Proteases: Multifunctional Enzymes in Life and Disease

Our view of proteases has come a long way since P. A. Levene reported his studies on “The Cleavage Products of Proteoses” in the first issue of The Journal of Biological Chemistry published October 1, 1905 (1Levene P.A. J. Biol. Chem. 1905; 1: 45-58Abstract Full Text PDF Google Scholar). Today, after more than 100 years and 350,000 articles on these enzymes in the scientific literature, proteases remain at the cutting edge of biological research. Proteases likely arose at the earliest stages of protein evolution as simple destructive enzymes necessary for protein catabolism and the generation of amino acids in primitive organisms. For many years, studies on proteases focused on their original roles as blunt aggressors associated with protein demolition. However, the realization that, beyond these nonspecific degradative functions, proteases act as sharp scissors and catalyze highly specific reactions of proteolytic processing, producing new protein products, inaugurated a new era in protease research (2Neurath H. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3825-3832Crossref PubMed Scopus (327) Google Scholar). The current success of research in this group of ancient enzymes derives mainly from the large collection of findings demonstrating their relevance in the control of multiple biological processes in all living organisms (3López-Otín C. Overall C.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 509-519Crossref PubMed Scopus (611) Google Scholar, 4Ehrmann M. Clausen T. Annu. Rev. Genet. 2004; 38: 709-724Crossref PubMed Scopus (167) Google Scholar, 5Sauer R.T. Bolon D.N. Burton B.M. Burton R.E. Flynn J.M. Grant R.A. Hersch G.L. Joshi S.A. Kenniston J.A. Levchenko I. Neher S.B. Oakes E.S. Siddiqui S.M. Wah D.A. Baker T.A. 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Thus, proteases regulate the fate, localization, and activity of many proteins, modulate protein-protein interactions, create new bioactive molecules, contribute to the processing of cellular information, and generate, transduce, and amplify molecular signals. As a direct result of these multiple actions, proteases influence DNA replication and transcription, cell proliferation and differentiation, tissue morphogenesis and remodeling, heat shock and unfolded protein responses, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, hemostasis, blood coagulation, inflammation, immunity, autophagy, senescence, necrosis, and apoptosis. Consistent with these essential roles of proteases in cell behavior and survival and death of all organisms, alterations in proteolytic systems underlie multiple pathological conditions such as cancer, neurodegenerative disorders, and inflammatory and cardiovascular diseases. Accordingly, many proteases are a major focus of attention for the pharmaceutical industry as potential drug targets or as diagnostic and prognostic biomarkers (12Turk B. Nat. Rev. Drug Discov. 2006; 5: 785-799Crossref PubMed Scopus (1008) Google Scholar). Proteases also play key roles in plants and contribute to the processing, maturation, or destruction of specific sets of proteins in response to developmental cues or to variations in environmental conditions (13Garcia-Lorenzo M. Sjodin A. Jansson S. Funk C. BMC Plant Biol. 2006; 6: 30Crossref PubMed Scopus (121) Google Scholar). Likewise, many infectious microorganisms require proteases for replication or use proteases as virulence factors, which has facilitated the development of protease-targeted therapies for diseases of great relevance to human life such as AIDS (12Turk B. Nat. Rev. Drug Discov. 2006; 5: 785-799Crossref PubMed Scopus (1008) Google Scholar). Finally, proteases are also important tools of the biotechnological industry because of their usefulness as biochemical reagents or in the manufacture of numerous products (e.g. Ref. 14Saeki K. Ozaki K. Kobayashi T. Ito S. J. Biosci. Bioeng. 2007; 103: 501-508Crossref PubMed Scopus (129) Google Scholar). This outstanding diversity in protease functions directly results from the evolutionary invention of a multiplicity of enzymes that exhibit a variety of sizes and shapes. Thus, the architectural design of proteases ranges from small enzymes made up of simple catalytic units (∼20 kDa) to sophisticated protein-processing and degradation machines, like the proteasome and meprin metalloproteinase isoforms (0.7–6 MDa) (15Bertenshaw G.P. Norcum M.T. Bond J.S. J. Biol. Chem. 2003; 278: 2522-2532Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In terms of specificity, diversity is also a common rule. Thus, some proteases exhibit an exquisite specificity toward a unique peptide bond of a single protein (e.g. angiotensin-converting enzyme); however, most proteases are relatively nonspecific for substrates, and some are overtly promiscuous and target multiple substrates in an indiscriminate manner (e.g. proteinase K). Proteases also follow different strategies to establish their appropriate location in the cellular geography and, in most cases, operate in the context of complex networks comprising distinct proteases, substrates, cofactors, inhibitors, adaptors, receptors, and binding proteins, which provide an additional level of interest but also complexity to the study of proteolytic enzymes. This work aims at serving as a primer to a minireview series on proteases to be published in forthcoming issues of this Journal. This introductory article will focus on the discussion of the large and growing complexity of proteolytic enzymes present in all organisms, from bacteria to man. We will first show the results of comparative genomic analysis that have shed light on the real dimensions of the proteolytic space. The levels of protease complexity and mechanisms of protease regulation will then be addressed. Finally, we will discuss current frontiers and future perspectives in protease research. Proteases are the efficient executioners of a common chemical reaction: the hydrolysis of peptide bonds (16Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. Oxford University Press, London2001Google Scholar). Most proteolytic enzymes cleave α-peptide bonds between naturally occurring amino acids, but there are some proteases that perform slightly different reactions. Thus, a large group of enzymes known as DUBs (deubiquitylating enzymes) can hydrolyze isopeptide bonds in ubiquitin and ubiquitin-like protein conjugates; γ-glutamyl hydrolase and glutamate carboxypeptidase target γ-glutamyl bonds; γ-glutamyltransferases both transfer and cleave peptide bonds; and intramolecular autoproteases (such as nucleoporin and polycystin-1) hydrolyze only a single bond on their own polypeptide chain but then lose their proteolytic activity. Notably and under some conditions, proteases can also synthesize peptide bonds. Proteases were initially classified into endopeptidases, which target internal peptide bonds, and exopeptidases (aminopeptidases and carboxypeptidases), the action of which is directed by the NH2 and COOH termini of their corresponding substrates. However, the availability of structural and mechanistic information on these enzymes facilitated new classification schemes. Based on the mechanism of catalysis, proteases are classified into six distinct classes, aspartic, glutamic, and metalloproteases, cysteine, serine, and threonine proteases, although glutamic proteases have not been found in mammals so far. The first three classes utilize an activated water molecule as a nucleophile to attack the peptide bond of the substrate, whereas in the remaining enzymes, the nucleophile is an amino acid residue (Cys, Ser, or Thr, respectively) located in the active site from which the class names derive (supplemental Fig. 1). Proteases of the different classes can be further grouped into families on the basis of amino acid sequence comparison, and families can be assembled into clans based on similarities in their three-dimensional structures. Bioinformatic analysis of genome sequences has been decisive for establishingthedimensionsofthe complexity of proteolytic systems operating in different organisms (Fig. 1). The last release of MEROPS (merops.sanger.ac.uk), a comprehensive data base of proteases and inhibitors, annotates 1008 entries for human proteases and homologs, although it includes a large number of pseudogenes and protease-related sequences derived from endogenous retroviral elements embedded in our genome. A highly curated data base, the Degradome Database, which does not incorporate protease pseudogenes or these retrovirus-derived sequences, lists 569 human proteases and homologs classified into 68 families (17López-Otín C. Matrisian L.M. Nat. Rev. Cancer. 2007; 7: 800-808Crossref PubMed Scopus (634) Google Scholar). Metalloproteases and serine proteases are the most densely populated classes, with 194 and 176 members, respectively, followed by 150 cysteine proteases, whereas threonine and aspartic proteases contain only 28 and 21 members, respectively. The recent availability of the genome sequence of different mammals has allowed the identification of their entire protease complement (termed degradome) and their detailed comparison with humans (Fig. 1). The chimpanzee degradome is very similar to the human degradome, although it exhibits some remarkable differences in immune defense proteases like caspase-12 (18Puente X.S. Gutierrez-Fernandez A. Ordonez G.R. Hillier L.W. López-Otín C. Genomics. 2005; 86: 638-647Crossref PubMed Scopus (50) Google Scholar). Interestingly, mice and rats contain more protease genes (644 and 629, respectively) compared with humans despite the fact that their genomes are smaller (19Puente X.S. Sanchez L.M. Overall C.M. López-Otín C. Nat. Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (725) Google Scholar, 20Puente X.S. López-Otín C. Genome Res. 2004; 14: 609-622Crossref PubMed Scopus (158) Google Scholar). These differences derive mainly from the expansion in rodents or the inactivation in humans of members of protease families (such as kallikreins and placental cathepsins) involved in immunological and reproductive functions (21Pampalakis G. Sotiropoulou G. Biochim. Biophys. Acta. 2007; 1776: 22-31PubMed Google Scholar, 22Mason R.W. Placenta. 2008; 29: 385-390Crossref PubMed Scopus (33) Google Scholar). The recent analysis of the degradome of other mammals such as the duck-billed platypus (Ornithorhynchus anatinus) has revealed some interesting findings on protease evolution. This fascinating monotreme also has more than 500 protease genes but lacks all genes encoding gastric pepsins, which are the archetypal digestive proteases widely conserved in all mammals (23Ordonez G.R. Hillier L.W. Warren W.C. Grutzner F. López-Otín C. Puente X.S. Genome Biol. 2008; 9: R81Crossref PubMed Scopus (38) Google Scholar). Birds, amphibians, and fish also contain large numbers of protease genes (382 in Gallus gallus, 278 in Xenopus tropicalis, and 503 in Danio rerio), although the protease annotation work in these species has not been as detailed as in mammals. Surprisingly, analysis of the protease content of invertebrates such as Drosophila melanogaster (a model organism with a gene content considerably lower than that in vertebrates) has shown the presence of more than 600 protease genes (24Shah P.K. Tripathi L.P. Jensen L.J. Gahnim M. Mason C. Furlong E.E. Rodrigues V. White K.P. Bork P. Sowdhamini R. Gene (Amst.). 2008; 407: 199-215Crossref PubMed Scopus (27) Google Scholar). The model plant Arabidopsis thaliana contains at least 723 protease-encoding genes, whereas a total of 955 protease genes have been annotated in the tree Populus trichocarpa. These marked differences are linked to the expansion of some protease families in Populus, especially the copia transposon endopeptidase family of aspartic proteases, which has 20 components in Arabidopsis and 123 in Populus (13Garcia-Lorenzo M. Sjodin A. Jansson S. Funk C. BMC Plant Biol. 2006; 6: 30Crossref PubMed Scopus (121) Google Scholar). Genomic analyses have also shown that plants share with prokaryotes a set of serine proteases absent in other eukaryotes, which may be an indication of ancient endosymbiotic events leading to evolution of chloroplasts (25Tripathi L.P. Sowdhamini R. BMC Genomics. 2006; 7: 200Crossref PubMed Scopus Google Scholar). Finally, there is a growing interest in the degradome of and as of strategies to targets for C. B. P. M. Annu. Rev. 2006; 1: PubMed Scopus Google Scholar, H. 2005; PubMed Scopus Google Scholar, R. B. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). In this the MEROPS annotates more than 100 protease genes in the genome of bacteria such as and or in the which human diseases. In the derived from the analysis of proteolytic systems is of diversity and These comparative genomic studies have also into the and relevance of this group of enzymes. Thus, it has that, in to proteolytic conserved in all organisms, there are also specific roles by unique proteases in different further studies will be necessary to the and molecular basis the evolutionary differences in the complex protease of all living Proteolytic enzymes are not catalytic in in their for substrates to be Thus, many proteases their catalytic to a variety of or that provide specificity, their cellular localization, their and their to endogenous These archetypal that direct these enzymes to their that and that or with other proteins, substrates, receptors, or of these the have been very in their into proteases and are present in a variety of enzymes from different whereas other (such as the of have the long S. J. 2005; PubMed Scopus Google Scholar). proteases, members of the serine protease exhibit a complex with up to six distinct located a single polypeptide chain R. J. Cell Biol. 2008; PubMed Scopus Google Scholar). This of and has also to the of very or proteases with different catalytic units embedded in the polypeptide chain S. A. López-Otín C. Biosci. 2007; PubMed Scopus Google Scholar). is very likely that the activity in protease genes has been a in the protease from nonspecific primitive enzymes to highly for proteolytic events that are at the of multiple biological The complexity of proteases is further events such as and of genes encoding proteases S. T. N. Biosci. 2008; PubMed Scopus Google Scholar, J.M. I. M. Sanchez L.M. R. J. López-Otín C. J. Biol. Chem. Full Text PDF PubMed Google by the of gene number variations or that may contribute to the of protease functions or their mechanisms C. G.R. J. S. S. S. D.N. J.M. C. 2008; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, G.L. G. W.C. Bond J.S. Genet. 2005; PubMed Scopus Google or by such as and Finally, we that, in many cases, proteases act in the context of complex and comprising many protein that to the protease U. A. 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Cell. 2007; PubMed Scopus Google Scholar). The of protease can be or by other proteases, although in some cases, protease additional or such as the which the of Nat. Rev. Mol. Cell Biol. 2007; 8: PubMed Scopus Google Scholar). may also be by protein such as the tissue that to serine protease and the 2006; PubMed Scopus Google Scholar). mechanisms of protease have also been for some W.C. Cell Dev. Biol. 2008; PubMed Scopus Google Scholar). known endogenous protease are proteins, although some microorganisms small that the proteolytic activity of the number of endogenous is considerably lower than that of As an a total of genes encoding protease have been annotated in the which with the more than 600 protease genes present in this species X.S. López-Otín C. Genome Res. 2004; 14: 609-622Crossref PubMed Scopus (158) Google Scholar). This derives in from the specificity of toward their target proteases, although there are also many proteases that are not by endogenous as their proteolytic are at other have been classified into families of members or to the catalytic class of proteases by this classification is by the of both that contain units of different protease classes and (such as that target enzymes of different classes a after by the protease A.J. of Proteolytic Press, Scholar). can also be classified into to their mechanism of (12Turk B. Nat. Rev. Drug Discov. 2006; 5: 785-799Crossref PubMed Scopus (1008) Google Scholar, R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The inhibitors, protease the active site of their target proteases binding in a like and some a to the active to this but directly the catalytic A group of protease inhibitors, of an mechanism based on a of the and Finally, (such as of a a that is located from the active but this binding of the target protease and activity. In to these may also be or by in the of protease genes, control of and degradation by such as proteins and and protease with protease homologs that act as protease of cellular and reactions that to the of proteolytic these mechanisms operate in a manner to that the substrates are at the and in the appropriate the of proteases on living the years, our of mechanisms at the level of proteases has considerably but information is on the regulation of proteolytic The of for proteases in different organisms will contribute to the mechanisms operating in the and control of the protease the of the a large of information is the and of proteolytic systems in many living organisms. These genomic have revealed that the protease is and it is very likely that the of the different will in the as new enzymes with structural and catalytic mechanisms are and The recent of and conserved cysteine proteases and an of work that has to the of that to G.R. M. H. K. M. K. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus (85) Google Scholar). proteases remain as in for activity. A major for the future will be to for these The comparative genomic studies have also interesting information and events in the protease Thus, the expansion of reproductive proteases in rodents may to some of the reproductive differences between whereas in proteases may evolutionary of defense mechanisms in response to new environmental conditions X.S. Sanchez L.M. Gutierrez-Fernandez A. G. López-Otín C. 2005; PubMed Scopus Google Scholar). In to the relevance of proteases for human genomic studies will contribute to the of diseases by in protease as as to the identification of protease gene associated with an to diseases. These will provide to design new of inhibitors, such as for the proteasome in multiple A. M. K. T. P. N. 2008; PubMed Scopus Google or in Biosci. 2008; PubMed Scopus Google Scholar). in different have also in the development of strategies to the and activity levels of the multiple proteases present in complex cellular and different have been for and protease levels and activity. are also to the in substrates by enzymes, a toward the of proteases for which a biological is The strategies for proteases and their in functions are as as the proteases although a first toward this is based on the of for a protease by peptide or protein C.M. Nat. Rev. Mol. Cell Biol. 2007; 8: PubMed Scopus Google Scholar). these only provide information peptide sequences that can be but not that are in their necessary the of additional for a protease to specific substrates. These can be classified into and in C.M. Nat. Rev. Mol. Cell Biol. 2007; 8: PubMed Scopus Google Scholar). The studies to in protease substrates are based on the of substrates in of mice in specific studies in other model organisms such as C. and A. thaliana have also allowed the identification of in substrates of proteases C.M. Nat. Rev. Mol. Cell Biol. 2007; 8: PubMed Scopus Google although these strategies are by the in most proteolytic systems of and A to for identification derives from the of to the protease N. S. R. C. M. G. Z. 2007; PubMed Scopus Google Scholar). it is that a single will be to the substrates by specific proteases under in A (3López-Otín C. Overall C.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 509-519Crossref PubMed Scopus (611) Google and the of biochemical and will be necessary in the for the substrates of the multiple proteases present in all organisms. studies will also be essential to the and between all different components of proteolytic systems that the protease Finally, the detailed analyses of complex processes such as proteolytic regulation of protein and are to be in the a series of articles focused on structural and of proteolytic systems as as on the analysis of biological processes by this minireview series will provide a current view of this complex group of protein that influence the of cell life and death in all living We all members of our for on the with