蛋白酶及其抑制剂关键活性位点研究进展

蛋白酶广泛存在于生物体中,参与分解蛋白质,维持生物体正常的生命活动。蛋白酶抑制剂通过与蛋白酶活性位点结合调控靶蛋白酶活性,从而影响蛋白质代谢。蛋白酶及其抑制剂关键氨基酸的突变,可以影响其生理功能、稳定性、催化活性、抑制特异性等。通过挖掘自然界蛋白酶及其抑制剂的各种突变体,分析它们的关键活性位点,并运用蛋白质工程手段改造和设计活性更强、稳定性更高、特异性更好、环境更友好、成本更低的蛋白酶及其抑制剂,已成为当前的热点研究之一。文中对近年来蛋白酶及其抑制剂关键活性位点研究进行了简要综述,以期深化人们对蛋白酶及其抑制剂活性作用机制的认识,并为蛋白酶及其抑制剂的生物学活性改造研究提供理论参考。     

Towards the control of intracellular protein turnover: mitochondrial Lon protease inhibitors versus proteasome inhibitors

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.     

Basidiomycetes harbour a hidden treasure of proteolytic diversity

This survey is the first to investigate the proteolytic potential of a large number of basidiomycetes. Aqueous extracts of 43 basidiomycetes were investigated for their content of proteolytic activities, using gelatin zymography. The activities were characterised qualitatively using class specific inhibitors. All four catalytic classes of proteases were present, with 4% of all activities classified as aspartic, 5% as cysteine, 6% as metallo and 22% as serine proteases, while the remaining activities could not be assigned unambiguously. The majority of the latter were not inhibited by any of the inhibitors used and were termed insensitive. Different proteolytic activities are evenly distributed among members of all orders of basidiomycetes, although some taxa are a richer source of proteases than others. A significant number of the cysteine protease activities shown here have not previously been reported in basidiomycetes. The fungal cysteine and serine protease inhibitors, clitocypin and CNSPI (Clitocybe nebularis serine protease inhibitor), both inhibited a number of activities and even a few activities that were otherwise insensitive to all other inhibitors used, hence indicating their potential for a regulatory role. The number and diversity of proteases in basidiomycetes are seen to be remarkable and encourage further investigation.     

Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process.

To induce exocellular proteolytic enzyme from carbon-starved exponential-phase cells of Neurospora crassa, both a protein substrate and an activating protease of certain specific properties must be present at the same time. The cells must be capable of protein synthesis, since cycloheximide inhibits the process, but cell growth, as determined by increase in cell mass, does not appear to be required. Both soluble (bovine serum albumin, myoglobin) and insoluble protein substrates (collagen, corn zein) will affect protease induction, although certain soluble, globular proteins (egg white globulin, bovine gamma globulin) will not. In most cases, rates of protease induction are proportional to protein concentration, regardless of the nature of the inducing protein. All activating proteases capable of affecting induction in a manner similar to that of N. crassa exocellular protease were of bacterial origin and were exoproteases. Mammalian proteases and peptidases had little or no effect on the induction process.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

Characterization and partial purification of an insulinase from Neurospora crassa.

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.     

Regulated serine proteinase lytic system on mammalian sperm surface: There must be a role

Serine proteases play key roles in many biological processes, regulating surface proteins that are key-points in signaling pathways. Several studies have reported the presence of members of this protease family in sperm from various species. The precise regulation of their activity is thought to be performed by specific endogenous or extrinsic inhibitors. The contribution of the sperm serine to proteases to fertilization has been demonstrated by synthetic inhibitors and several single knock out experiments, but to date, there is no evidence that links a single enzyme to a single step of fertilization. The explanation for the failure in the understanding of the “one-enzyme-one-process” hypothesis may be that sperm have multiple serine proteases as a mechanism to ensure the success of fertilization. In addition to the classical purification and expression studies, we summarized recent advances in proteomics and performed a bioinformatics search of proteases and inhibitors, providing support for the idea of redundancy. This review summarizes current knowledge about serine proteases and their inhibitors in sperm capacitation and maturation, identifies questions that need to be answered, and provides a reference for future research.     

Regulation of two extracellular proteases of Neurospora crassa by induction and by carbon-nitrogen and sulfur-metabolite repression.

Synthesis and release by Neurospora crassa 74A (FGSC 262) of a neutral and an alkaline protease have been studied by experiments in which mycelia grown in Vogel's minimal medium were transferred to media containing protein inducer and deficient in carbon, nitrogen, and/or sulfur sources. The kinetics of accumulation of each of the two proteases in filtrates of induced, metabolite-deprived (derepressed) cultures were found to be similar and the same two electrophoretically separable enzymes were elicited by each of the three induction and derepression treatments. Moreover, antiserum specific for the major protease (Protease 2) elicited by one of the treatments gave a reaction of identity with the major proteases elicited by the other two treatments. It would therefore appear that these two proteases are coordinately regulated by a single system of induction and repression in which successful induction by exogenous protein depends upon the lifting of any one of carbon, nitrogen, or sulfur metabolite repressions.     

Serine protease activity in developmental stages of Eimeria tenella

A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)-sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.     

Digestive proteases during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae).

The evolution of digestive proteases during larval development of Rhynchophorus ferrugineus (Olivier, 1790) has been studied. A progressive increase of protease activity has been found. The optimum pH for proteolytic activity against azocasein was determined. Caseinograms revealed an active complex of alkaline proteases from the early stages of the development. From the apparent molecular masses, three groups of proteases have been found - high molecular-mass proteases, medium molecular-mass proteases, and low molecular-mass proteases. Studies using specific protease inhibitors showed the major presence of serine proteases in gut extracts. The results obtained from larvae reared on different substrates have made possible a comparative assessment of the influence of diet on the development of the digestive enzymatic system. Larvae fed on an artificial diet showed a complete pattern of digestive proteases. Data suggest that this diet seems to be suitable for future research with this insect pest.     

Serine protease inhibitors in plants: nature’s arsenal crafted for insect predators

Plant serine protease inhibitors are defense proteins crafted by nature for inhibiting serine proteases. Use of eco-friendly, sustainable and effective protein molecules which could halt or slow down metabolism of nutrients in pest would be a pragmatic approach in insect pest management of crops. The host-pest complexes that we observe in nature are evolutionary dynamic and inter-depend on other defense mechanisms and interactions of other pests or more generally speaking symbionts with the same host. Insects have co-evolved and adapted simultaneously, which makes it necessary to investigate serine protease inhibitors in non-host plants. Such novel serine protease inhibitors are versatile candidates with vast potential to overcome the host inhibitor-insensitive proteases. In a nutshell exploring and crafting plant serine proteinase inhibitors (PIs) for controlling pests effectively must go on. Non-host PI seems to be a better choice for coevolved insensitive proteases. Transgenic plants expressing wound inducible chimaeric PIs may be an outstanding approach to check wide spectrum of gut proteinases and overcome the phenomenon of resistance development. Thus, this article focuses on an entire array of plant serine protease inhibitors that have been explored in the past decade, their mode of action and biological implications as well as applications.     

Identification and characterization of midgut proteases in <Emphasis Type="Italic">Achaea janata</Emphasis> and their implications

Insect midgut proteases are excellent targets for insecticidal agents such as Bacillus thuringiensis Cry toxins and protease inhibitors. The midgut proteases of Achaea janata have been characterized and Casein zymograms indicated at least five distinct activities corresponding to approx 17, 20, 29 and 80, and 90 kDa. Using a combination of synthetic substrates and specific inhibitors in casein zymograms, photometric assays and activity blots, three trypsin-like and one elastase-like serine proteases were identified but no chymotrypsin-like activity. Various proteinase inhibitors displayed differential inhibitory effects towards the midgut proteases.     

Protection from tumor necrosis factor cytotoxicity by protease inhibitors

Tumor necrosis factor (TNF) is cytocidal for human and murine cells when protein synthesis is inhibited by cycloheximide, but some protease inhibitors completely protect these cells from TNF cytotoxicity. Inhibitors of chymotrypsin-like proteases are active at lower concentrations than inhibitors of trypsin-like proteases. Both irreversible inhibitors, such as alkylating compounds, and reversible inhibitors, such as substrates of proteases, protect cells from the cytocidal activity of TNF. This protection is most effective when the cells are pretreated with these inhibitors before addition of TNF. When the protease inhibitors are removed, the cells gradually lose resistance to TNF cytotoxicity. The inhibitors do not interfere with the functioning of TNF-receptor complexes, since SK-MEL-109 melanoma cells treated with a protease inhibitor synthesize a TNF-induced protein. These findings suggest that a protease in involved in the cytocidal action of TNF.     

Interactions of serine proteases with cultured fibroblasts

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.     

Purification and characterization of a novel protease from the latex of Pedilanthus tithymaloids

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.     

Microsphere-based protease assays and screening application for lethal factor and factor Xa.

BACKGROUND: Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific. METHODS: We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere-based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCyt flow cytometry system. RESULTS: We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition of the proteases, and demonstrated high throughput detection of these inhibitors. CONCLUSIONS: The assay developed here is adaptable to any site-specific protease, compatible with high throughput flow cytometry systems, and multiplexable. Coupled with flow cytometry, which provides continuous time resolution and intrinsic resolution of free vs. bound fluorophores, this assay will be useful for high throughput screening of protease inhibitors in general and could simplify assays designed to determine protease mechanism.     

Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease

Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.     

Molecular and enzymatic characterization of the porcine endogenous retrovirus protease

The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M(r) of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.     

Secreted aspartic proteases as virulence factors of Candida species

Candida infections have emerged as a significant medical problem during the last few decades. Among the different virulence traits of C. albicans, secreted proteolytic activity has been intensively investigated. Pathogenesis of the various forms of candidiasis was shown to be associated with the differential and temporal regulation of the expression of genes coding for secreted aspartic proteases (Sap). These enzymes act as cytolysins in macrophages after phagocytosis of Candida, are present in tissue penetration and are also involved in adherence to epithelial cells. Since the introduction of new antiretroviral therapeutics such as HIV protease inhibitors, oropharyngeal candidiasis is less often observed in AIDS patients. Different HIV aspartic protease inhibitors were able to inhibit the C. albicans Saps involved in adherence. The lower rates of oropharyngeal candidiasis observed in individuals receiving antiretroviral combination therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida Saps by HIV protease inhibitors. Therefore, the development of specific aspartic protease inhibitors might be of interest for the inhibition of candidiasis.