Table Of ContentKlaudia Brix · Walter Stöcker Editors
Proteases:
Structure
and
Function
Proteases: Structure and Function
ThiSisaFMBlankPage
Klaudia Brix (cid:129) Walter Sto¨cker
Editors
Proteases: Structure
and Function
Editors
KlaudiaBrix WalterSto¨cker
SchoolofEngineeringandScience InstituteofZoology
JacobsUniversity JohannesGutenbergUniversity
Bremen Mainz
Germany Germany
ISBN978-3-7091-0884-0 ISBN978-3-7091-0885-7(eBook)
DOI10.1007/978-3-7091-0885-7
SpringerWienHeidelbergNewYorkDordrechtLondon
LibraryofCongressControlNumber:2013956226
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Foreword
The first chapter “Protease Families, Evolution, and Mechanism of Action”
providesuswithacomprehensiveandrefreshingviewoverproteaseandpeptidase
family relationships. It is written by Neil Rawlings, THE expert, when it comes
to proteolytic enzyme classification. He has pioneered and initiated with Alan
Barrett MEROPS, an invaluable database for protease researchers, that has been
maintained and updated for several years now. The first chapter of this book
therefore introduces and explains the logics of this exceptional information
resourceandcompendiumonproteases.TheconclusionofNeilRawlings’chapter
considersclassificationbasedonthecomparisonofsequencesandstructuralmotifs
ofproteasesthatevolvedovermillionsofyearswiththeir(substratespecificitiesand)
typesofcatalysisduringproteolysis.
Thesecondchapter“KineticsoftheInteractionofPeptidaseswithSubstrates
andModifiers”focusesonthetoolsprovidedbyenzymekineticsforcharacterizing
peptidase-mediated turnover of substrates. Enzymatic reactions eventually take
place in the context of interactions of the specific enzymes with their modifiers,
which may function as activators and inhibitors. Thus, the modifiers of specific
enzymes—here,proteases—areofhighrelevancewhendiscussingenzymekinetics
conceptually.Poisedbytheireducationalanddidacticaltalents,theexpertsAntonio
Baici,MarkoNovinec,andBrigitaLenarcˇicˇteamedupinwritingachapterthatwill
serveasanexcellentguideforresearchersinvestigatingthebiologyofproteolysis
andthebiochemistryofproteolyticenzymesencounteringsubstratesinthecellular
environment. This chapter is an invaluable source to the quantitative kinetic
analysis of proteases for both the well-versed researcher and beginner in the field
ofpeptidases,theirsubstrates,andmodifiers.
Inthethirdchapter“CompartmentalizationofProteolysis,”thecellbiologists
KlaudiaBrix,ChristopherJ.Scott,andMargareteM.Heckdiscussthechallenges
that proteolytic enzymes face when acting in various compartments of eukaryotic
cells.Theseenzymesneedtobepreciselytargetedandadaptedtotheirindividual
work space. In this context, the authors explain the blueprint of demands and
necessities of cells in coping with proteolysis for maintenance of protein homeo-
stasis.Theyoutlinethebiochemicalconditionsforthecleavingenvironment(s)and
v
vi Foreword
the spatial concept of cellular compartmentalization required for intracellular and
extracellular juxta-membrane proteolytic processing. They address the important
issueofradicaldecisionmaking,whichisessentialattimesofbusytraffickingand
turnoverofsubstrates.Finally,thecontrolofproteolysisbyendogenousinhibitors
andnewdevelopmentsincludingintra-membraneproteolysisarepresented.
Thefourthchapter“Cathepsins:GettinginShapeforLysosomalProteolysis”
isdedicatedtodescribethemolecularandcellularrequirementsofcorrecttargeting,
sorting,andtraffickingoflysosomalcathepsins.Disordersaffectingthebiosynthesis
andtransportofcathepsinsmanifestwithseveresymptomsrangingfromlysosomal
storage disorders of several kinds to osteoporosis and Papillon–Lefe`vre syndrome.
TheauthorsAnnH.Erickson,CiroIsidoro,LukasMach,andJohnS.Mortcombine
biochemical, cell biological, and structural expertise to outline most instructively
how different cell types have found solutions for loading the compartments of the
endocytic pathway with aspartic and cysteine cathepsins. The authors also explain
comprehensivelytheveryindividualandspecificrolesoftheseproteolyticenzymes
beyondgeneralproteindegradationandturnover.
The fifth chapter “Limited and Degradative Proteolysis in the Context of
Posttranslational Regulatory Networks: Current TechnicalandConceptional
Advances” explains technologies that allow assessing protease functions in
degradomics approaches which aim at characterization of protease activities in the
complexity of their network partners—substrates and inhibitors—and with regard
to physiological and pathophysiological backgrounds. Thus, in vivo validation
techniquesofsubstratesidentifiedbyinvitrodegradomicsareoutlined,forinstance
by employing activity-based probes or knockout mouse models. In addition, the
interrelatedness of proteolytic cleavage as a crucial posttranslational modification
is discussed with respect to diverse other posttranslational modifications like
glycosylation, phosphorylation, or ubiquitinylation. This chapter, written by the
next-generation experts Stefan Tholen, Maria M. Koczorowska, Zon Weng Lai,
Joern Dengjel, and Oliver Schilling, also revisits important traditional concepts
ranging from cellular protein turnover to protein fates and protein homeostasis in
general.
The sixth chapter “Exploring Systemic Functions of Lysosomal Proteases:
The Perspective of Genetically Modified Mouse Models” deals with a specific
proteolytic network by highlighting experimental approaches in which cysteine
cathepsins and/or their endogenous inhibitors have been tested by targeted gene
knockout and phenotypic rescue experiments by knockin studies. The seniors in
the list of authors Martina Gansz, Ursula Kern, Christoph Peters, and Thomas
Reinheckel belong to the pioneers in generating genetic mouse models in which
cysteinecathepsinsarelackingorover-expressedsystemicallyorinacelltype-and
tissue-specific manner. Specific examples depicted in this chapter concern the
biological significance of cathepsins for epidermal keratinocyte homeostasis in
the skin and for the cardiovascular system including pathological features ofboth
atherosclerosis and heart development. Of note, effects of cathepsin deficiencies,
overexpression, compensatory functional redundancy, and misbalance with anti-
proteolyticfactorssuchastheendogenouscystatins/stefinsillustratestrikinglyhow
Foreword vii
importantitistoinvestigateproteasesandtheirinhibitorsintheircelltype-specific
context of a given tissue. Even more important, the genetically modified mouse
modelsuncoverhowsystemsbiologyisurgentlyneededtoprovideuswithinsights
into the importance of proteolysis for specific processes, regardless of whether
lookingatdevelopmental,physiological,pathophysiological,orregenerativeroles
ofproteasesandtheirinhibitors.
In the seventh chapter “Astacins: Proteases in Development and Tissue
Differentiation”structuralbiologymeetsfunctionofaversatilegroupofproteases,
namely the astacins. These proteases act from the very beginning of life right
starting with sperm–egg fusion, early embryonic development, organogenesis,
andhatchingprocesses.Moreover,astacinsarecrucialintheadultforperi-cellular
signaling,tissuehomeostasis,andregeneration,implicatingthatmalfunctionsmay
cause neurodegenerative diseases, fibrosis, or cancer. This astonishing group of
proteasescomprisessmall,singledomainenzymesuptogiantproteaseslikemeprin
multimers reaching MDa size. The versatility of astacins has been studied in a
variety of biological systems throughout the animal kingdom. This chapter
interprets this fascinating group of enzymes combining molecular biology and
zoology, as provided by Walter Sto¨cker, with the beauty and explanatory power
of structural biology, as contributed by the protein crystallographer F. Xavier
Gomis-Ru¨th.
The eighth chapter “Proteases in Death Pathways” takes us from the
very beginning of life of a multicellular organism through the crucial phases of
developmentandtissuehomeostasis,whereprogrammedcelldeathisanessential
prerequisite. The responsible proteases, caspases, are an extremely well-studied
proteasefamily,buttheybearmysteriesandsecretsthatneedtobeuncoveredsince
their dysfunction may initiate a most dangerous landslide of severe pathologies
ranging from inflammation and cancer to neurodegenerative diseases. Again,
structuremeetsfunctioninthischapter,andtheyoungresearcherAndreasFlu¨tsch
hasteamedupwiththemostexperiencedresearcherMarkusGru¨tterinanapproach
that combines cell and structural biology and which links underpinning topics
related to caspases with application and therapeutic potential of, e.g., DARPins
that overcome the problems of specific targeting of caspases. Programmed cell
deathanditsseveralmodalitiescomealonglivelyinthischapter.
The ninth chapter “ADAM Proteases in Physiology and Pathophysiology:
Cleave to Function in Health or to Cause Disease” covers the sheddases
ADAM17 and ADAM10. The authors Joachim Gro¨tzinger and Stefan Rose-John
provide us with deep insights into the structure–function relationships of these
multi-substrateproteases.ADAMscleaveahugevarietyofsolubleproteinsubstrates
likecytokines,growthfactors,oradhesionmoleculesbesidesbeingseeminglymade
for shedding of transmembrane receptor proteins. These proteolytic enzymes are
multi-domaintransmembraneproteinsthemselves,anditisbelievedthatconforma-
tionalchanges,i.e.,bendingoftheactivesidebacktowardsplasmamembrane-near
regions,facilitatetheirbroadcleavagerepertoirethatincludeseventheprocessingof
transmembrane domains. This chapter highlights once more the importance of
creativeanimalmodeldesignincaseswhereknockoutapproachesarenotapplicable.
viii Foreword
TheauthorsintroduceanapproachcalledEXITS(exoninducedtranslationalstop)to
generate animals with strongly reduced ADAM17 activity throughout all tissues.
Such hypomorphicanimal models bearthe promise toclarify how ADAMs realize
their functions in development as sheddases of numerous receptors initiating vital
signaling pathways like for instance those operating via the EGF receptor. It goes
withoutsayingthattheADAMproteasesfulfilluncountablefunctionsinregulating
development, tissue homeostasis, and regeneration. Likewise it is still more than
difficulttotherapeuticallytargetADAMSfortreatmentofdiseasesinwhichtheyare
involved, be it during the initial or progressive phases of inflammation or other
immunesystemrelateddisorders,andincancer.
Thetenthchapter“ProteasesintheNervousSystem”coverscomprehensively
the proteolytic enzymes with a major but not exclusive function in the nervous
system. The expert authors Holger Cynis, Stefan Lichtenthaler, Leona Wagner,
and Hans-Ulrich Demuth begin with a survey of APP processing enzymes and
discuss evidences and contradictory findings when claiming specific proteolytic
enzymesasalpha-,beta-,andgamma-secretases.Whilethereisgeneralagreement
withinthescientificcommunityaboutwhichenzymesarealpha-secretases,namely
ADAM10, ADAM17, and other metallopeptidases, the issue of beta-secretases is
a lot more controversial. Gamma-secretase is an unusual aspartyl protease acting
as heterotetrameric complex in catalyzing regulated intra-membrane proteolysis
of TMPs such as APP and another dozens of substrates, many of them belonging
to the receptors that initiate signaling during developmental processes. However,
inamoreprovocativeviewconsideringtheenormousturnoverofAPP(i.e.,8%per
hour),itisnotthegenerationofAβbutitsdegradationwhichwillbecomecrucial
for understanding AD and other neurodegenerative disorders. Thus, the authors
dealwithavarietyofenzymesinvolvedinAβcatabolism.Anin-depthdescription
of the structural and functional features, tissue distribution, regulation, and thera-
peutic potentials of targeting proline-specific peptidases reveals thorough insights
into neuropeptide processing enzymes as well as their involvement in general
peptide processing tasks. A number of enzyme families are highlighted which
are often not covered due to their uniqueness such as prolylcarboxypeptidase,
X-prolylaminopeptidases,prolidase,andprolinase.
The eleventh chapter “Proteases in the Mammalian Digestive System” takes
us to another aspect of protein and peptide substrate processing, namely in the
mammaliandigestivesystem.Thelong-standingexpertiseoftheauthorsS.Gaylen
Bradley,ToniM.Antalis,andJudithS.Bondintroducesandhighlightstheproteo-
lyticenzymesactingalongthealimentarytractinthisverywell-digestibleandthus
“tasty”chapter.Differentialexpressionpatternsandpeculiaractivitiescharacterize
the gastrointestinal proteases which comprise a variety of enzymes ranging from
enteropeptidasetomeprinsandincludingmatriptaseasoneofthecrucialmarkers
of colon cancer. However, a number of other proteases like DPs or the serine
protease TMPRSS4 are also discussed. Without doubt, it is the importance of the
epithelialliningoftheGItractasabarrierbetweentheoutsideworldandthebody’s
interior which implies that the tasks and teamwork of proteolytic enzymes in the
mammaliandigestivetractareupformorethansimpledegradation.Itisthebeauty
Foreword ix
of cleaving with precision and maintaining the body healthy and well protected
fromintrudersthatisenabledbyafine-tuneddifferentialexpressionandactivityof
proteolyticenzymesharboredinthedigestivesystem.
Thetwelfthchapter“CalpainsinHealthandDisease”introducesaremarkable
family of cytosolic enzymes that are activated upon increased calcium concentra-
tion, i.e., typically following traumatic injury or upon prolonged chemical and
mechanical stress. The activity of one of the family members, skeletal muscle
specific CAPN3/p94, even depends on both calcium and sodium ions. Calpains
have recently experienced a nomenclature reform, which was integrated into this
chapterwhileitwasinpress.Calpainsarefoundthroughouttheanimalsandplant
kingdoms. Calpains and their endogenous inhibitors, the calpastatins, function
in such diverse processes as cell motility and cell death, vesicular transport, and
even pH-sensing in yeast or body size determination of nematodes. Calpains are
considered parts of signal transduction cascades and cleave a brought range of
cytosolic substrates, thereby releasing smaller fragments or peptides that can,
in turn, serve as biomarkers of the site of the insult. Thus, while calcium
hyperactivated calpains contribute to massive degradation of signaling receptors
and enzymes, transcription factors, as well as cytoskeletal and other structural
proteinsin,e.g.,braininjury/stroke,theybearanenormouspotentialastheranostics.
Instroke, calpain inhibitors may even prove neuro-protectiveagents. Calpains also
exhibit vital tasks in skeletal muscle and the so-called calpainopathies are covered
comprehensively.Moreover,calpainsbeartasksintheGItractwheretheyprotect
the stomach from stress-induced ulcer formation, and they are considered
contributing to glucose control in diabetes and obesity. The underlying theme
of this chapter is to shed light on a protease family which is both protective
and destructive. Therefore, activators and inhibitors of calpains must both be
considered in physiological and pathological situations. The expert authors
John Anagli, Kevin K.W. Wang, Yasuko Ono, and Hiroyuki Sorimachi, who
have acquired a wealth of knowledge about this intriguing family of proteolytic
enzymes, suggest novel therapeutic approaches by not only discussing novel
biomarkersfordiagnosticsbutalsoconsideringcalpaininhibitionfortreatmentof
ischemicortraumaticcellandtissueinjury.
Thethirteenthchapter“MetalloproteinasesinCartilageMatrixBreakdown:
TheRolesinRheumatoidArthritisandOsteoarthritis”byHideakiNagaseand
Gillian Murphy summarizes and critically discusses the impressive knowledge of
theECM-degradingenzymefamiliescomprisingMMPs,ADAMs,andADAMTSs
in the context of rheumatoid arthritis and osteoarthritis. Thus, destructive ECM
degradation as a response to inflammation destroys cartilage upon stress, injury,
orexcessive load butalso during aging. Numerousproteases have been attributed
to function in this severe pathophysiology that is a major burden not only for
aging patient populations. The metalloproteinases discussed in this chapter are
considered key players in degradation of a variety of substrates ranging from
triple-helical collagen to proteoglycans like aggrecans. The ADAM proteases are
viewed here from a different angle compared to previous chapters of this book,
inthattheirdestructiveratherthanregulatorynatureofproteolyticactionbecomes
