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This paper is the introduction to the following papers, which were presented at the National Academy of Sciences colloquium “Proteolytic Processing and Physiological Regulation,” held February 20–21, 1999, at the Arnold and Mabel Beckman Center in Irvine, CA.

Proteolytic enzymes, past and future

HANS NEURATH*

Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195

ABSTRACT Today’s knowledge is based on yesterday’s research, which, for me, started some 60 years ago. In the introduction to this colloquium, the past history of proteolytic enzymes is briefly reviewed against the background of simultaneously developing concepts and methodologies in protein chemistry. This history is followed by a sketch of more recent developments of the role of proteolytic enzymes in physiological regulation and an outlook of future trends apparent from current research.

The history of proteolytic enzymes is intimately interwoven with that of protein chemistry. In the very early days, proteolytic enzymes were considered an impediment that had to be removed in the isolation of proteins generally. When I entered the field some 60 years ago, Northrop, Kunitz, and Herriott (1) had published the first edition of their treatise Crystalline Enzymes and demonstrated that, contrary to some prevailing notions, the crystalline proteolytic enzymes and protease inhibitors that they had isolated were chemical entities of constant solubility and hence obeyed the thermodynamic criteria of pure compounds. These compounds included pepsinogen, pepsin, and pepsin inhibitor, chymotrypsin, trypsin, their zymogens and inhibitors, carboxypeptidase, ribonuclease, hexokinase, diphtheria antitoxin, and a few others. Because these proteins were commercially unavailable, anyone interested in studying them had to isolate them the hard way. The field lay relatively dormant and awaited the development of more effective and specific methods of isolation, purification, and characterization of proteins, which came some 20 years later, including the methods of chromatography, gel electrophoresis, gel filtration, ultracentrifugation, amino acid analysis, and protein sequencing (2). In an effort to avoid the complexity of protein substrates, low molecular-weight synthetic peptides and their ester analogs were synthesized and found to simulate the specificity requirements of these proteases. Other landmarks included the discovery of natural and synthetic protease inhibitors such as disopropylfluoro phosphate, which introduced an organic phosphate label into the active site of serine proteases. Chemical characterization of active sites together with x-ray structure analysis of proteases showed that they can be grouped into families of common mechanism, similar structural features, and hence common evolutionary origin. They included the well known families of serine, cysteine, aspartic, and metallo endo- and exopeptidases.

The number of proteases under investigation in the early days is minuscule compared with the current inventory of several thousand proteolytic enzymes that are coded by 2% of the structural gene pool (3). Interest in proteases was considerably stimulated by the recognition that, aside from their digestive action, proteases are involved in the regulation of a great many physiological processes. In many cases, regulation is mediated by the association of proteases with nonproteolytic domains that confer specificity to their interaction with receptor sites. The most studied among them are the proteases involved in blood coagulation, fibrinolysis, the complement system, and the processing of protein hormone precursors by specific convertases. A telling case of such an association is enterokinase, a protease that fulfills the simple but specific task of cleaving the amino-terminal hexapeptide during the activation of trypsinogen. Although enterokinase was discovered more than 50 years ago, it was only recently that its x-ray structure was elucidated by cloning and expressing the heavy chain (4). Surprisingly, it was found to be composed of a trypsin-like catalytic domain covalently bound to a series of nonprotease domains that also exist in unrelated proteins. One of these resembles the low density lipoprotein receptor, another resembles meprin, a third occurs in complement C1r, and yet another occurs in a macrophage receptor. The functional significance of these specific combinations is unknown.

The term “limited proteolysis” was coined by Linderstrom-Lang to differentiate the restricted specificity of certain enzymes under certain conditions from the random proteolysis accompanying protein degradation. Proteolytic processing can be limited by the specificity of the protease, the accessibility of the susceptible peptide bond of the substrate, the obligatory activation of an enzyme precursor, the action of protease inhibitors, or a combination of these factors.

By far the best characterized and perhaps most versatile proteolytic enzymes are the serine proteases. Together with their inhibitors, they regulate a great variety of physiological events. Whereas initially the different specificities of trypsin and chymotrypsin were exclusively ascribed to differences in the sequence and structure of the primary substrate-binding site (aspartic acid in trypsin vs. serine in chymotrypsin), this simple explanation had to be abandoned when Craik and coworkers (5) demonstrated that, in addition, two surface loops are changed, indicating that conformational changes at distant secondary binding sites are also required. It has also been shown that the introduction of a metal binding site by site-directed mutagenesis allows the interconversion of a protease belonging to the serine family into another that can be regulated like a zinc metallo protease (6, 7). However, the metal inhibits the serine protease but is essential for metalloprotease activity. A relative newcomer in the families of proteases are the caspases, which resemble each other in amino acid sequence, structure, and substrate specificity, as will be discussed in a paper to follow [G.S.Salvesen and V.M.Dixit (8)]. Another important recent advance is the isolation and characterization of proteasomes [R.Huber (9)].

One of the earliest and best understood cases of proteolytic processing is zymogen activation. It underlies a great variety of physiological regulations, particularly when coupled to consecutive activation reactions as in the cascades of blood

   

PNAS is available online at www.pnas.org.

*  

To whom reprint requests should be addressed. E-mail: neurath@u.washington.edu.



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OCR for page 10962
Colloqium on Proteolytic Processing and Physiological Regulation This paper is the introduction to the following papers, which were presented at the National Academy of Sciences colloquium “Proteolytic Processing and Physiological Regulation,” held February 20–21, 1999, at the Arnold and Mabel Beckman Center in Irvine, CA. Proteolytic enzymes, past and future HANS NEURATH* Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195 ABSTRACT Today’s knowledge is based on yesterday’s research, which, for me, started some 60 years ago. In the introduction to this colloquium, the past history of proteolytic enzymes is briefly reviewed against the background of simultaneously developing concepts and methodologies in protein chemistry. This history is followed by a sketch of more recent developments of the role of proteolytic enzymes in physiological regulation and an outlook of future trends apparent from current research. The history of proteolytic enzymes is intimately interwoven with that of protein chemistry. In the very early days, proteolytic enzymes were considered an impediment that had to be removed in the isolation of proteins generally. When I entered the field some 60 years ago, Northrop, Kunitz, and Herriott (1) had published the first edition of their treatise Crystalline Enzymes and demonstrated that, contrary to some prevailing notions, the crystalline proteolytic enzymes and protease inhibitors that they had isolated were chemical entities of constant solubility and hence obeyed the thermodynamic criteria of pure compounds. These compounds included pepsinogen, pepsin, and pepsin inhibitor, chymotrypsin, trypsin, their zymogens and inhibitors, carboxypeptidase, ribonuclease, hexokinase, diphtheria antitoxin, and a few others. Because these proteins were commercially unavailable, anyone interested in studying them had to isolate them the hard way. The field lay relatively dormant and awaited the development of more effective and specific methods of isolation, purification, and characterization of proteins, which came some 20 years later, including the methods of chromatography, gel electrophoresis, gel filtration, ultracentrifugation, amino acid analysis, and protein sequencing (2). In an effort to avoid the complexity of protein substrates, low molecular-weight synthetic peptides and their ester analogs were synthesized and found to simulate the specificity requirements of these proteases. Other landmarks included the discovery of natural and synthetic protease inhibitors such as disopropylfluoro phosphate, which introduced an organic phosphate label into the active site of serine proteases. Chemical characterization of active sites together with x-ray structure analysis of proteases showed that they can be grouped into families of common mechanism, similar structural features, and hence common evolutionary origin. They included the well known families of serine, cysteine, aspartic, and metallo endo- and exopeptidases. The number of proteases under investigation in the early days is minuscule compared with the current inventory of several thousand proteolytic enzymes that are coded by 2% of the structural gene pool (3). Interest in proteases was considerably stimulated by the recognition that, aside from their digestive action, proteases are involved in the regulation of a great many physiological processes. In many cases, regulation is mediated by the association of proteases with nonproteolytic domains that confer specificity to their interaction with receptor sites. The most studied among them are the proteases involved in blood coagulation, fibrinolysis, the complement system, and the processing of protein hormone precursors by specific convertases. A telling case of such an association is enterokinase, a protease that fulfills the simple but specific task of cleaving the amino-terminal hexapeptide during the activation of trypsinogen. Although enterokinase was discovered more than 50 years ago, it was only recently that its x-ray structure was elucidated by cloning and expressing the heavy chain (4). Surprisingly, it was found to be composed of a trypsin-like catalytic domain covalently bound to a series of nonprotease domains that also exist in unrelated proteins. One of these resembles the low density lipoprotein receptor, another resembles meprin, a third occurs in complement C1r, and yet another occurs in a macrophage receptor. The functional significance of these specific combinations is unknown. The term “limited proteolysis” was coined by Linderstrom-Lang to differentiate the restricted specificity of certain enzymes under certain conditions from the random proteolysis accompanying protein degradation. Proteolytic processing can be limited by the specificity of the protease, the accessibility of the susceptible peptide bond of the substrate, the obligatory activation of an enzyme precursor, the action of protease inhibitors, or a combination of these factors. By far the best characterized and perhaps most versatile proteolytic enzymes are the serine proteases. Together with their inhibitors, they regulate a great variety of physiological events. Whereas initially the different specificities of trypsin and chymotrypsin were exclusively ascribed to differences in the sequence and structure of the primary substrate-binding site (aspartic acid in trypsin vs. serine in chymotrypsin), this simple explanation had to be abandoned when Craik and coworkers (5) demonstrated that, in addition, two surface loops are changed, indicating that conformational changes at distant secondary binding sites are also required. It has also been shown that the introduction of a metal binding site by site-directed mutagenesis allows the interconversion of a protease belonging to the serine family into another that can be regulated like a zinc metallo protease (6, 7). However, the metal inhibits the serine protease but is essential for metalloprotease activity. A relative newcomer in the families of proteases are the caspases, which resemble each other in amino acid sequence, structure, and substrate specificity, as will be discussed in a paper to follow [G.S.Salvesen and V.M.Dixit (8)]. Another important recent advance is the isolation and characterization of proteasomes [R.Huber (9)]. One of the earliest and best understood cases of proteolytic processing is zymogen activation. It underlies a great variety of physiological regulations, particularly when coupled to consecutive activation reactions as in the cascades of blood     PNAS is available online at www.pnas.org. *   To whom reprint requests should be addressed. E-mail: neurath@u.washington.edu.

OCR for page 10962
Colloqium on Proteolytic Processing and Physiological Regulation coagulation, fibrinolysis, the complement reaction, and others. The key point here is that a signal can be specifically and irreversibly amplified every time a downstream inactive enzyme precursor is activated. Recent work, to be presented in the paper to follow (8), has demonstrated a specific role of the pro segment of activation, which early on was regarded a throwaway piece but in certain cases can act as an intramolecular inhibitor and as an intramolecular chaperon that assures proper folding of the active enzyme (10). Certain generalizations have emerged from these and related investigations. If I may borrow a page from Nancy Thornberry (11), most proteases are synthesized as inactive precursors (zymogens) that require limited proteolysis for activation. Because proteolysis is irreversible under physiological conditions, the generation of the uncleaved precursor requires de novo synthesis. All active proteases, including those that activate zymogens, are regulated by specific inhibitors. However, some protease precursors can regulate their own activation, e.g., trypsinogen, whereas others, e.g., plasminogen, do not require peptide bond cleavage for their activation. Proteolytic processing, like all proteolytic reactions, requires unique combinations of primary, secondary, and tertiary structures to permit interaction with substrate so as to form the reactive enzyme-substrate intermediate. Let me now make a major leap in time and discuss in brief how we reached the current era of research on proteolytic enzymes and what we can expect in the millennium that we are about to enter. Two major factors have expanded our conceptual horizons and endowed us with experimental tools of previously unimaginable powers of resolution. One factor is the application of the newly emerging concepts and methodologies of molecular and cell biology, such as DNA cloning and sequencing, site-directed mutagenesis, gene amplification, gene knockouts, phage display, and the wealth of information yielded by genomics research generally. The other major impetus came from a group of newly developed concepts and experimental approaches to the structure and function of proteins by mass spectroscopy (12), multidimensional NMR, and the use of computers for the prediction of protein structure based on various types of algorithms. To these one might add the methods of combinatorial chemistry as applied to proteins to scan and identify protein ligands of physiological significance. Although we are still far from understanding the rules of the in vivo folding of nascent polypeptide chains, the challenge lies in deriving the function of a protein from its known chemical and biological parameters and in learning how to design proteins of predetermined physiological properties. All of these developments, singly and in combination, expand our horizons and the goals that we are setting for their application to biology and medicine. The importance of proteolytic enzymes to the understanding of vital biological tasks is perhaps best illustrated by current trends in the study of viral proteases (13). In every known instance, the timing, placement, and mode of action of the virus encoded protease are somehow adapted to the conditions under which it operates within the viral environment. Two examples follow: in herpes viruses such as cytomegalovirus, the structure of the protease reveals a catalytic triad of His/His/Ser instead of the conventional Asp/His/Ser of the mammalian serine proteases and a single beta barrel structure per monomer instead of two in the mammalian serine proteases (13). Analogously, in adeno viruses the cysteine protease contains a Glu/His/Cys catalytic triad characteristic of cysteine proteases, but the seven alpha helices and a single five-stranded beta sheet are not seen in the parent protease (papain). In either case, the examples given demonstrate the ability of the virus proteases to adapt themselves to the evolution of functions within the limits of compatible protein structures (13). Other rapidly expanding areas of biological research involving well known proteases include those of apoptosis, the mediation of thrombin signaling by protease activated receptors, proteolytic processing in cholesterol metabolism, in the cell cycle, and the many others included in this issue of the Proceedings. It is no coincidence that industry and academia are almost equally represented in this audience, because intense cooperation between both is essential if we are to reap the full benefits of the advances and discoveries in both basic and applied research. 1. Northrop, J.H., Kunitz, M. & Herriott, R.M. (1938) Crystalline Enzymes (Columbia Univ. Press, New York). 2. Neurath, H. (1995) Protein Sci. 4, 1939–1943. 3. Barrett, A.J., Rawlings, N.D. & Woessner, J.F. (1998) in Handbook of Proteolytic Enzymes (Academic, New York), pp. xiii–xxix. 4. Kitamoto, Y., Yuan, X., Wu, Q., McCourt, D.W. & Sadler, J.E. (1994) Proc. Natl. Acad. Sci. USA 91, 7588–7592. 5. Perona, J.J. & Craik, C.S. (1995) Protein Sci. 4, 337–360. 6. Higaki, J.N., Fletterick, R.J. & Craik, C.S. (1992) Trends Biochem. Sci. 17, 100–104. 7. Klemba, M., Gardner, K.H., Marino, S., Clarke, N.D. & Regan, L. (1995) Struct. Biol. 2, 368–373. 8. Salvesen, G.S. & Dixit, V.M. (1999) Proc. Natl. Acad. Sci. USA 96, 10964–10967. 9. Groll, M., Heinemeyer, W., Jäger, S., Ullrich, T., Bochtler, M., Wolf, D.H. & Huber, R. (1999) Proc. Natl. Acad. Sci. USA 96, 10976–10983. 10. Cunningham, E.L., Jaswal, S.S. & Agard, D.A. (1999) Proc. Natl. Acad. Sci. USA 96, 11008–11014. 11. Thornberry, N.A. & Lazebnik, Y. (1998) Science 281, 1312–1316. 12. Cohen, S.L. (1996) Structure (London) 4, 1013–1016. 13. Babé, L.M. & Craik, C.S. (1997) Cell 91, 427–430.