Intracellular Serine Protease Research Articles

Differences in the regulation of the intracellular Ca2+-dependent serine proteinase activity between Bacillus subtilis and B. megaterium.

A rise of the intracellular serine proteinase activity (ISP) during postexponential growth of Bacillus subtilis was decreased by a temperature upshift from 35 degrees to 42 degrees C. However, the amount of both molecular forms of the major intracellular serine proteinase ISP1 determined by immunoblotting was similar at both temperatures or even slightly increased at 42 degrees C. The evolution of the ISP activity in B. megaterium showed an opposite temperature dependence, being faster during growth at 42 degrees C. The amount of immunologically detected ISP1 again did not correlate well with the enzyme activity. Moreover, most of the ISP1 molecules in cell-free extracts from B. megaterium were inactive and were activated by increasing the CaCl2 concentration up to 30 mM--unlike B. subtilis, where the enzymic activity was unaffected by Ca2+ concentration. These data suggest that the ISP1 activity in the two bacillar species during postexponential growth is regulated posttranscriptionally, but that the regulatory mechanisms differ.

Homodimerization via a Leucine Zipper Motif Is Required for Enzymatic Activity of Quiescent Cell Proline Dipeptidase

Homodimerization via a Leucine Zipper Motif Is Required for Enzymatic Activity of Quiescent Cell Proline Dipeptidase

Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase.

Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.

The role of cysteine proteases in intracellular pancreatic serine protease activation.

Autodigestion by proteolytic enzymes is thought to represent the critical mechanism by which acute pancreatitis is initiated. Where and why pancreatic proteases, which are physiologically stored and secreted as inactive precursor zymogens, are activated within the pancreas has remained controversial. Here we present data which indicate that: the lysosomal protease cathepsin B can activate trypsinogen in vitro in a manner that is similar to trypsinogen activation by enterokinase; that cathepsin B colocalizes with trypsinogen in the secretory compartment of the rat pancreas and of the human pancreas; that trypsinogen activation begins in a secretory compartment that is distinct from mature zymogen granules; and that the inhibition of cathepsin B can either increase or decrease premature trypsinogen activation depending on the concentration of the inhibitor, its specificity and its site of action in the pancreatic acinar cell. These observations elucidate some of the complex relations between cysteine and serine proteases in the pancreas with respect to their mechanisms of activation, their subcellular sites of action, and their possible role in the onset of pancreatitis.

Sequence, Purification, and Cloning of an Intracellular Serine Protease, Quiescent Cell Proline Dipeptidase

We recently observed that specific inhibitors of post-proline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.

Mechanisms of intracellular zymogen activation

The pancreatic acinar cell is potentially the initial site of injury that begins the series of events leading to acute pancreatitis. Pathological intrapancreatic zymogen activation occurs in experimental pancreatitis in animals and in human pancreatitis. Intracellular activation has been clearly linked to aberrant zymogen processing in one form of hereditary pancreatitis; in this genetic disease a mutation in cationic trypsinogen may eliminate the degradation of any trypsin activated in the acinar cell. Recent studies have also provided the first direct evidence that trypsinogen activation takes place early in the course of caerulein-induced pancreatitis; parallel studies have used isolated pancreatic acini and conditions that simulate those that cause pancreatitis in vivo to demonstrate that zymogens can be pathologically activated in isolated cells. A unique acinar cell pathway regulates the intracellular proteinase processing of zymogens to their active forms. Stimulating the acinar cell with supramaximal concentrations of cholecystokinin (CCK) or carbamylcholine can activate this pathway. The activation depends on a low pH compartment within the acinar cell and activation of an intracellular serine protease. A marker of trypsinogen processing, the trypsinogen activation peptide (TAP), is generated in acinar cell compartments that do not overlap with secretory granules. This compartment overlaps with a marker of recycling endosomes and lysosomes. Thus, zymogen processing within the acinar cell proceeds in a distinct subcellular compartment and is dependent on a low pH environment and activation of serine proteases.

Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins.

Caspase activation and apoptosis can be initiated by the introduction of serine proteinases into the cytoplasm of a cell. Cytotoxic lymphocytes have evolved at least one serine proteinase with specific pro-apoptotic activity (granzyme B), as well as the mechanisms to deliver it into a target cell, and recent evidence suggests that other leucocyte granule proteinases may also have the capacity to kill if released into the interior of cells. For example, the monocyte/granulocyte proteinase cathepsin G can activate caspases in vitro, and will induce apoptosis if its entry into cells is mediated by a bacterial pore-forming protein. The potent pro-apoptotic activity of granzyme B and cathepsin G suggests that cells producing these (or other) proteinases would be at risk from self-induced death if the systems involved in packaging, degranulation or targeting fail and allow proteinases to enter the host cell cytoplasm. The purpose of the present review is to describe recent work on a group of intracellular serine proteinase inhibitors (serpins) which may function in leucocytes to prevent autolysis induced by the granule serine proteinases.

Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1.

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5-10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90 degrees C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85min at 100 degrees C and 12 min at 110 degrees C.

A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B

We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374-amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS-stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.

Disruption of three acid proteases in Aspergillus niger--effects on protease spectrum, intracellular proteolysis, and degradation of target proteins.

Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger. Northern-blot analysis showed the absence of wild-type protease mRNAs in the disruptants while western-blot analysis proved the absence of the encoded proteases. Characterization of the residual proteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16% and 94% for the delta pepA and the delta pepB disruptants, repectively. In the delta pepE disruptant, the total intracellular proteolytic activity was reduced to 32%. Apart from the reduced intracellular pepstatin-inhibitable aspartyl protease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the delta pepE strain. This may indicate the presence of a cascade activation mechanism for several vacuolar proteases, triggered by the PEPE protein, similar to the situation in Saccharomyces cerevisiae. Disruption of a single protease gene had no effects on the transcription of other non-disrupted protease genes in A. niger. In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was observed. By recombination, we also constructed delta pepA delta pepB, delta pepB delta pepE and delta pepA delta pepE double disruptants as well as a delta pepA delta pepB delta pepE triple disruptant, lacking all three acid protease activities. The in vitro residual PELB activity was the highest in the triple disruptant and the delta pepA delta pepB recombinant.

Cloning and expression of an intracellular alkaline protease gene from alkalophilic Thermoactinomyces sp. HS682.

An intracellular alkaline serine protease gene of alkalophilic Thermoactinomyces sp. HS682 was cloned and expressed in Escherichia coli. Sequence analysis showed a putative promoter region, a putative transcriptional termination signal, and an open reading frame of 963 bases, coding for a polypeptide of 321 amino acids. The protease expressed in E. coli was purified by DEAE-Toyopearl 650M and Sephadex G-75 chromatography. The N-terminal sequence (30 amino acids) of the purified protein was coincident with Asp16-Val45 of the deduced amino acid sequence of the ORF. Fifteen amino acids in the N-terminal region were removed during the purification procedures. The deduced amino acid sequence showed high similarity with microbial intracellular serine proteases. The molecular mass of this enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme was stable at pH 6.0-12.0 and below 60 degrees C in the presence of Ca2+. The temperature and pH optima of the enzyme were 65 degrees C and pH 11.0, respectively. The enzyme was inhibited by DFP and PMSF, but not by MIA and EDTA.

P-646 - Expression of an intracellular serine proteinase inhibitor, SPI-3, in murine brain and its alteration on neuronal degeneration

P-646 - Expression of an intracellular serine proteinase inhibitor, SPI-3, in murine brain and its alteration on neuronal degeneration

Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool

Proteinase 3, which is known as an intracellular serine protease of neutrophils, was detected at the surface of a subpopulation of freshly isolated PMN. The proportion of PR3-positive and -negative PMN, observed by flow cytometry with anti-PR3 mAbs or ANCA autoantibodies, varies among individuals but is extremely stable for each individual over prolonged time periods. After PMN degranulation by FMLP with cyt. B, membrane PR3 expression increases but the proportion of low and high PR3-expressing cells remains stable. The existence of a subset of PMN which spontaneously expresses PR3 and varies among individuals, may be relevant to the pathogenesis of anti-PR3 ANCA autoantibody-related vasculitis.

Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris

The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants ( k a) were derived for the interaction with trypsin (1.8 · 10 6 M −1s −1), thrombin (1.2 · 10 5 M −1 s −1), urokinase plasminogen activator (4.0 · 10 4 M −1s −1), plasmin (1.3 · 10 6 M −1s −1), and activated protein C (7.5 · 10 3 M −1 s −1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35 degrees to 40-42 degrees C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27 degrees C in the growth medium to 35 degrees C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T0) or at T3, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45-47 degrees C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24-27 degrees C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.

A new alkaline serine protease from alkalophilic Bacillus sp.: Cloning, sequencing, and characterization of an intracellular protease

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.

The Complete Amino Acid Sequences of Two Serine Proteinase Inhibitors from the Fruiting Bodies of a Basidiomycete, Pleurotus ostreatus

The complete amino acid sequences of two isomeric endogenous inhibitors, IA-1 and IA-2, both of which specifically inhibit an intracellular serine proteinase (proteinase A) purified from the fruiting bodies of a higher basidiomycete, Pleurotus ostreatus, were determined. Both inhibitors are acidic polypeptides with respective molecular masses of 8307 and 8244 Da, as determined by plasma desorption mass spectral analyses, and their N-terminal serine residue is blocked by acetylation. The fragments generated from the inhibitors by proteolytic and chemical cleavages were subjected to amino acid composition, sequence, and mass spectral analyses. The sequence and molecular mass information for the peptides established that the inhibitors both consisted of 76 amino acid residues and differed from each other in that aspartic acid and glutamic acid residues at residues 12 and 15 of IA-1 were replaced by glycine and aspartic acid in 1A-2, respectively, The molecular masses of IA-1 and IA-2 were calculated to be 8309 and 8237, based on the sequence data. The action of carboxypeptidase Y on IA-1 resulted in a complete loss of the inhibitory activity along with successive release of glutamine and threonine from the C-terminus. Cyanogen bromide cleavage of Met38-Pro39 and Met41-Lys42 in IA-1 and hydroxylamine degradation of IA-2 completely abolished their inhibitory activity. These results suggest that the whole molecules of both inhibitors are essential to their inhibitory activities. Their structural resemblance to propeptides of subtilisin family proteinases revealed their mechanism of action.

Dual effect of amino acids on the development of intracellular proteolytic activity in the irreversible sporulation phase ofBacillus megaterium

Amino acids added to a population ofBacillus megaterium immediately after its transfer to a sporulation medium stimulated growth, delayed sporulation by 1 h, and delayed the development of intracellular cytoplasmic serine proteinase (ISP) activity. However, the ISP activity in late sporulation stages exceeded twice that of the control population. Amino acids supplemented at T3, i.e., at the time when engulfed forespores were developing, caused a decrease of specific ISP activity. The course of the phenylmethane sulfonyl fluoride (PMSF)-resistant activity in the cytoplasm was not affected by amino acids. Intracellular degradation of proteins prelabeled at the end of the growth phase was decreased by amino acids during the reversible sporulation phase but was only slightly affected later.

Complementary DNA cloning and kinetic characterization of a novel intracellular serine proteinase inhibitor: mechanism of action with trypsin and factor Xa as model proteinases.

The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majority of structural motifs conserved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplasmic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino acid sequence identified three potential N-glycosylation sites and Arg341-Cys342 as the reactive site P1-P1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mechanism of bovine trypsin and human factor Xa inhibition by the novel cytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic antiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization step to a more stable, tight complex. The binding of the cytoplasmic antiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M-1 s-1 and an overall equilibrium constant of 22.5 pM, demonstrating that the factor is a potent inhibitor of this proteinase. Under the appropriate conditions, the tight complex between trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stabilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic antiproteinase followed a single-step binding mechanism. The apparent first-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. Formation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M-1 s-1 and a equilibrium constant of 3.7 nM. These findings suggests that the cytoplasmic inhibitor may initially encounter significant energy barriers for proper alignment with the substrate binding cleft of factor Xa. However, once aligned, the reaction proceeds rapidly to a tight factor Xa.inhibitor complex that dissociates at a slow rate.

Horseshoe crab (1,3)-beta-D-glucan-sensitive coagulation factor G. A serine protease zymogen heterodimer with similarities to beta-glucan-binding proteins.

Horseshoe crab factor G is an intracellular serine protease zymogen that initiates the (1,3)-beta-D-glucan-sensitive hemolymph clotting pathway. Unlike other known serine protease zymogens, which are composed of a single subunit, factor G consists of two distinct subunits, alpha and beta, which are autocatalytically converted to active factor G in the presence of (1,3)-beta-D-glucan. We have now cloned and sequenced cDNAs encoding both subunits of factor G. The subunits are derived from separate mRNA species and thus encoded by different genes. Subunit beta is a serine protease zymogen which consists of 278 residues with a calculated molecular mass of 30,846 Da; it exhibits homology to the serine protease domain of horseshoe crab factor B. Subunit alpha, on the other hand, is a new type of mosaic protein with intriguing features. The mature protein consists of 654 residues with a calculated molecular mass of 73,916 Da. The NH2-terminal portion of this subunit is similar to bacterial beta-1,3-glucanases. Its 126 amino acid COOH terminus exhibits a repetitive sequence having partial homology to xylanases. Between these regions are three repeating units of 47 amino acids, whose similarity to carbohydrate-binding proteins suggests that these may be the (1,3)-beta-D-glucan-binding domain(s) of factor G. Factor G, thus, is a structurally unique heterodimeric serine protease zymogen and as such may represent a new class of active defense proteins.