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Proteases: History, discovery, and roles in health and disease

2019; Elsevier BV; Volume: 294; Issue: 5 Linguagem: Inglês

10.1074/jbc.tm118.004156

1083-351X

Judith Bond,

Blood Coagulation and Thrombosis Mechanisms

The Journal of Biological Chemistry (JBC) has been a major vehicle for disseminating and recording the discovery and characterization of proteolytic enzymes. The pace of discovery in the protease field accelerated during the 1971–2010 period that Dr. Herb Tabor served as the JBC’s editor-in-chief. When he began his tenure, the fine structure and kinetics of only a few proteases were known; now thousands of proteases have been characterized, and over 600 genes for proteases have been identified in the human genome. In this review, besides reflecting on Dr. Tabor’s invaluable contributions to the JBC and the American Society for Biochemistry and Molecular Biology (ASBMB), I endeavor to provide an overview of the extensive history of protease research, highlighting a few discoveries and roles of proteases in vivo. In addition, metalloproteinases, particularly meprins of the astacin family, will be discussed with regard to structural characteristics, regulation, mechanisms of action, and roles in health and disease. Proteases and protein degradation play crucial roles in living systems, and I briefly address future directions in this highly diverse and thriving research area. The Journal of Biological Chemistry (JBC) has been a major vehicle for disseminating and recording the discovery and characterization of proteolytic enzymes. The pace of discovery in the protease field accelerated during the 1971–2010 period that Dr. Herb Tabor served as the JBC’s editor-in-chief. When he began his tenure, the fine structure and kinetics of only a few proteases were known; now thousands of proteases have been characterized, and over 600 genes for proteases have been identified in the human genome. In this review, besides reflecting on Dr. Tabor’s invaluable contributions to the JBC and the American Society for Biochemistry and Molecular Biology (ASBMB), I endeavor to provide an overview of the extensive history of protease research, highlighting a few discoveries and roles of proteases in vivo. In addition, metalloproteinases, particularly meprins of the astacin family, will be discussed with regard to structural characteristics, regulation, mechanisms of action, and roles in health and disease. Proteases and protein degradation play crucial roles in living systems, and I briefly address future directions in this highly diverse and thriving research area. In the very first issue of the Journal of Biological Chemistry (JBC) 2The abbreviations used are: JBCJournal of Biological ChemistryMMPmatrix metalloproteinaseADAMa disintegrin and metalloproteinaseASBMBAmerican Society for Biochemistry and Molecular BiologyMet-turnmethionine-containing turnBMPbone morphogenetic proteinEGFepidermal growth factor. in 1905, P. A. Levene published studies on “The Cleavage Products of Proteoses” (1Levene P.A. The cleavage products of proteoses.J. Biol. Chem. 1905; 1: 45-58Abstract Full Text PDF Google Scholar). The Journal continually published state-of-the-art work on proteases over the years, but the pace of discovery in the field accelerated during the 39 years that Herb Tabor served as Editor of the JBC. When Herb began his tenure as Chief Editor of the JBC (1971), we knew the fine structure and a substantial amount about the kinetics of only a few proteases. Some examples of the major classes of proteolytic enzymes (aspartic, serine, cysteine, metallo) that were well studied before 1970 are as follows. •Pepsin, an aspartic protease of the stomach, was one of the first enzymes to be discovered, characterized, and named (in 1825), and it was crystallized in 1930 (2Northrup J.H. Crystalline pepsin. I. Isolation and tests of purity.J. Gen. Physiol. 1930; 13 (19872561): 739-76610.1085/jgp.13.6.739PubMed Google Scholar). Studies of pepsin’s action can be found in the JBC as far back as in 1907 (3Robertson T.B. Note on the synthesis of a protein through the action of pepsin.J. Biol. Chem. 1907; 3: 95-99Abstract Full Text PDF Google Scholar), and mechanistic studies were well on the way in the 1970s.•The serine proteases, trypsin and chymotrypsin from pancreatic secretions, were also discovered in the 1800s and crystallized in the 1930s (4Northrop J.H. Kunitz M. Isolation of protein crystals processing tryptic activity.Science. 1931; 73 (17755302): 262-26310.1126/science.73.1888.262Crossref PubMed Scopus (30) Google Scholar). Studies of the action of trypsin appeared in the JBC in 1907 (5Robertson T.B. Studies in the chemistry of the ion-proteid compounds: IV. On some chemical properties of casein and their possible relation to the chemical behavior of other protein bodies, with especial reference to hydrolysis of casein by trypsin.J. Biol. Chem. 1907; 2: 317-383Abstract Full Text PDF Google Scholar), whereas those for chymotrypsin appeared in the 1930s (6Bergman M. Fruton J.S. On proteolytic enzymes: XIII. Synthetic substrates for chymotrypsin.J. Biol. Chem. 1937; 118: 405-415Abstract Full Text PDF Google Scholar).•Papain, the cysteine protease from papaya, was also discovered in the 1800s, and pure forms were reported in the JBC as early as 1954 (7Kimmel J.R. Smith E.L. Crystalline papain: I. Preparation, specificity, and activation.J. Biol. Chem. 1954; 207 (13163037): 515-531Abstract Full Text PDF PubMed Google Scholar).•Thermolysin, an extracellular metalloprotease from thermophilic bacteria, was the first metalloendoproteinase to be crystallized and to have its structure solved (8Matthews B.W. Jansonius J.N. Colman P.M. Schoenborn B.P. Dupourque D. Three dimensional structure of thermolysin.Nature. 1972; 238 (18663849): 37-41Crossref PubMed Scopus (27874) Google Scholar).•Carboxypeptidase A, isolated in 1937 (9Anson M.L. The preparation of crystalline carboxypeptidase.J. Gen. Physiol. 1937; 20 (19873019): 663-66910.1085/jgp.20.5.663Crossref PubMed Scopus (40) Google Scholar), was kinetically characterized in 1970 (10Auld D.S. Vallee B.L. Kinetics of carboxypeptidase A. II. Inhibitors of the hydrolysis of oligopeptides.Biochemistry. 1970; 9 (5461217): 602-60910.1021/bi00805a022Crossref PubMed Scopus (105) Google Scholar).•Carboxypeptidase B was isolated in 1960 (11Folk J.E. Piez K.A. Carroll W.R. Gladner J.A. Carboxy-peptidase B: IV. Purification and characterization of the porcine enzyme.J. Biol. Chem. 1960; 235 (13823740): 2272-2277Abstract Full Text PDF PubMed Google Scholar), and bacterial collagenase, now known as part of the matrixin family, matrix metalloproteinase 1 (MMP-1), was isolated in 1957 (12Gallop P.M. Seifter S. Meilman E. Studies on collagen: I. The partial purification, assay, and mode of activation of bacterial collagenase.J. Biol. Chem. 1957; 227 (13463011): 891-906Abstract Full Text PDF PubMed Google Scholar). Journal of Biological Chemistry matrix metalloproteinase a disintegrin and metalloproteinase American Society for Biochemistry and Molecular Biology methionine-containing turn bone morphogenetic protein epidermal growth factor. There are many excellent reviews available for individually characterized proteases and for clans and families of proteases, as well as for general insights into functional aspects of proteases (e.g. see Ref. 13López-Otín C. Bond J.S. Proteases: multifunctional enzymes in life and disease.J. Biol. Chem. 2008; 283 (18650443): 30433-3043710.1074/jbc.R800035200Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). A comprehensive database, MEROPS, of the more than 1000 individual proteases is available to all and contains a wealth of information on the characterization and evolutionary relationships of the proteases and the current literature (https://www.ebi.ac.uk/merops/) 3Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. (98Rawlings N.D. Barrett A.J. Thomas P.D. Huang X. Bateman A. Finn R.D. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database.Nucleic Acids Res. 2018; 46 (29145643): D624-D63210.1093/nar/gkx1134Crossref PubMed Scopus (922) Google Scholar). A degradome database of human proteases (14López-Otín C. Matrisian L.M. Emerging roles of proteases in tumour suppression.Nat. Rev. Cancer. 2007; 7 (17851543): 800-80810.1038/nrc2228Crossref PubMed Scopus (679) Google Scholar) and the Handbook of Proteolytic Enzymes (15Rawlings, N. D., and Salvesen, G., (eds) (2013) Handbook of Proteolytic Enzymes, 3rd Ed., Academic Press, Inc., New YorkGoogle Scholar) are also valuable resources. There was ample new information coming forth in the 1960s and early 1970s on protease structure and function about small (20–35-kDa), secreted proteases (as those cited above), but little to nil was known about cell-associated proteases, cellular functions of proteases, or protein turnover. In an era when there were great advances and interest in the mechanisms of protein synthesis (the 1950s and 1960s), there was a comparative dearth of information and effort devoted to studies of protein degradation. That said, it had been known since the pioneering studies of Schoenheimer (1942) (16Schoenheimer R. The Dynamic State of Body Constituents. Harvard University Press, Cambridge, MA1942Google Scholar) that there was continuous turnover (synthesis and breakdown) of cellular proteins in eukaryotic cells. The extent of that turnover (intracellular protein degradative process) and its importance to the vitality of the cell, however, was unappreciated. Cell death was recognized to involve proteases, as were wasting diseases (e.g. type 1 diabetes), and lysosomes (17Coffey J.W. De Duve C. Digestive activity of lysosomes: I. The digestion of protein by extracts of rat liver lysosomes.J. Biol. Chem. 1968; 243 (5656369): 3255-3263Abstract Full Text PDF PubMed Google Scholar) were thought to handle these “downhill” processes through autophagy. Studies with individual proteins indicated great differences in turnover of specific proteins (18Schimke R.T. Sweeney E.W. Berlin C.M. Studies of the stability in vivo and in vitro of rat liver tryptophan pyrrolase.J. Biol. Chem. 1965; 240 (5846982): 4609-4620Abstract Full Text PDF PubMed Google Scholar, 19Berlin C.M. Schimke R.T. Influence of turnover rates on the responses of enzymes to cortisone.Mol. Pharmacol. 1965; 1 (4378655): 149-156PubMed Google Scholar), and the concept of short- and long-lived proteins grew with studies of many individual cellular proteins. There was expanding interest in intracellular protein degradation in the 1970s, and one of the first conferences in the United States that heralded that interest was organized by Bob Schimke (an Associate Editor of the JBC) and Nobuhiku Katunuma (a prominent biochemist in Japan) in 1973, the Conference on Protein Turnover in Palo Alto, California (20Schimke R.T. Katumuma N. Intracellular Protein Turnover. Academic Press, New York1975Google Scholar). Intracellular protein degradation was clearly of international interest and activity, leading to several conferences in Europe in the 1970s. For example, Alan Barrett organized a meeting at Strangeways Research Laboratory in Cambridge, England, in 1970 on tissue proteinases; in 1973, a group of scientists at the Martin Luther University in Halle, German Democratic Republic (GDR), organized a symposium on intracellular protein catabolism in Reinhardsbrunn, GDR; Vito Turk organized a meeting in 1975 in Lubljana, Yugoslavia (now Slovenia); and Professors Horst Hanson and Peter Bohley organized additional conferences on intracellular proteolytic enzymes and protein turnover in vivo in 1977 and 1981. The 1970s were times in which GDR scientists could not leave their country for meetings, so scientists in Western countries went to the GDR, placing science above politics. This interest resulted in the formation of committees to increase communication among scientists who work on proteases and protein turnover. First there was ECOP, the European Committee on Proteolysis, in 1981, followed by ACOP (the American Committee on Proteolysis, which organized the 5th International Symposium on Intracellular Protein Catabolism) and then JCOP, the Japanese Committee on Proteolysis, and finally ICOP, the International Committee on Proteolysis. These were forerunners of the current International Proteolysis Society formed in 1999. Before the 1970s, there were several myths, or misconceptions, regarding proteolytic enzymes and protein turnover. •There were many who thought the only function of proteases was to totally degrade proteins at certain stages of life (particularly end-stages) or that their only function was to be secreted in order to degrade extracellular proteins, thereby releasing amino acids so that other proteins could be synthesized.•It was thought that there were very few proteases in cells and that they could handle a great variety of degradative functions, similar to the trypsins and chymotrypsins along with some exopeptidases that could degrade almost anything in the intestinal tract.•There were bacteriologists who argued that protein degradation did not occur in growing procaryotes because there was no need to degrade proteins; it was thought that defective, damaged, or useless proteins could be diluted out as cells divided rapidly.•The known proteases were small (20–35 kDa), compact, uncomplicated (no carbohydrate, lipids, or cofactors) proteins, and it was assumed that this was generally true of all proteases.•Lysosomes were thought to be the primary or only site for degrading proteins in cells, as well as those taken up by endocytosis, and that this occurred through the merging of lysosomes and other cell components to form autophagic vacuoles. But now we know that there are a large number of proteases in and secreted from cells. Proteinases are the largest enzyme gene family in vertebrates. •There are 641 protease genes in the human and 677 in the mouse (i.e. ∼3% of the human and mouse genome).•Proteolysis occurs in virtually all stages of a cell’s life, in all cell compartments, and in many stages of a protein’s existence: from processing of preproproteins coincident or soon after protein synthesis to total destruction of the protein.•There are a great variety of protease structures, from small to large (20 kDa to 6 MDa), highly complex structures, some containing multiple domains with many posttranslational moieties, such as carbohydrates and lipids.•Lysosomal proteases are not the only intracellular proteases and, under many circumstances, are not the major proteases responsible for intracellular protein degradation.•Evolutionary clans and families of proteases have been identified, and the classification of individual proteases is highly developed.•Proteases regulate fate, localization, and activity of many proteins.•Proteases are key factors in the health and viability of cells, involved in multiple processes, such as replication, transcription, cell proliferation, differentiation, extracellular matrix remodeling, and processing of hormones and biologically active peptides.•Proteases are highly regulated (e.g. transcriptionally, post-translationally, activated, inhibited, and compartmentalized).•Proteases are involved in many diseases (e.g. cancer, Alzheimer’s, arthritis, blood clotting disorders, allergies, and infections, to name a few).•Protease inhibitors are useful medically (e.g. angiotensin-converting enzyme inhibitors for blood pressure, HIV inhibitors, proteasome inhibitors for myeloma, dipeptidyl peptidase IV inhibitors for type II diabetes).•Proteases are useful industrially (e.g. clarifying beer and wines, preparation of leather, tenderizing, and debraiding). The JBC has been a major vehicle for elucidating the structures and functions of proteases and especially the fundamental aspects of these enzymes. Herb was responsible for keeping the Journal focused on fundamental/basic science, not the “hot science” of the day. His emphasis was on high-quality science that stood the test of time and had the potential of long-range importance and impact. Herb also has had a strong commitment to and influence on the ASBMB. I know this through my role as an Associate Editor of the JBC from 1999 to 2012 and as a president of the ASBMB (2004–2006). Herb participated in many activities of ASBMB, including business and financial meetings, publication committee meetings, centennial planning meetings, and Associate Editor and editorial board member activities. When it was time for the centennial celebration, he felt strongly that both the Society and the JBC should be celebrated together, even though the Journal started in 1905, one year before the Society was established (1906). He gave strong support to the Associate Editors and staff. He was always thinking ahead about issues, best ways to communicate, and new emerging areas. Herb always listened to various viewpoints, considered alternatives, and had an uncanny way of getting people to “agree” with his view. He has always been forward-looking and especially encouraged the online version of the Journal; the JBC was the first of the life science journals to appear online (in 1995). There has been great excitement about proteases and their functions in the last half-century. A few examples of discoveries that created that excitement will be mentioned here. •The discovery of proteasomes and the ATP-ubiquitin proteolytic pathway certainly changed our view of the world of protein degradation. The role of ubiquitin and the proteasome in intracellular protein breakdown began to unfold in the 1970s (e.g. see Refs. 21Ciechanover A. Hod Y. Hershko A. A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.Biochem. Biophys. Res. Commun. 1978; 81 (666810): 1100-110510.1016/0006-291X(78)91249-4Crossref PubMed Scopus (438) Google Scholar, 22Rose I.A. Warms J.V. Hershko A. A high molecular weight protease in liver cytosol.J. Biol. Chem. 1979; 254 (468813): 8135-8138Abstract Full Text PDF PubMed Google Scholar23DeMartino G.N. Goldberg A.L. Identificaton and partial purification of an ATP-stimulated alkaline protease in rat liver.J. Biol. Chem. 1979; 254 (35530): 3712-3715Abstract Full Text PDF PubMed Google Scholar) and expanded rapidly in the 1980s (e.g. see Refs. 24Wilk S. Orlowski M. Cation-sensitive endopeptidase: isolation and specificity of the bovine pituitary enzyme.J. 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Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates.J. Biol. Chem. 1987; 262 (3029081): 2451-2457Abstract Full Text PDF PubMed Google Scholar).•Signal peptidases that cleave signal peptides from secretory and membrane-associated proteins as they are translocated across membranes and into the endoplasmic reticulum were discovered in the 1970s and 1980s (e.g. see Refs. 29Milstein C. Brownlee G.G. Harrison T.M. Mathews M.B. A possible precursor of immunoglobulin light chains.Nat. New Biol. 1972; 239 (4507519): 117-12010.1038/newbio239117a0Crossref PubMed Scopus (382) Google Scholar, 30Wolfe P.B. Silver P. Wickner W. The isolation of homogeneous leader peptidase from a strain of Escherichia coli which overproduces the enzyme.J. Biol. Chem. 1982; 257 (6282859): 7898-7902Abstract Full Text PDF PubMed Google Scholar31Evans E.A. Gilmore R. Blobel G. Purification of microsomal signal peptidase as a complex.Proc. Natl. Acad. Sci. U.S.A. 1986; 83 (3511473): 581-58510.1073/pnas.83.3.581Crossref PubMed Scopus (212) Google Scholar).•Caspases, proteases involved in programmed cell death (apoptosis), were discovered in Caenorhabditis elegans in the 1980s, and the complexity of the caspase family in humans and the role of these enzymes in apoptosis and cytokine processing was revealed in the 1990s (e.g. see Refs. 32Ellis H.M. Horvitz H.R. Genetic control of programmed cell death in the nematode C. elegans.Cell. 1986; 44 (3955651): 817-82910.1016/0092-8674(86)90004-8Abstract Full Text PDF PubMed Scopus (1398) Google Scholar and 33Orth K. O'Rourke K. Salvesen G.S. Dixit V.M. Molecular ordering of apoptotic mammalian CED-3/ICE-like proteases.J. Biol. Chem. 1996; 271 (8702858): 20977-2098010.1074/jbc.271.35.20977Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar).•The HIV-1 protease, the retroviral aspartic protease that is essential for the maturation of the AIDS virus, was discovered in the 1980s (see Ref. 34De Clercq E. The design of drugs for HIV and HCV.Nat. Rev. Drug Discov. 2007; 6 (18049474): 1001-101810.1038/nrd2424Crossref PubMed Scopus (393) Google Scholar). This protease is a prime target for drug therapy, and inhibitors of the protease, along with other drugs, have greatly prolonged the lives of people infected with the virus. The development of inhibitors of the HIV-1 protease was accelerated by the large body of information available about aspartic proteases in many organisms, which allowed development of specific viral protease inhibitors. This is an example of the importance of basic science for therapeutic advances.•The great variety of cysteine proteases (e.g. cathepsins and calpains) and their diverse functions have come to light in recent decades (see, for example, Ref. 35Turk V. Stoka V. Vasiljeva O. Renko M. Sun T. Turk B. Turk D. Cysteine cathepsins: from structure, function and regulation to new frontiers.Biochim. Biophys. Acta. 2012; 1824 (22024571): 68-8810.1016/j.bbapap.2011.10.002Crossref PubMed Scopus (907) Google Scholar). They participate in a variety of processes, including autophagy, the lysosomal degradation of cellular constituents. The discovery of the molecular players in the autophagic process has enhanced our understanding of this process in health and disease (36Tsukada M. Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae.FEBS Lett. 1993; 333 (8224160): 169-17410.1016/0014-5793(93)80398-ECrossref PubMed Scopus (1466) Google Scholar, 37DeMartino G.N. Introduction to the Thematic Minireview series: autophagy.J. Biol. Chem. 2018; 293 (29467224): 5384-538510.1074/jbc.TM118.002429Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Metalloproteases have emerged as a fascinating group of enzymes. They are present in all kingdoms of living organisms and have expanded widely during evolution. In 1980, 11 metalloproteinases were identified (38Barrett A.J. McDonald J.K. Mammalian Proteases: A Glossary and Bibliography. 1. Academic Press, Inc., New York1980: 359-378Google Scholar). Now we know that the mouse and human genomes encode ∼200 metalloproteinases, the largest group in the proteolytic enzyme realm (39Sterchi E.E. Stöcker W. Bond J.S. Meprins, membrane-bound and secreted astacin metalloproteinases.Mol. Aspects Med. 2008; 29 (18783725): 309-32810.1016/j.mam.2008.08.002Crossref PubMed Scopus (163) Google Scholar). Most of these enzymes are secreted from cells or plasma membrane–bound, and they act pericellularly and extracellularly. They are involved in tissue differentiation and remodeling during embryogenesis and in processing biologically active peptides and cytokines in adult tissues. Angiotensin-converting enzyme inhibitors to control blood pressure are among the most widely used inhibitors for humans. Metalloproteinases are also involved in many diseases, such as cancer and inflammatory diseases. They and their inhibitors (e.g. TIMPs (tissue inhibitors of metalloproteinases)) are of great medical interest and have provided optimism and disappointment in clinical trials. The use of synthetic inhibitors of metalloproteinases to inhibit cancer cell mobility provides great promise but has not yet reached its potential. The metzincin superfamily contains most of the known metalloendoproteinases (zinc-containing enzymes that cleave peptide bonds internally on protein substrates) (39Sterchi E.E. Stöcker W. Bond J.S. Meprins, membrane-bound and secreted astacin metalloproteinases.Mol. Aspects Med. 2008; 29 (18783725): 309-32810.1016/j.mam.2008.08.002Crossref PubMed Scopus (163) Google Scholar). The superfamily is composed of six evolutionarily related families: a disintegrin and metalloproteinases (ADAMs), MMPs, pappalysins (pregnancy-associated plasma proteins), serralysins (bacterial enzymes), leishmanolysins (protozoan proteinases), and astacins (Fig. 1). Each of these families has multiple individual enzymes and fascinating stories of discovery and functions. Interestingly, there are relatively low amino acid sequence similarities between the protease domains of different families. However, all of the catalytic domains have strikingly similar three-dimensional structures as well as a conserved zinc binding domain (HEXXHXX(G/N)XX(H/D)) at the active site and a conserved methionine-containing turn (Met-turn) (40Bode W. Gomis-Rüth F.X. Stöcker W. Astacins, serralysins, snake venom and matrix metalloproteinases exhibit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) and topologies and should be grouped into a common family, the “metzincins”.FEBS Lett. 1993; 331 (8405391): 134-14010.1016/0014-5793(93)80312-ICrossref PubMed Scopus (656) Google Scholar). This review will focus on the astacin family (41Dumermuth E. Sterchi E.E. Jiang W.P. Wolz R.L. Bond J.S. Flannery A.V. Beynon R.J. The astacin family of metalloendopeptidases.J. Biol. Chem. 1991; 266 (1939172): 21381-21385Abstract Full Text PDF PubMed Google Scholar, 42Bond J.S. Beynon R.J. The astacin family of metalloendopeptidases.Protein Sci. 1995; 4 (7670368): 1247-126110.1002/pro.5560040701Crossref PubMed Scopus (360) Google Scholar), and particularly meprins of this family. The astacin family was recognized as a consequence of extensive cloning and sequencing that occurred in the 1980s and 1990s (see Fig. 1). The original members of the family, identified by sequence similarities, were as follows: the crayfish digestive enzyme astacin, bone morphogenetic protein-1 (BMP-1) from human bone, meprins from mouse kidney and human intestine, and UVS.2, a partial sequence from Xenopus laevis embryos (41Dumermuth E. Sterchi E.E. Jiang W.P. Wolz R.L. Bond J.S. Flannery A.V. Beynon R.J. The astacin family of metalloendopeptidases.J. Biol. Chem. 1991; 266 (1939172): 21381-21385Abstract Full Text PDF PubMed Google Scholar). The name “astacin family” was chosen because the crayfish Astacus astacus enzyme was the first to be sequenced and characterized (43Titani K. Torff H.J. Hormel S. Kumar S. Walsh K.A. Rödl J. Neurath H. Zwilling R. Amino acid sequence of a unique protease from the crayfish Astacus.Biochemistry. 1987; 26 (3548817): 222-22610.1021/bi00375a029Crossref PubMed Scopus (103) Google Scholar, 44Bode W. Gomis-Rüth F.X. Huber R. Zwilling R. Stöcker W. Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases.Nature. 1992; 358 (1319561): 164-16710.1038/358164a0Crossref PubMed Scopus (300) Google Scholar). Astacins are present in animals and bacteria; none have yet been found in plants and fungi. Hundreds of astacins have been identified as genome sequencing expands to many species (45Möhrlen F. Maniura M. Plickert G. Frohme M. Frank U. Evolution of astacin-like metalloproteases in animals and their function in development.Evol. Dev. 2006; 8 (16509900): 223-23110.1111/j.1525-142X.2006.00092.xCrossref PubMed Scopus (41) Google Scholar). In the human and mouse genomes, six astacin family genes have been identified, which includes two meprin genes, three BMP-1/tolloid-like genes, and one ovastacin gene. However, in Drosophila melanogaster, there are 16, and in C. elegans there are 40 astacins. The functions of most of the astacin genes in D. melanogaster and C. elegans have not been determined, but in parasitic nematodes, astacin enzymes are involved in moving through extracellular matrixes and in Hydra in head regeneration (46Yan L. Leontovich A. Fei K. Sarras Jr., M.P. Hydra metalloproteinase 1: a secreted astacin metalloproteinase whose apical axis expression is differentially regulated during head regeneration.Dev. Biol. 2000; 219 (10677259): 115-12810.1006/dbio.1999.9568Crossref PubMed Scopus (62) Google Scholar). Of the characterized astacins, the crayfish astacin is the smallest, containing a 200-amino acid residue catalytic domain. From cDNA sequencing, it is known that there is a prepro sequence that is cleaved off during protein synthesis. Pre or signal sequences are found in all of the astacin family members examined thus far, presumably to direct the protein into the endoplasmic reticulum and the secretory pathway. Pro sequences keep the enzymes inactive as a regulatory mechanism. Whereas the active crayfish protein contains only the ∼20-kDa catalytic domain, most of the astacin family members contain one or more noncatalytic domains, C-terminal to the protease domain. Many contain one or more copies of an epidermal growth factor (EGF)-like domain, and a CUB (complement subcomp