endolysosomal compartments and lysosome-related organelles

The endolysosomal compartment is a vesiculo-tubular network of sorting and degradative organelles that accepts,redirects and processes molecules from the exterior or interior of the cell (see figure). Moieties that move from the cellsurface in transport vesicles arrive at the early endosome, where they are either sorted into a recycling pathway back tothe cell surface through a recycling endosome or are retained in the early endosome which, through a process of fusionand fission, transforms into a multivesicular body (also known as a late endosome). The multivesicular body then maturesthrough further fusion and fission events to a lysosome or lysosome-related organelle (LRO). On receipt of a signal, LROs

fuse with the plasma membrane to release their contents. The maturation of early endosomes into lysosomes is markedby a progressive decrease in interior (luminal) pH, which is crucial for the activation of resident enzymes that degradeexogenous material trapped in the lumen of the lysosome. Integration of the secretory (biosynthetic) and endolysosomalsystems ensures that newly synthesizedresident enzymes are delivered to theappropriate endolysosomal compartment afterexiting the trans -Golgi network and thatcomponents of the delivery system arereturned from the endolysosomal system tothe trans -Golgi network for re-use. AlthoughLROs have many features in common withlysosomes (including low internal pH,distinctive membrane proteins such as thelysosome-associated membrane proteinsand similar maturation pathways in the

endolysosomal system), LROs differ in theirinternal protein constituents betweendifferent cell types, particularly in the immunesystem. For example, the LROs of mast cellscontain lipid mediators, histamine, heparin andtryptase and chymase proteases; the LROs of dendritic cells contain the antigen processingmachinery including dedicated cathepsinproteases; and the LROs of natural killer cellscontain porins and granzyme proteases.

Multivesicularbody

Early endosome

Recyclingendosome

Fusion

Fusion

Fusion

Fusion

Nucleus

Endoplasmicreticulum

Trans -Golginetwork

Golgistack

Lysosome

Transport vesicle

LRO

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APC that lack cathep i D, re pecti ely, eca e thepeptide are other i e degraded y the e protea e 14,15.The role of AEP i the pre e tatio of MbP i of par-tic lar i tere t eca e AEP i a da tly expre edi h ma thym ; it a therefore propo ed that lack of MbP pre e tatio i the thym might pre e t the

egati e electio of a toreacti e MbP- pecific T cell ,hich co ld the recog ize the epitope i the periph-

ery a d co tri te to the o et of m ltiple clero i14.Thi hypothe i i pported y the o er atio that iperipheral h ma APC , cathep i G, ot AEP, ha acr cial role i the pre e tatio of MbP16. The pict rethat emerge from the e t die i that protea e ha e

oth creati e a d de tr cti e role i MHC cla II-re tricted a tige pre e tatio 13, t altho gh thea e ce of a i di id al protea e ca alter the relati ele el of pre e tatio of m ltiple peptide , i mo t ca ethe e cha ge are ot fficie t to alter the o erallpatter of T cell recog itio .

MHC class I-restricted cross-presentation. I ome typeof DC, imported a tige ca e pre e ted y MHC cla Imolec le i a proce k o a cro -pre e tatio ,

hich e a le DC to prime CD8+ T cell agai t ir eor t mo r itho t expre i g the iral or t mo r a ti-ge them el e17. similar to MHC cla II-re tricteda tige pre e tatio , there i e ide ce for oth crea-ti e a d de tr cti e role of e doly o omal protea e iMHC cla I-re tricted cro -pre e tatio(FIG. 1). Thereare mero example of exoge o a tige ei gdegraded i e doly o omal compartme t to ge eratepeptide that are o d i the e ame compartme t y MHC cla I molec le a mecha i m k o a the

e do omal path ay of cro -pre e tatio 18. The protea ecathep i s ha ee gge ted to ha e a importa trole i thi acti ity 19. similarly, the e doly o omal exo-protea e i li -reg lated ami opeptida e (IRAP) arece tly implicated i cro -pre e tatio , ha i g a rolei peptide trimmi g a alogo to that of the e dopla -mic retic l m-a ociated peptida e (ERAP) i cla icalMHC cla I-re tricted a tige pre e tatio20.

The co tri tio of e doly o omal protea e to cro -pre e tatio i co tro er ial eca e it eem likely thatthe e compartme t ca recapit late the co ditio fora tige proce i g i the cytopla m a d peptide load-i g i the e dopla mic retic l m that are req ired forcla ical MHC cla I-re tricted a tige pre e tatio . If DC did ot ge erate y cro -pre e tatio the amepeptideMHC cla I complexe that i fected or t mo rcell prod ce i g the cla ical MHC cla I-re trictedpath ay, the cro -pre e ti g DC might primeCD8+ T cell that o ld later e i capa le of recog iz-i g their i te ded target . A alter ati e, al eit ill-defi ed, mecha i m of cro -pre e tatio , amely

the c yto olic path ay 18, eem more pro a le. Thipath ay i ol e the tra fer of imported a tigefrom the e do ly o omal et ork to the cytopla m,

here a tige acce the proce i g a d peptide loadi gmachi ery of the cla ical MHC cla I-re tricted path-

ay (FIG. 1). The cyto olic path ay eem to predomi atein vivo eca e the i ol eme t of cathep i s i cro -pre e tatio i o ly detecta le he the cyto olic path-

ay i impaired19. similarly, the IRAP-depe de t path ay of cro -pre e tatio eem to operate o ly i a -

et of DC that are recr ited to i flamed ti e a dco tri te little to cro -pre e tatio y teady- tateDC 21. ne erthele , the cyto olic path ay ca al o ei fl e ced y the de tr cti e pote tial of e doly o-

omal protea e , eca e the more a tige that idegraded i e do ome , the le ill reach the cyto-pla m. I deed, dr g that i hi it e do omal acidifica-tio a d he ce proteoly i ch a chloroq i epromote cro -pre e tatio 22, a d cro -pre e ti gDC acidify their phago ome more lo ly tha do

o -cro -pre e ti g DC23. F rthermore, DC eemto tore imported a tige for lo g period i pe-cialized o -acidified compartme t , from here thea tige are grad ally tra ferred to the cytopla m24,25.The e o er atio pport the otio that protecti gor di erti g a tige from e doly o omal degradatioi crea e the q a tity of a tige that i a aila le for

deli ery to the cyto olic cro -pre e tatio path ay.se eral t die ha e i dicated a role for protea e

i hi itor of the cy tati family i the mod latio of a tige pre e tatio , partic larly i DC .Cy tati C i differe tially expre ed y DC pop latio 26, a d itle el of expre io a d i tracell lar di tri tio cha ged ri g DC mat ratio 2628. Altho gh cy tati C i aefficie t i hi itor of cathep i s, o t dy ha clearly

ho a ig ifica t role for cy tati C i a tige pre -e tatio 2,26,28. similarly, the fi di g that cy tati F(al ok o a le kocy tati ) i mai ly expre ed y hae-matopoietic cell 29,30 a d i deli ered to e do ome31 i dicated that thi i hi itor might ha e a role i a tige

Box 2 | General properties of proteases

Proteases or peptidases catalyse the hydrolysis of peptide bonds. Exopeptidasesremove residues from the ends of polypeptides, whereas endopeptidases break bondswithin polypeptides. Proteases belong to four main groups according to thecharacteristics of their catalytic sites: serine proteases, cysteine (thiol) proteases,aspartyl proteases and metalloproteases. In broad terms, proteases can be classified aseither degradative, meaning that the hydrolysis of peptide bonds continues until the

target protein is essentially disassembled, or regulatory, meaning that hydrolysis of oneor more specific bonds alters the biological activity of the target protein while leavingit largely intact.

Because proteins cannot be repaired but only replaced, proteolysis irreversibly altersthe proteome. Therefore, proteolysis is stringently regulated in biological systems byvarious mechanisms. These include: recognition requirements such that a protease willonly cleave a particular bond flanked by specific amino acids that dictate the fit of thesubstrate into the catalytic cleft of the protease; the synthesis of proteases in inactiveforms known as zymogens, which acquire enzymatic activity only after cleavage by afunctional protease; the separation (compartmentalization) of a protease from itssubstrates until it is released by a specific signal; the modulation of substraterecognition or catalysis by binding of the protease to a cofactor such as an ion, protein,nucleic acid or sugar; and the reversible or irreversible suppression of protease activityby a cognate inhibitor, which binds in the catalytic cleft of the protease and therebyprevents access to substrates.

The inter-species diversity of protease and inhibitor genes makes the use of animalmodels difficult, particularly for the development of therapeutics. Approximately 2% of mammalian genes encode proteases or their inhibitors, and those involved in immunityand reproduction show the most divergence between species and the most evidenceof positive selection during evolution.

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Antigenor PAMP

Plasma membrane

Cytoplasm

Proteases

Proteases

Proteases

Proteases

TLR TLR

TLR

Proteasome

MHC class IImolecule

MHC class Imolecule

MHC class Imolecule

MHC class IImolecule ( Ii)

CLIPPeptide

Ii

CLIP

peptide

Golgistack

Endoplasmicreticulum

Ii

-chain -chain

TAP

?

c

b

a

Toll-like receptors(TLRs). A family of membrane-spanning proteinsthat recognize pathogen-associated molecular patternsthat are shared by variousmicroorganisms. Signallingthrough TLRs generally resultsin immune activation.

pre e tatio . no t dy ha yet pro ided e ide ce for cha f ctio , altho gh it remai po i le that cy tati Fha imm omod latory role thro gh the i hi itioof cathep i C32.

Endolysosomal protease-dependent activation of patternrecognition receptors. The role of e doly o omalprotea e i a tige degradatio a d acti atio of thepeptide- i di g f ctio of MHC cla II molec lemake the e e zyme importa t compo e t of theadapti e ra ch of the imm e y tem, a d rece tfi di g ho that they al o ha e importa t f c-tio i i ate imm ity thro gh the acti atio of Toll-like receptors (TLR )(FIG. 1) . The TLR recog izepathoge -a ociated liga d a d i itiate ig alli gca cade that lead to i flammatio 33. Altho gh the TLRare expre ed i m ltiple ti e , their f ctio ha ee

t died mai ly i cell of the imm e y tem, partic larly

i APC . O e co teri g TLR liga d , APC ecretecytoki e a d i crea e their capacity for a tige pre -e tatio a d T cell tim latio , hich allo TLR-acti ated DC to promote i flammatio a d adapti eimm ity 34. TLR ca al o recog ize elf compo e t

that are relea ed d ri g ti e damage, a d exce i eexpo re to ch liga d ca ca e a toimm ity 35,altho gh thi might o ly happe i pecific it atiorather tha reflecti g a ge eral role for TLR i therecog itio of elf compo e t 36.

TLR acti atio m t e trictly reg lated a d thi iachie ed y e eral mecha i m . O e ch mecha i mi the compartme talizatio of TLR to i tracell larlocatio i hich they are likely to e co ter theirliga d le i fectio or ti e damage occ r37. Thii the ca e forTLR3, TLR7, TLR8 a d TLR9, hich ithe teady tate are mo tly eq e tered i the e dopla -mic retic l m38,39. whe APC ecome acti ated a a

Figure 1 | E a ea e i a e e e i a a i e e e a i . a| Endosomal Toll-likereceptors (TLRs) are synthesized in an inactive form. Proteolysis of their luminal portion by endolysosomal proteasesgenerates TLR forms that can mediate signalling after recognition of their pathogen-associated molecular patterns(PAMPs). b | Similarly, newly synthesized MHC class II molecules assemble in the endoplasmic reticulum with the invariantchain (Ii), a chaperone that needs to be proteolytically degraded to enable antigen peptide binding to the MHC class IImolecules. The antigenic peptides are generated by endolysosomal proteolysis of endocytosed material. | Such materialcan also be delivered to the MHC class I-restricted cross-presentation pathway. The route followed by antigens andMHC class I molecules in the cross-presentation pathway is still unclear (reviewed in REF. 18 ). Some reports indicatethat the cross-presented peptides might be generated in endosomal compartments and loaded onto MHC class Imolecules that are recycled from the cell surface. Other reports indicate that antigens are transferred to the cytoplasmand degraded by the proteasome, thereby accessing the canonical MHC class I-restricted antigen presentationpathway; transfer of antigens to the cytoplasm might occur from the endoplasmic reticulum or from a hybrid endosomeendoplasmic reticulum fusion vesicle. For simplicity, the various possible cross-presentation pathways are not shown.CLIP, class II-associated invariant chain peptide; TAP, transporter for antigen processing.

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Chagas diseaseA disease that is caused byinfection with the tropicalparasite Trypanosoma cruzi,which is transmitted throughthe skin by the faeces of blood-feeding triatomine bugs.In chronic cases, Chagasdisease is associated withautoimmune damage tovarious organs and potentiallyfatal cardiac and neuraldamage.

re lt of pathoge e i g, the protei unC93b1 directthe tra port of TLR3, TLR7 a d TLR9 to the e doly o-

omal compartme t 4043. A additio al le el of co trolha al o ee reported: f ll le gth TLR9 ca i d itliga d t ca ot trigger acti atio ig al itho t fir t

ei g proce ed y e doly o omal protea e to remo ea portio of it l mi al, n-termi al regio 4446 (FIG. 1) .so, TLR9 i y the ized, imilar to other e doly o omalcompo e t , a a q ie ce t pro-form that ha to eproteolytically acti ated to carry o t it f ctio . Thedo le- tra ded RnA receptor TLR7 a al o oted io e report to dergo proteolytic proce i g44, altho ghthi a ot o er ed i a differe t t dy 46.

A aly e of e eral protea e-deficie t cell a d of theeffect of protea e i hi itor ha e co cl ded that o i gleprotea e i re po i le for TLR7 or TLR9 proce i g, i di-cati g that there i red da cy i thi reactio 4446. Thico tra t ith a other report i dicati g a req ireme tfor cathep i Ki TLR9 ig alli g or it do treameffect , amely cytoki e ecretio 47. The e appare tly co tradictory re lt ca e reco ciled if the f ctio of

cathep i K i ot to acti ate TLR9 t to facilitate the i i-tiatio of ig alli g or cytoki e prod ctio . The pecificrole of e doly o omal protea e i ch i ate imm ere po e therefore remai to e f lly el cidated.

Hostpathogen interactionsPathoge y the ize protea e of ario cla e thatre em le the e doly o omal protea e of the ho t

oth f ctio ally a d tr ct rally 48,49. The e patho-ge protea e ha e f dame tal role i the life cyclea d ir le ce of pathoge , oth i tracell larly a d ithe extracell lar e iro me t. For example, para itichelmi th ( orm ) ecrete protea e i large q a titie50 to degrade the extracell lar matrix d ri g ho t i a ioa d i tracell lar protozoa , ch a Leishmania pp. , Plasmodium pp. a dToxoplasma pp., req ire protea efor i a io , tritio a d egre from ho t cell51. Athoro gh de criptio of all of the f ctio that ha e

ee a cri ed to the protea e ecreted y para ite a dother pathoge i eyo d the cope of thi Re ie a dfor thi e refer the reader to other excelle t article 51;here, e highlight ome rece t ad a ce that ill tratethe role of the e e zyme a imm omod latory fac-tor . s ch imm omod latory f ctio ca e impor-ta t determi a t of ir le ce. For example,Entamoebahistolytica, hich ca e amoe ia i the third mo tdeadly para itic di ea e after malaria a d chi to omia i

i more ir le t tha the related pecie Entamoebadispar ; thi ha ee attri ted i part to differe ce ithe type a d amo t of e doly o omal papai -likecathep i protea e ith imm omod latory prop-ertie that are ecreted y the t o pecie 52. similarly,Leishmania mexicana para ite lacki g the cathep i -like protea e b (CPb) complex are le ir le t thatheir ild-type co terpart eca e they elicit a moreeffecti e type of imm e re po e ( ee elo )5355.

The tra d cer of the imm omod latory effectof pathoge protea e are till poorly der tood.Commo target of ecreted pathoge protea e are IgA,IgG a d/or IgE, hich are clea ed y eri e protea e

of chi to ome orm 56 a d the papai -like cathep iof E. histolytica57. Imm oglo li degradatio i al o

ed y acteria a a mecha i m of imm e e cape:for example, Streptococcus pyogenes ecrete the pro-tea e Ides ( trictly, ot a e doly o omal protea e),

hich i a importa t ir le ce factor that ha trictprotea e pecificity for imm oglo li 58. striki gly,Ides adopt a ca o ical papai fold de pite the lack of

eq e ce homology ith papai 59. F rthermore, Ideshijack the mecha i m that are deployed y the ho tto i hi it pathoge protea e (cy tati , ee elo ), y

i g cy tati C a a cofactor to i crea e it acti ity 60.Other target of pathoge protea e i cl de comple-me t compo e t 51,52, i terle ki -18 (IL-18)61 a d thereceptor for IL-2 a d the Fc regio of IgE54. The targetof pathoge protea e are ot o ly fo d i the extra-cell lar e iro me t:Leishmania para ite re idi g imacrophage phago ome relea e CPb protei , hichacce the ho t cell cytopla m a d ppre the nF-bpath ay i i fected cell , i t r re lti g i the do -reg latio of IL-12 prod ctio 62. It i pro a le that ma y

more target of pathoge protea e ill e di co ered ithe f t re a i e tigatio i thi area i crea e .

Gi e the complexity of the i teractio et eepathoge a d their ho t , it i ot yet po i le to e ta -li h a clear correlatio et ee the acti itie of pecificpathoge protea e a d their imm omod latory effect .Certai acti itie might eem to e paradoxical, ch ai d ci g the prod ctio of pro-i flammatory cytoki e

y Entamoeba pp. protea e , hich promote rather thadecrea e the imm e re po e63. Ho e er, the i d ctioof a partic lar type of imm e re po e co ld e efitthe pathoge if the effector mecha i m a ociated iththat re po e are ot properly tailored to the i fectio .A commo co eq e ce of the ecretio of papai -likeprotea e y para ite i the i d ctio of T helper 2(TH2)-type imm e re po e , hich are optimalagai t certai pathoge . similarly, the CPb proteithat L. mexicana relea e i to the cytopla m of i fectedcell promote deleterio TH2-type, i tead of protecti eTH1-type, imm e re po e

5355 (FIG. 2) . Cr zipai , apapai -like protea e ecreted y Trypanosoma cruzi (theca al age t of Chagas disease ), elicit tro g TH2-typere po e that e efit the para ite64. Therefore, it might

e more appropriate to de cri e the role of pathoge pro-tea e or i hi itor i e a io of the imm e re po e aimm ode iatio rather tha imm o ppre io .

why doe the imm e y tem re po d to acterial

or protozoa papai -like protea e ith i effecti e TH2-type re po e ? The a er co ld e that ch protea e

ormally alert the imm e y tem to the pre e ce of m lticell lar para ite . Helmi th orm ch aFasciola hepatica (the li er fl ke) a dSchistosoma mansoni ecretelarge amo t of protea e 4850. beca e TH2-type i flam-matory re po e are mo t efficie t agai t orm , theimm e y tem might ha e e ol ed mecha i m to detectthe acti ity of pathoge protea e a a ig al of ormi fectio . I other ord , para ite protea e acti ity i theextracell lar milie might e recog ized y the ho t a atype of pathoge -a ociated molec lar patter or da ger

ig al to acti ate TH2-type imm e re po e , imilar to

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TH1 cell

TH1 cell

Naive CD4+ T cell

Bacteriaand viruses

Helminthworms

TH2 cell

TH2 cell

Naive CD4+T cellDC

DCTLR orother PRR

ProteasesPAMP

Basophil

C-type lectin receptorsA large family of receptors thatbind glycosylated ligands andhave multiple functions, suchas cell adhesion, endocytosis,target recognition by naturalkiller cells and dendritic cellactivation, as well as antigencapture and presentation.

the i d ctio of TH1-type imm ity y compo d thatare recog ized y TLR a dC-type lectin receptors 34 (FIG. 2).I pport of thi model, i jectio of the cy tei e pro-tea e papai promote the acti atio of a ophil , hichthe pre e t a tige to CD4+ T cell a d i d ce TH2 cellre po e thro gh the ecretio of IL-4 a d thymic tro-mal lymphopoieti 6569, hich re em le the type of reac-tio that i triggered y helmi th . The e fi di g pro idea mecha i tic expla atio for the ell-k o allerge icity of protea e , a exemplified y DERP1, a cy tei e protea ethat i excreted i the faece of d t mite70. Therefore,imm ity agai t helmi th orm , imm e e a io y

ome protozoa para ite a d hyper e iti ity to protea eallerge co ld all e triggered y a commo mecha i mof recog itio of extracell lar protea e .

The a ility of cy tei e protea e to i d ce TH2-typeimm e re po e depe d o their protea e acti ity,

hich i dicate that they are ot i tri ically pro-i flammatory t i tead are i ol ed i the prod ctio of pro-i flammatory mediator . It i ot yet clear hat the emediator are. se eral DC molec le that are i ol ed iimm e mod latio ca e clea ed y DERP1 a d, pre-

ma ly, y other papai -like protea e70, t a ophil ,

ot DC , are reportedly the mai APC i ol ed i theprotea e-mediated i d ctio of i flammatory T H2-typere po e68 (FIG. 2) . It i po i le that a ophil directly detect the pre e ce of protea e prod ct ; the e co ld e

imilar to tho e prod ct ge erated y acterial a d pro-tozoa para ite to e ade the imm e y tem ( ee a o e),

t thi i till pro ed. Ide tificatio of the e mediatori a major goal of o goi g re earch a they co ld re eal

e approache for the treatme t of pathological T H2 cellreactio . Ho e er, thi doe ot imply that the aller-ge icity of protea e i o ly a re lt of their e zymaticacti ity; other mecha i m , ch a direct i di g a dacti atio of TLR , co ld al o co tri te71,72.

I li e ith the f ctio of their target e zyme ,cy tati of oth ho t a d pathoge origi ha e eeimplicated i imm ity to i fectio . For the ho t, type Ia d II (cytopla mic a d e doly o omal or ecreted,re pecti ely) cy tati pre e t herpe implex ir repli-catio a d the i d ctio of apopto i i i fected cell7375.Thi co ld e the re lt of i terfere ce ith iral pro-tea e a d ith pro-apoptotic reactio that are i itiated

y e doly o omal e zyme , re pecti ely ( ee elo ), tthe exact mecha i m are ot yet k o . Mo t t dieof the role of cy tati i i fectio di ea e ha e foc edo protozoa or orm i fectio . striki gly, i jectio of cy tati C protect mice agai t lethal i ceral lei hma i-a i 76. Ho e er, thi protectio i mediated y the acti a-tio of macrophage effector re po e y cy tati C a d

ot y the i hi itio of Leishmania or ho t protea e 77.I deed, cy tati C ha imm oreg latory propertie thatare i depe de t of protea e i hi itio 78. More ork ireq ired to f lly characterize the role of cy tati a dother protea e i hi itor of the ho t i the reg latio of imm e re po e .

with regard to protea e i hi itor expre ed y path-oge , the e t t died are tho e of filarial ematode79.

The imm oreg latory propertie that ha e ee attri -ted to the e molec le i cl de the i hi itio of a tige

pre e tatio 80,81, i d ctio of IL-10 expre io 81 a dmacrophage tim latio 82, imilar to the f ctio of ho tcy tati 7678. Leishmania pp. expre a iq e cy tati ,i hi itor of cy tei e peptida e (ICP), hich decrea e the ir le ce of the pathoge he o erexpre ed i recom-

i a t para ite , pro a ly a a re lt of the i hi itio of CPb protei ( ee a o e)83. Ho e er, the para ite i af-fected y the a e ce of ICP, o it phy iological role iimm omod latio remai certai 83. The otiothat pathoge cy tati might ha e imm omod la-tory role y affecti g ho t protea e make e e i

Figure 2 | I a e a a i e ea e .Helminth worms secrete proteases that activatehost-protective T helper 2 (T H2)-type immune responses through dendritic cells (DCs) or basophils. Endogenous parasitesand bacteria induce host-protective T H1 cell responses mediated by DCs activated through Toll-like receptors (TLRs) orother pattern recognition receptors (PRRs). T H1 cell responses are effective against these pathogens, but the pathogenscan also secrete proteases that cause immune deviation towards T H2 cell responses and inhibition of the T H1 cell response.PAMP, pathogen-associated molecular pattern.

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Intrinsic apoptotic pathwayA cell death pathway that istriggered by stressors such asgrowth factor deprivation,cytotoxic drugs and radiation.By contrast, the extrinsicapoptotic pathway is triggeredby death receptor signalling.

the light of the f ctio that ha e ee de cri ed forpapai -like protea e i imm e reg latio . Ho e er,the e ide ce that ha ee o tai ed o far to pport thif ctio i circ m ta tial a d a ed o a aly ein vitro.F rthermore, a mecha i m to explai the effect of cy tat-i o cytoki e prod ctio i lacki g, a d there i e i-de ce that thi effect might e i depe de t of protea ei hi itio . A clearer pict re ill e o tai ed a morepathoge lacki g i di id al cy tati ecome a aila lefor te t in vivo. It i al o ece ary to characterize the

iochemical path ay that are mod lated y cy tati ,hich i t r ill req ire ide tificatio of the trate

of their target protea e .

Endolysosomal proteases in cytotoxic cellsThe LRO of lymphocyte a d gra locyte co tai

mero eri e protea e that are re po i le forextracell lar matrix remodelli g, chemotaxi , cytoki emod latio a d the de tr ctio of microorga i ma d compromi ed cell . The e t k o protea eare e trophil ela ta e, cathep i G, trypta e a d

chyma e, hich are fo d i gra locyte , a d thegra zyme that are fo d i cytotoxic T lymphocyte(CTL )84,85. Altho gh pre io ork o gra zymeha foc ed mai ly o their role i killi g da gero cell (a de cri ed riefly here), more rece t fi di gi dicate that gra zyme ha e other importa t f c-tio . Gra zyme are more idely di tri ted thapre io ly tho ght, ei g expre ed i reprod cti e ti -

e a d y gra locyte , ma t cell , CD4+ T cell a db cell 86,87. no el f ctio of the gra zyme i cytoki eproce i g, direct i terfere ce ith iral pathoge a dextracell lar matrix clea age are ecomi g appare t, adi c ed elo .

The i tracell lar milie pro ide o ligate i tracel-l lar para ite ch a ir e ith oth the opport ity to hijack cell lar machi ery for their meta olic p r-po e a d a ha e from phagocytic cell a d op o iz-i g a ti odie . The re lti g di t r a ce i cell larhomeo ta i o ld ormally trigger apopto i , tmo t ir e ha e de eloped mecha i m to delay celldeath til they ca pread to a i fected cell88. Todeal ith thi pro lem, the imm e y tem deployCTL (ge erally CD8+) a d at ral killer (nK) cell tokill i fected cell . O er e ol tio ary time, the ig al-li g path ay that trigger cell death ha e ecome morepo erf l a d er atile to o ercome the large m erof molec lar trategie de eloped y differe t ir e

to tall apopto i .Altho gh CTL a d nK cell rface receptor i ter-

act ith i fected cell i a differe t ma er, oth celltype e highly co er ed mecha i m to kill targetcell . LRO co tai i g gra zyme ith ario -

trate pecificitie e the microt lar apparat tomigrate to ard the target cell, f e ith the pla mamem ra e a d relea e gra zyme i to the arro a dtra ie t i tercell lar y ap e89. The target cell the die

y apopto i , hich i typically completed i mi teor (at the mo t) 12 ho r . The gra zyme exert theireffect y clea i g importa t trate i the targetcell cytopla m a d cle . The preci e mecha i m

derpi i g cell death are complex a d till co tro- er ial, a de cri ed i detail el e here84,85. There i aclear co e that gra zyme ca e ter the target cell

y e docyto i irre pecti e of their proteolytic acti ity or the req ireme t for pecific receptor 9092. Ho e er,acce to cytopla mic trate i trictly depe de to the ( o -proteolytic) mem ra e-di r pti e proteiperfori , hich i co-packaged i LRO a d relea edtogether ith gra zyme . O i g to the m ltiplicity of cell death path ay that are acti ated y i di id algra zyme , the a e ce of a i gle gra zyme doe otge erally a oli h or e e e erely i hi it target celldeath. by co tra t, gra zyme-depe de t cell death itotally a oli hed i perfori -deficie t mice, re lti gi e ere imm e ppre io , cepti ility to ma y ir e a d po ta eo haematological ca cer late ilife93,94. There are o report of gra zyme-deficie t miceor h ma ith i crea ed cepti ility to po ta eoca cer. O ce perfori ha ee deli ered to a LRO, theacidic pH mea that it ca ot i d calci m or poly-merize, hich e re that tored perfori remai

q ie ce t to protect the killer cell from i ad erte t dam-age efore perfori a d gra zyme relea e95. The f ctioof perfori are calci m depe de t a d calci m i di gi totally a oli hed elo pH 6.2(REF. 96) .

why are there o ma y gra zyme ? There i lit-tle do t that the proteolytic repertoire of gra zyme arie et ee differe t orga i m , depe di g o thera ge of pathoge ic ir e that the pecie e co ter .no ethele , all mammal ha e at lea t o e gra zyme

ith the capacity to clea e after acidic, typically a par-tic acid, re id e (gra zyme b) or after a ic re id e(gra zyme A a d gra zyme K), a d at lea t e eralgra zyme that clea e after hydropho ic re id e(gra zyme M a d ortholog e of h ma gra zyme H).The ca pa e-like clea age pecificity of gra zyme b iimporta t i i d ci g apopto i , ch that DnA frag-me tatio i cell targeted y gra zyme b-deficie tCTL i markedly delayed97. The effector ca pa e 3,6 a d 7 ca e directly proce ed y i itiator ca pa e ,

herea the pro-apoptotic bCL-2 family mem er bH3-i teracti g death domai ago i t (bID) al o co tai a

ite that i clea ed excl i ely y gra zyme b98. Clea ageof bID re lt i mitocho drial o ter mem ra e permea-

ilizatio thro gh the tra locatio of bCL-2-a ociatedX protei (bAX) a d bCL-2 a tago i t killer (bAK), a ddeath thro gh the intrinsic apoptotic pathway 99. Ho e er,gra zyme b protei from differe t pecie ha e o er-

lappi g t ig ifica tly differe t trate prefere ce ,ch that h ma a d rat gra zyme b prefere tially

clea e bID, herea i mo t cell type , mo e gra zymeb trigger cell death thro gh direct pro-ca pa e proce -i g100,101. The importa ce of the gra zyme b-depe de tcell death path ay ha o ee ho i g i tactCTL a ell a p rified reage t102,103.

The tle t ig ifica t ariatio i gra zymef ctio et e pecie , a d the polymorphic at reof gra zyme b i o t red mo e pop latio 104, i di-cate that the gra zyme ha e dergo e adaptatioi re po e to e ol tio ary pre re y pathoge .Perhap al o co i te t ith thi otio i the fi di g

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Proteases

Proteaseinhibitor

Proteaseinhibitor

LRO

a

b

c

Release signal orstress response

Lysososomalpermeabilization

Caspase-dependentcell death

Caspase-independent

cell death

Immunologicalsynapse

Target cell

Cytotoxickilling

Immune signallingand tissue remodelling

AnoikisThe apoptosis of anchorage-dependent cells followingdetachment from theirsupporting extracellular matrix.

that mo t gra zyme ca trigger cell deathin vitro hedeli ered y perfori 105108, t the relati e importa ceof thi f ctio in vivo i till clear109. The path ayleadi g to target cell death i re po e to gra zymeother tha gra zyme b are ge erally poorly der-

tood, t they typically do ot req ire ca pa e acti a-tio i in vitro t die . whether thi i al o tr ein vivo remai to e determi ed, t i diffic lt to approachexperime tally o i g to ge e compe atio . For exam-ple, gra zyme A-deficie t CTL ha e o o ert cytotoxicdefect, t it i po i le that gra zyme K, hich ha im-ilar trate pecificity to gra zyme A, ca compe atefor the lack of gra zyme A 110.

It i al o ecomi g i crea i gly clear that gra zymeha e ma y f ctio other tha i d ci g cell death111.For example, h ma gra zyme b a d H f ctio coop-erati ely to directly i terfere ith iral DnA replicatioa d irio a em ly, a ell a facilitati g cell death112.Gra zyme b ca al o i d ce anoikis i a chorage-depe de t cell li e a d ca facilitate lymphocyte traf-ficki g y clea i g extracell lar matrix protei 113. The e

extracell lar effect do ot req ire gra zyme deli ery tothe i terior of a target cell, t o ld req ire the f ioof LRO ith the pla ma mem ra e a d eq e trelea e of gra zyme i to the extracell lar pace. F ioof ly o ome or LRO ith the pla ma mem ra e i a

ell doc me ted a d reg lated proce that occ r i ario it atio , ch a mem ra e o d heali g.

I a fa ci ati g rece t t dy, it a ho thatgra zyme A-deficie t mice are partially re i ta t tolipopoly accharide (LPs) challe ge a d that thi effectmight e mediated thro gh decrea ed proce i g a d/or relea e of IL-1 y APC 114. A the LPs ig al i medi-ated thro gh TLR4 o DC a d macrophage , a d the ecell do ot typically expre gra zyme , it ill e i ter-e ti g to ra el the mecha i m y hich gra zyme Amight i fl e ce pro-IL-1 proce i g.

Cell death and the endolysosomal systemI their role a degradati e orga elle , ly o ome a d

ome LRO co tai a ra ge of e zyme that are dedi-cated to di ma tli g a d recycli g cell lar compo e t .The i ad erte t relea e of the e ly o omal e zyme i tothe cytopla m o ld ha e detrime tal effect o or-mal cell lar f ctio a d the pote tial to ca e death(a toly i )115 (FIG. 3) . I deed Chri tia de D e116, hodi co ered ly o ome , recog ized thi pro lem a dtermed ly o ome icide ac . That ch relea e ofly o omal e zyme act ally occ r i gge ted y the

pre e ce of cytopla mic i hi itor of the e e zyme ithe i terior of cell . si ce de D e time, m ch ork ha ee do e to der ta d he a d ho ly o omemight e de ta ilized or damaged fficie tly to relea etheir co te t a d ho cell re po d to the egre of ly o omal e zyme . M ch of hat ha ee lear t a o tly o omal ta ility ca e applied to LRO , o i g totheir imilaritie , partic larly the role of protea e ii d ci g cell damage a d death.

Ho are ly o ome damaged a d hat are the co -eq e ce ? Damage refer to a reach of ly o omal

mem ra e i tegrity (permea ilizatio ), hich allothe egre of ly o omal protei i to the cytopla m115.Mo t damage re lt from cell tre , ca ed y fac-tor ra gi g from expo re to oxida t to radiatio(FIG. 3) . Ho e er, i ome cell type , death receptor-mediated ig alli g eem to i d ce ly o ome permea-

ilizatio , rai i g the po i ility that ly o ome or LROcompo e t co ld e tra d cer or effector of pro-grammed cell death ig al (re ie ed i REF. 117 ). Theco eq e ce of ly o omal damage for the affectedcell depe d o the degree of tre : lo le el of treca e limited permea ilizatio of a fe ly o ome a dmight e co tered y defe ce a d repair mecha i m ,

herea higher le el of tre ca e ide pread per-mea ilizatio affecti g more ly o ome , hich lead toirre er i le damage a d cell death.

Of the ly o omal e zyme , protea e eem toca e the mo t damage he they are relea ed i tothe cytopla m118. Co trary to the cla ical ie thatly o omal e zyme f ctio o ly at acidic pH, ma y of the protea e co tai ed i ly o ome remai f c-tio al at e tral pH a d alter ati ely pliced i oformof the e protea e might ha e ge i e f ctio ithe cytopla m (BOX 3) . Relea e of cathep i b, cathep-

i D a d cathep i L from ly o ome , i partic lar, ia ociated ith cell death thro gh m ltiple, cell type-

pecific path ay 119. Cathep i b a d cathep i L caclea e the pro-apoptotic tra d cer bID to acti atethe i tri ic apoptotic path ay, herea cathep i D

Figure 3 | E a ea e a e ea .Proteases are stored inlysosome-related organelles (LROs), in either a quiescent (granzymes) or active(cathepsins) form. During an immune response, LROs are signalled to releaseproteases into the environment ( a ) or, in the case of cytotoxic T lymphocytes, intothe interior of an abnormal target cell (through an immunological synapse) toinduce target cell death ( b ). Under conditions of stress, or possibly after receipt of a receptor-mediated signal, lysosomes or LROs can be destabilized and undergopermeabilization, releasing proteases into the cytoplasm ( ). Unless opposed by acognate cytoplasmic inhibitor (such as a serpin or cystatin), the released proteasescan trigger caspase-dependent or -independent cell death.

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Activation-induced cell deathA process by which fullyactivated T cells undergoprogrammed cell deathfollowing binding of the T cellreceptor by antigen ormitogen.

acti ate bAX to relea e apopto i -i d ci g factor(AIF) from mitocho dria. There i al o e ide ce for thecathep i -mediated acti atio of ca pa e-i depe de t

apopto i path ay , t a yet the e path ay arepoorly defi ed 117. similar to ly o ome , LRO co taicathep i t ma y LRO al o co tai additio alprotea e that co ld co tri te to cell damage a ddeath if relea ed i to the cytopla m of the ho t cell.It ho ld e oted that the eri e protea e that arefo d i lymphocyte a d gra locyte LRO f ctiooptimally at e tral pH o o ld operate effecti ely ithe ho t cell cytopla m. For example, the LRO of CTLco tai gra zyme that are pote t apopto i i d c-er he relea ed to kill target cell , a d there i e i-de ce of gra zyme-mediated elf de tr ctio of othT cell a d nK cell a a re lt of LRO de ta ilizatio .Gra locyte tore protea e ch a cathep i G a dcathep i D i LRO k o a az rophilic gra le .Cathep i D i relea ed from de ta ilized az rophilicgra le i e trophil i a reacti e oxyge pecie(ROs)-depe de t ma er, leadi g to cell death 120.Cytopla mic cathep i G ha the pote tial to ca e ho tcell death y clea i g pro-apoptotic protei 121,122.

Regulated release of LRO proteases into the cytoplasm? A co tro er ial i e i hether ly o ome a dLRO de ta ilizatio a d permea ilizatio occ r o ly i re po e to pecific tre or hether they are al oi tri ic compo e t of a apopto i path ay 117 (FIG. 3) .I the latter ce ario, ( defi ed) ly o omal permea-

ilizer a d the relea ed protea e o ld act a ig altra d cer co pli g a death receptor ith co er eddo tream eleme t of the cell death path ay, cha bH3 family mem er or ca pa e . I itial e ide cefor cell death path ay i ol i g receptor-co pled ly -o ome permea ilizatio came from in vitro t die of hepatocyte 123 a d t mo r cell 124, i hich acti atioof the t mo r ecro i factor (TnF) receptor ge erateca pa e 8, hich promote the relea e of cathep i bfrom ly o ome , there y i itiati g or amplifyi g celldeath. Cathep i b-deficie t mo e hepatocyte re i tTnF-mediated ly o ome permea ilizatio . Ho e er,i ome etti g , TnF-mediated cathep i b relea e

appare tly precede ca pa e acti atio , i dicati g thatthe po itio of ly o omal protea e a d ca pa e i thecell death path ay might e cell type depe de t.

I the co text of the imm e y tem, there ie ide ce that receptor-co pled ly o ome permea iliza-tio ca occ r a d that it co ld co tri te to le kocytehomeo ta i . For example, CD95-mediated apopto iof germi al ce tre b cell req ire cathep i acti ity a d i a ociated ith ly o ome de ta ilizatio125. Al o,model of peripheral T cell deletio i d ced y higha tige load i dicate that T cell death i ol e ly o omepermea ilizatio a d the relea e of cathep i b a dcathep i L126. A d i macrophage , i hi itio of nF-b promote TnF-mediated cathep i b relea e a dcell death127. I additio , after the acti atio of nK cellthro gh CD2, mo t cell die rapidly a d gra zyme bi pre e t i the cytopla m, i dicati g that ly o omepermea ilizatio ha occ rred128. Gra zyme b i al oo er ed i the cytopla m of h ma CD8+ T cellafter tim latio of the T cell receptor in vitro, a dthi i a ociated ithactivation-induced cell death 129.

Gra zyme b i al o implicated i the co tractioof TH2 cell pop latio d ri g the re ol tio of aimm e re po e: TH2 cell dergoi g apopto i ha egra zyme b i the cytopla m a d gra zyme b-deficie tmice ha e i crea ed cepti ility to a thma o i g toa fail re to re ol e TH2 cell re po e

130.

Dealing with the release of LRO proteases into thecytoplasm. If tre elicit ly o ome permea ilizatioa d the i te ded relea e of toxic protea e , cellm t ha e de eloped oppo i g defe ce y tem(FIG. 3) .I deed, mo t cell prod ce cytopla mic protea ei hi itor : for example, ly o omal thiol cathep i arei hi ited y the cytopla mic (type 1) cy tati tefi A a d tefi b131. The importa ce of ch reg lator i

ho y the i crea ed e iti ity of tefi b-deficie tmice to e ro al apopto i , hich i ameliorated

he the mice are cro ed to cathep i b-deficie ta imal 132. If ig alli g req ire ly o ome permea i-lizatio a d the relea e of cytotoxic protea e a pe-cific effector of cell death, cell might e protea ei hi itor to pro ide a ffer that allo protectiofrom lo le el of ly o ome permea ilizatio re lti gfrom tre or ackgro d ig alli g, t hich ca eo ercome he a a the tic ig al i recei ed a d thele el of protea e relea ed i to the cytopla m reachea acti atio thre hold.

some eri e protea e a d thiol cathep i areal o co trolled y i tracell lar eri e protea e i hi i-tor ( erpi )133. A good example of ch a reg latori erpi b9, a i hi itor of gra zyme b134. In vitro

t die i dicate that thi cleocytopla mic erpiprotect cytotoxic lymphocyte from the leakage of gra zyme b from their LRO 135. Co i te t ith thihypothe i , mice lacki g erpi b9 ha e lo er m-

er of acti ated, gra zyme b-prod ci g CD8+ T cellafter i fectio , a d the T cell that are pre e t ha efe er LRO a d expre le gra zyme b. by co tra t,mice lacki g oth gra zyme b a d erpi b9 ha e

ormal m er of T cell 136. I a other example, the

Box 3 | Extra-lysosomal functions of cathepsins

The conventional view of cathepsins is that they are enzymes that function inlysosomes at acidic pH. This view has been challenged by the demonstration thatcathepsins are released from cells and can function in environments at neutral pHin processes such as extracellular matrix degradation or the processing of thyroglobulin 144,145 .

In addition, it has been shown recently that cathepsin L is present in the cell nucleus

and that during S phase of the cell cycle it processes the transcription factor cut-likehomeobox 1 (CUX1; also known as CDP) 146 , which is a homeodomain protein thatrepresses developmentally regulated lymphoid and myeloid genes. Cux1 -knockoutmice have decreased thymic cellularity and decreased numbers of bone marrow B cellsas a result of increased apoptosis, which indicates that CUX1 controls cell survivalsystems 147,148 . Both CUX1-deficient and cathepsin L-deficient 149 mice also have defectsin hair follicle morphogenesis. Furthermore, cleavage of histone H3 by cathepsin Loccurs during mouse embryonic stem cell differentiation 150 .

Taken together, these findings indicate that nuclear cathepsins and their inhibitorsmight have roles in haematopoiesis and immune cell survival.

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mo e erpi A3G, a cathep i b i hi itor, protectcell agai t TnF- or ROs-i itiated ly o ome perme-a ilizatio a d cathep i b-i d ced death137,138, a d

erpi A3G i propo ed to facilitate the de elopme tof cytotoxic T cell to memory T cell 139.

so, e eral li e of e ide ce i dicate that permea i-lizatio of LRO a d e doly o omal protea e-mediatedcell death ca occ r i re po e to tre a d receptor-mediated ig alli g. At pre e t, the pathophy iologicalco eq e ce of alteratio i LRO ta ility or permea i-lizatio co pled ith alteratio i protea e or i hi itorf ctio are diffic lt to determi e eca e of differe ce

et ee pecie i term of the m er of protea ea d i hi itor a d their pecificity. For example, miceha e te gra zyme compared ith fi e i h ma , a dorthologo gra zyme i the t o pecie ha e differ-e t pecificitie 100. Mice ha e ma y more cathep itha h ma , mai ly i the reprod cti e y tem, tthey lack cathep i v fo d i h ma 140. Exampleof differe ce i i hi itor i cl de the ix homolog eof h ma erpi b9 that are fo d i mice141, the three

mo e homolog e of h ma tefi A142, a d mo eerpi A3G, hich ha o h ma co terpart. A mice

ge erally ha e larger m er of protea e a d i hi i-tor tha h ma , the pote tial for co fo di g effect imo e k ocko t model a a re lt of ge e compe atio

eed to e caref lly co idered.

Conclusions and future directionsImm ologi t are familiar ith the f ctio of e doly o-

omal protea e i a tige pre e tatio , t the t diee ha e re ie ed here ha e ho that the e e zyme

a d their i hi itor ha e additio al role i i ate imm -ity, reg latio of cell death a d co trol of pathoge

i a io , amo g other importa t f ctio . we thi k iti importa t to dedicate more atte tio to thi ject

eca e of the pote tial to ge erate pote t, pecific, y -thetic i hi itor of the protea e i large q a titie143. Thetherape tic pote tial of the e compo d i e ormo ,

t ca o ly e realized if e o tai a acc rate de crip-tio of the protea e that are implicated i each partic larf ctio a d ide tify their rele a t trate .

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AcknowledgementsThe authors are supported by the National Health andMedical Reseach Council, Australia. J.A.V. is a Leukemia andLymphoma Society scholar.

DATABASESUniProtKB: http://www.uniprot.orgAEP | cathepsin B | cathepsin C | cathepsin D | cathepsin F |cathepsin G | cathepsin K | cathepsin L | cathepsin S |chymase | cruzipain | cystatin C | cystatin F | DERP1 | ERAP |granzyme A | granzyme B | granzyme H | granzyme K |granzyme M | ICP | IdeS | Ii | IRAP | neutrophil elastase |perforin | serpin A3G | serpin B9 | stefin A | stefin B | TLR3|TLR7| TLR8| TLR9 | UNC93B1

FURTHER INFORMATIONMEROPS peptidase database: http://merops.sanger.ac.uk/

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