Revisiting catalysis by chymotrypsin family serine proteases using peptide substrates and inhibitors with unnatural main chains.

Chymotrypsin family serine proteases play essential roles in key biological and pathological processes and are frequently targets of drug discovery efforts. This large enzyme family is also among the most advanced model systems for detailed studies of enzyme mechanism and structure/function relationships. Productive interactions between these enzymes and their substrates are widely believed to mimic the "canonical" interactions between serine proteases and "standard" inhibitors observed in numerous protease-inhibitor complexes. To test this central hypothesis we have synthesized and characterized a series of peptide analogs, based on model substrates and inhibitors of trypsin, that contain unnatural main chains. These results call into question a long accepted theory regarding the interaction of chymotrypsin family serine proteases with substrates and suggest that the canonical interactions observed between these enzymes and standard inhibitors may represent nonproductive rather than productive, substrate-like interactions.     

Pancreatic and intestinal carbohydrases are matched to dietary starch level in wild passerine birds

Evolutionary shifts in diet composition are presumably accompanied by simultaneous changes in digestive physiology. The adaptive modulation hypothesis predicts that activities of digestive enzymes should match the relative levels of their substrates in an animal's diet so that available membrane space and synthetic energy are not wasted on enzymes in excess of need. However, previous studies on captive passerine birds showed high intraspecific phenotypic flexibility only in proteases but not in carbohydrases in response to varying diet composition. In this study, we measured the activities of pancreatic, intestinal, and hepatic enzymes in six wild-caught passerine species. We predicted that if the adaptive modulation hypothesis holds during evolutionary shifts in diet composition in birds, then mass-specific activities of digestive enzymes should be correlated positively with the content of their relevant substrates in species' diets. Whereas mass-specific activities of proteases (aminopeptidase-N, trypsin, chymotrypsin, alanine aminotransferase) were not correlated with estimated dietary protein content, mass-specific activities of all studied carbohydrases (amylase, maltase, sucrase) were positively correlated with estimated dietary starch content. We conclude that activities of carbohydrases but not proteases are evolutionarily matched to diet composition in passerine birds. We hypothesize that the need for nitrogen and essential amino acids can prevent the evolution of a low activity of proteases, even in species feeding on a low-protein diet.     

A comparison of staphostatin B with standard mechanism serine protease inhibitors

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus. We have previously shown that staphostatins A and B are competitive, active site-directed inhibitors that span the active site clefts of their target proteases in the same orientation as substrates. We now report the crystal structure of staphostatin B in complex with wild-type staphopain B at 1.9 A resolution. In the complex structure, the catalytic residues are found in exactly the positions that would be expected for uncomplexed papain-type proteases. There is robust, continuous density for the staphostatin B binding loop and no indication for cleavage of the peptide bond that comes closest to the active site cysteine of staphopain B. The carbonyl carbon atom C of this peptide bond is 4.1 A away from the active site cysteine sulfur Sgamma atom. The carbonyl oxygen atom O of this peptide bond points away from the putative oxyanion hole and lies almost on a line from the Sgamma atom to the C atom. The arrangement is strikingly similar to the "ionmolecule" arrangement for the complex of papain-type enzymes with their substrates but differs significantly from the arrangement conventionally assumed for the Michaelis complex of papain-type enzymes with their substrates and also from the arrangement that is crystallographically observed for complexes of standard mechanism inhibitors and their target serine proteases.     

The isolation and properties of collagenolytic proteases from crab hepatopancreas

A mixture of collagenolytic proteases has been isolated from the Kamchatka crab hepatopancreas. The four individual enzymes were further separated with FPLC and partially characterized. Crab collagenolytic proteases possess a high activity against different types of collagen, especially against calf skin collagen Type III and bovine lens capsule collagen Type IV, which is resistant to the microbial Clostridium sp. collagenases. In contrast with microbial collagenases the crab enzymes are good general proteases, able to cleave standard synthetic and protein substrates and possess a chymotrypsin-, trypsin- and elastase-like specificity. N-Terminal sequence analysis revealed that crab collagenolytic proteases had evolved from a trypsin-like ancestor. Crab proteases, structurally belonging to the trypsin-like enzymes, nevertheless, possess the unique ability, among this class of enzymes, to cleave the native insoluble collagen. It seems that crab collagenolytic proteases and true metalloenzyme vertebrate and microbial collagenases have certain common structural features particularly in the regions of their substrate binding site.     

Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines.     

ATP-dependent proteases of bacteria: recognition logic and operating principles

ATP-powered AAA+ proteases degrade specific proteins in intracellular environments occupied by thousands of different proteins. These proteases operate as powerful molecular machines that unfold stable native proteins before degradation. Understanding how these enzymes choose the "right" protein substrates at the "right" time is key to understanding their biological function. Recently, proteomic approaches have identified numerous substrates for some bacterial enzymes and the sequence motifs responsible for recognition. Advances have also been made in elucidating the mechanism and impact of adaptor proteins in regulating substrate choice. Finally, recent biochemical dissection of the ATPase cycle and its coupling to protein unfolding has revealed fundamental operating principles of this important, ubiquitous family of molecular machines.     

Substrate specificity of the ubiquitin and Ubl proteases

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families.     

植物类Caspase及其调控细胞程序性死亡的研究进展

植物细胞程序性死亡（PCD）在植物生长发育和逆境适应中发挥重要作用。半胱氨酸蛋白酶（caspase）调控动物PcD的启动、执行及信号转导。通过人工合成底物、动物caspase抑制剂等方法已证实在植物中存在类caspase,可分为metacas．pases、VPEs（vacuolar processing enzymes）和saspases等。本文综述了植物类caspase的种类、结构、定位、功能及其调控PCD的研究进展,提出植物PCD中类caspase作用的调控途径,为深入研究植物PCD提供参考。     

Protease signalling: the cutting edge

Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research.     

Advances in Proteomics in Cancer Research

Proteases are a family of proteolytically active enzymes whose dysfunction is implicated in a wide variety of human diseases. Although an estimated 2% of the human genome encodes for proteases, only a small fraction of these enzymes have well-characterized functions. Identification of the specificity and natural substrates of proteases in complex biological samples is challenging, but proteomic screens for proteases are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, fluorescent 2D difference gel electrophoresis with mass spectrometry, differential isotope labeling in combination with mass spectrometry, quantitative degradomics analysis of proteolytically generated neo-N-termini, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify protease specificity, cleavage sites and natural substrates with a particular focus on the cytotoxic lymphocyte granule serine proteases granzymes.     

DeSUMOylating enzymes--SENPs

Modification of proteins by ubiquitin and SUMO (small ubiquitin-like modifiers) is a dynamic and reversible process. Similar to the ubiquitin pathway, where the action of deubiquitinating enzymes removes ubiquitin from ubiquitin-adducts, SUMO is also removed intact from its substrates by proteases belonging to the sentrin-specific proteases (SENPs) family. In addition to their isopeptidase activity, SENPs also execute another essential function as endopeptidases by removing the short C-terminal extension from immature SUMOs. The defining characteristics of SENPs are their predicted conserved molecular scaffold-defined as members of peptidase Clan CE, conserved catalytic mechanism, and their reported activity on SUMO or Nedd8 conjugated proteins (or the respective precursors). We discuss recent progress on the human SENPs and their substrates.     

A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family

ADAMTS proteases are complex secreted enzymes containing a prometalloprotease domain of the reprolysin type attached to an ancillary domain with a highly conserved structure that includes at least one thrombospondin type 1 repeat. Known functions of ADAMTS proteases include processing of procollagens and von Willebrand factor as well as catabolism of aggrecan, versican and brevican. They have been demonstrated to have important roles in connective tissue organization, coagulation, inflammation, arthritis, angiogenesis and cell migration. ADAMTS can be grouped into distinct clades within which there is conservation of modular organization, protein sequence, gene structure and possibly, of substrate preference. ADAMTS proteases are synthesized as zymogens, with constitutive proprotein convertase removal of the propeptide occurring prior to secretion. Their enzymatic specificity is heavily influenced by their ancillary domain, which plays a critical role in directing these enzymes to their substrates, the cell surface and the extracellular matrix.     

Microbial keratinases and their prospective applications: an overview

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.     

Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties

Two extracellular alkaline proteases produced by an alkalophilic Bacillus isolate were purified and characterized using acetone precipitation, DEAE- and CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chromatographic techniques. Analysis of the purified proteases by SDS–PAGE revealed that both proteases, AP-1 and AP-2 were homogenous with molecular weight estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 and AP-2 were at temperatures of 50 and 55°C and pHs of 11 and 12, respectively. The enzymes were also stable in the pH range of 6.0–12.0 for a period of 4 h with and without Ca²⁺ (5 mM) and temperatures of up to 50°C. The half-lives of the enzymes recorded at 50°C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native protein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA. The K_m values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, respectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also indicated that small aliphatic and aromatic amino acids were the preferred residues at the P₁ position.     

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Involvement of proteases in apoptosis

Genetically programmed (apoptotic) cell death plays a key role in cell and tissue homeostasis and in pathogenesis of various diseases. However, the mechanisms involved in apoptotic cell death are poorly understood. At present, the role of proteases in key events of apoptosis is intensively studied and discussed and the involvement of various proteolytic enzymes in the induction and development of the cell death is well-recognized. Proteases of various classes participating in apoptosis have been identified as well as some substrates of these proteases whose cleavage is critical to cell viability; specific protease inhibitors which prevent the cell death have been synthesized. This review summarizes new data on proteolytic enzymes involved in apoptosis and considers the mechanisms of activation of proteases upon induction of apoptosis and the pathways of their involvement in the cell death. The participation of nuclear proteolytic enzymes in the destabilization of chromatin structure and regulation of DNA fragmentation by endonucleases in apoptotic cells is discussed.     

Immobilization of enzymes on alumina by means of pyridoxal 5′-phosphate

A number of proteases have been immobilized on alumina in a two-step procedure: the first step converted them into semisynthetic phosphoproteins which, in the second step, spontaneously bonded to alumina through their phosphate function. The immobilized enzymes thus obtained showed the physical properties typical of the inorganic carrier and a high activity on low molecular weight substrates.     

Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Three distinct alkaline serine proteases (named CTSP-1, -2, and -3) were purified from the polychaete Cirriformia tentaculata and characterized in terms of their enzymatic properties and kinetics. The estimated molecular masses of CTSP-1, -2, and -3 enzymes were found to be 28.8, 30.9, and 28.4 kDa, respectively. The enzymes were active at the temperature range of 50–60 °C under pH 8.5–9.0 and completely inactivated by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, but not by 1,10-phenanthroline and bestatin, suggesting that they are all typical serine proteases and not metalloproteases or cysteine proteases. CTSP-1 and -2 cleaved arginine, whereas CTSP-3 digested tyrosine residue at the carboxyl sides in their peptide substrates. A typical hepta-sequence (I-X-X-G-X-X-A) conserved in serine proteases from annelid species was found in N-termini of all CTSPs. CTSP-2 was the most active enzyme among the proteases purified as shown by kinetic values. The enzymes cleaved all chains of fibrinogen within 20 min and also hydrolyzed actively fibrin polymer as well as cross-linked fibrin. In addition, the enzymes could actively digest the fibrin clot in blood plasma milieu. Taken together, the results obtained demonstrate that CTSP enzymes have a potential of becoming therapeutic agents for thrombus dissolution.     

Identification and characterisation of two extracellular proteases of Streptococcus mutans

Abstract Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.