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PubMed2000

Nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2A through S-nitrosylation: A protective mechanism against enteroviral cardiomyopathy.

BACKGROUND: Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirus-induced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A. METHODS AND RESULTS: In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC(50), 51 micromol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S:-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S:-nitrosylation of this site was confirmed with electron spin resonance spectroscopy. CONCLUSIONS: These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S:-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.

Animals↗
PubMed2004

Focal cerebral ischemia induces active proteases that degrade microvascular matrix.

BACKGROUND AND PURPOSE: Focal cerebral ischemia causes microvessel matrix degradation and generates proteases known to degrade this matrix. However, proof that the proteases generated do indeed degrade vascular matrix is lacking. Here we demonstrate that active proteases derived from ischemic tissue after middle cerebral artery occlusion (MCAO) and transferred to normal tissue can degrade vascular matrix. METHODS: In an ex vivo bioassay, the effects of supernatants from ischemic and normal basal ganglia of nonhuman primates, proteases, and control buffer on the immunoreactivity of vascular matrix constituents in normal brain tissue sections were quantified. Protease families were identified with specific inhibitors. RESULTS: Plasmin, active matrix metalloproteinase (MMP)-2, and active MMP-9 significantly reduced microvessel-associated collagen, laminin, and heparan sulfate proteoglycans (HSPG). The vascular HSPG perlecan was more sensitive than collagen or laminin in the bioassay and in the ischemic core 2 hours after MCAO. Two-hour and 7-day ischemic tissue samples significantly degraded matrix perlecan and collagen. Inhibitor studies confirmed that while active MMPs were generated, active cysteine proteases significantly degraded microvessel perlecan. The cysteine proteases cathepsins B and L were generated in the microvasculature and adjacent neurons or glial cells 2 hours after MCAO and decreased perlecan in the bioassay. CONCLUSIONS: This is the first direct evidence that active proteases are generated in ischemic cerebral tissues that are acutely responsible for vascular matrix degradation. Degradation of vascular perlecan, the most sensitive matrix component thus far identified, may be due to cathepsins B and L, generated very rapidly after MCAO.

Animals↗
PubMed2006

Inducible cardiac-restricted expression of enteroviral protease 2A is sufficient to induce dilated cardiomyopathy.

BACKGROUND: Enterovirus infection is a cause of cardiomyopathy. We previously demonstrated that enteroviral protease 2A directly cleaves the cytoskeletal protein dystrophin. However, the direct effect of protease 2A in enteroviral cardiomyopathy is less clear because other viral proteins are also expressed with viral infection. METHODS AND RESULTS: A transgenic mouse with inducible cardiac-restricted expression of enteroviral protease 2A was generated. In the transgenic mouse, a tamoxifen-regulated Cre-loxP system, MerCreMer (MCM), was used to induce genetic recombination in cardiac myocytes, which led to protease 2A expression. Protease 2A and MCM double transgenic (2AxMCM) mice were treated with tamoxifen; the controls included 2AxMCM mice treated with diluents for tamoxifen and tamoxifen-treated MCM littermates. Protease 2A activity was significantly induced after tamoxifen in the 2AxMCM mice compared with controls. Echocardiographic analysis demonstrated an increase in left ventricular end-diastolic and end-systolic chamber size, with decreased fractional shortening in tamoxifen-treated 2AxMCM mice. There was an increase in heart weight-to-body weight ratio in 2AxMCM mice treated with tamoxifen. Only a small increase in interstitial fibrosis and inflammation was found in tamoxifen-treated 2AxMCM mice; however, ultrastructural analysis demonstrated myofibrillar collapse with abnormalities of intercalated discs and sarcolemmal membranes. Evans blue dye-positive myocytes with disruption of dystrophin were present in 2AxMCM mice treated with tamoxifen. Disruption of dystrophin was also found in cultured myocytes isolated from 2AxMCM mice with Cre in the nucleus. CONCLUSIONS: Protease 2A has a significant role in enteroviral cardiomyopathy and alone is sufficient to induce dilated cardiomyopathy, which is associated with disruption of the sarcolemmal membrane and cleavage of dystrophin with protease 2A expression.

Animals↗
PubMed1999

Migration of neutrophils across human pulmonary endothelial cells is not blocked by matrix metalloproteinase or serine protease inhibitors.

It has long been speculated that neutrophils deploy proteases to digest subendothelial matrix as they migrate from the bloodstream. Direct evidence for the involvement of proteases in neutrophil transendothelial migration is, however, lacking. To address this issue we used transmission electron microscopy to verify the presence of continuous basal lamina beneath pulmonary endothelial cells grown on microporous filters, and then examined the effects of protease inhibitors on neutrophil migration through the endothelial cells and their associated subcellular matrix. Inhibitors of the two major matrix-degrading protease groups present in neutrophils, the matrix metalloproteinases (MMPs) and serine proteases, were assessed for their ability to modulate neutrophil transendothelial migration in response to the chemoattractant n-formylmethionyl leucylphenylalanine (FMLP). Neither the naturally occurring MMP inhibitor, tissue inhibitor of metalloproteinase-1, nor the hydroxamic acid-based inhibitors GM-6001, BB-3103, or Ro 31-9790 had any significant effect on FMLP-stimulated neutrophil migration across endothelial cells and associated basal lamina, with >/= 80% of neutrophils migrating through the system, even in the presence of inhibitors, at concentrations that totally inhibited all the gelatinase B (MMP-9) released upon stimulation with FMLP. Similarly, with serine protease inhibitors no significant inhibition of neutrophil migration was observed with a naturally occurring inhibitor, secretory leukocyte protease inhibitor, or a low molecular-weight synthetic inhibitor, Pefabloc SC. These results indicate that neither MMP nor serine protease digestion of sub-endothelial matrix is required for successful neutrophil transendothelial migration.

Cell Movement↗
PubMed2005

Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions.

PURPOSE: The purpose of this study was to identify a new Pseudomonas protease and determine its possible role in keratitis. METHODS: Concentrated culture supernatants of the Pseudomonas aeruginosa strains PA103 and ATCC 19660 were analyzed by zymography. P. aeruginosa small protease (PASP) was purified from strain PA103, and modified elastase B (LasB) was purified from strain ATCC 19660. SDS-PAGE and Western blot analysis were performed on purified PASP and modified LasB. PASP was further analyzed by mass spectrometry and amino-terminal sequencing. The Pasp gene was cloned and expressed, affinity-purified in denatured form from inclusion bodies, and refolded by removal of the denaturant. Purified recombinant PASP was analyzed by zymography for protease activity. PASP and heat-inactivated PASP were injected into rabbit corneas, and the corneas were monitored for erosions caused by protease activity. RESULTS: Each strain produced a protease with a molecular mass of 80 kDa on zymograms. LasB antiserum identified the ATCC 19660 protease as modified LasB. Mass spectrometry defined the PA103 protease as having a molecular mass of 18.5 kDa. Amino-terminal sequencing and analysis of the P. aeruginosa genome sequence determined that the PA103 Pasp gene sequence was >99% identical with the PA0423 sequence of strain PAO1. Recombinant PASP was proteolytic, with a zymogram mass of 50 kDa. PASP purified from PA103 produced extensive corneal epithelial erosions, whereas heat-inactivated PASP produced no erosions. CONCLUSIONS: PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.

Amino Acid Sequence↗
PubMed1980

Effect of secreted Bacteroides proteases on human intestinal brush border hydrolases.

Selected bacteroides species secreted various amounts of protease and glycosidase into their growth medium. Bacteroides vulgatus, distasonis, and ovatus secreted the most (31-60% of total). The secreted protease was similar in action to the protease within the organism, in that it had a broad pH optimum of 6-9, a K(m app.) for casein of 0.1 muM, and was inhibited by benzamidine, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate (DIFP), and by an elastase inhibitor, Ac(Ala)(3)AlaCH(2)Cl. Exposure of human brush border preparations to the secreted protease reduced maltase and sucrase activities; the reduction could be prevented by DIFP. In contrast, brush border alkaline phosphatase activity either did not change or increased after exposure to bacterial secretions. >90% inhibition of secreted glycosidase using EDTA and p-chloromercuribenzoic acid did not prevent the reduction of brush border maltase and sucrase activity, suggesting that glucosidases were not likely to be involved in the destruction of brush border enzymes. Moreover, the bacterial proteases caused only a small net release of active maltase or sucrase from the brush border. Most of the loss of activity was due to destruction of the enzyme. Proximal bowel fluid of three patients with overgrowth contained DIFP-inhibitable protease that destroyed sucrase in isolated brush borders. A Bacteroides species was isolated from each sample that secreted protease and destroyed brush border sucrase. We conclude that in bacterial overgrowth syndromes, brush border damage may occur from protease(s) secreted by Bacteroides.

Alkaline Phosphatase↗
PubMed1987

Induction of a novel Ca2+-dependent serine protease in rat liver treated with various promoters of liver carcinogenesis.

The induction of a novel Ca2+-dependent protease in rat liver treated with various liver promoters, as well as its increase in preneoplastic lesions during liver carcinogenesis, was demonstrated. Six groups of male Fischer 344 rats (150 g body weight) were fed separately diets containing one of the following promoters: 0.05% phenobarbital (PB), 0.05% dichlorodiphenyltrichloroethane (DDT), 0.25% ethyl-alpha-chlorophenoxyisobutyrate (CPIB), 0.5% butylated hydroxytoluene (BHT), 10 ppm 17-alpha-ethynylestradiol (EE), and 0.05% of the non-promoter diphenylhydantoin (DH). After feeding the indicated diets for 1 week, rats were killed and protease activity in the microsomal fraction of liver tissue was determined using N-benzoyl-L-tyrosine ethyl ester as substrate. The activity of protease increased 3- to 5-fold after treatment with the promoters and compared with normal liver; the non-promoter (DH) induced a slight increase in activity. Hyperplastic nodules were induced according to the method of Solt and Farber. The activity of protease was significantly high in these preneoplastic lesions compared with the surrounding liver tissue. Biochemical characterization of this protease revealed the following properties: high Ca2+ dependency, different molecular weight and optimum pH from previously reported proteases, and preferential distribution in the SER fraction. These results suggest that a novel type of protease is induced specifically in the liver by promoters of liver carcinogenesis. The possible importance of this protease in the carcinogenic process is discussed.

Animals↗
PubMed2006

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain.

BACKGROUND: We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. RESULTS: Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the alpha-chain of fibrinogen as well as pro-urokinase-type plasminogen activator (pro-uPA). Northern blot analysis showed that polyserase-3 exhibits a unique expression pattern among human polyserases, being predominantly detected in testis, liver, heart and ovary, as well as in several tumor cell lines. CONCLUSION: These findings contribute to define the growing group of human polyserine proteases composed at present by three different proteins. All of them share a complex structural design with several catalytic units in a single polypeptide but also show specific features in terms of enzymatic properties, expression patterns and post-translational maturation mechanisms.

Amino Acid Sequence↗
PubMed2004

CD8+ T lymphocyte responses target functionally important regions of Protease and Integrase in HIV-1 infected subjects.

BACKGROUND: CD8+ T cell responses are known to be important to the control of HIV-1 infection. While responses to reverse transcriptase and most structural and accessory proteins have been extensively studied, CD8 T cell responses specifically directed to the HIV-1 enzymes Protease and Integrase have not been well characterized, and few epitopes have been described in detail. METHODS: We assessed comprehensively the CD8 T cell responses to synthetic peptides spanning Protease and Integrase in 56 HIV-1 infected subjects with acute, chronic, or controlled infection using IFN-gamma-Elispot assays and intracellular cytokine staining. Fine-characterization of novel CTL epitopes was performed on peptide-specific CTL lines in Elispot and 51Chromium-release assays. RESULTS: Thirteen (23%) and 38 (68%) of the 56 subjects had detectable responses to Protease and Integrase, respectively, and together these targeted most regions within both proteins. Sequence variability analysis confirmed that responses cluster largely around conserved regions of Integrase, but responses against a large, highly conserved region of the N-terminal DNA-binding domain of Integrase were not readily detected. CD8 T cell responses targeted regions of Protease that contain known Protease inhibitor mutation residues, but strong Protease-specific CD8 T cell responses were rare. Fine-mapping of targeted epitopes allowed the identification of three novel, HLA class I-restricted, frequently-targeted optimal epitopes. There were no significant correlations between CD8 T cell responses to Protease and Integrase and clinical disease category in the study subjects, nor was there a correlation with viral load. CONCLUSIONS: These findings confirm that CD8 T cell responses directed against HIV-1 include potentially important functional regions of Protease and Integrase, and that pharmacologic targeting of these enzymes will place them under both drug and immune selection pressure.

Journal Article↗
PubMed2005

Neutrophil elastase, an acid-independent serine protease, facilitates reovirus uncoating and infection in U937 promonocyte cells.

BACKGROUND: Mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. During enteric infections, proteolysis of the reovirus outer capsid protein sigma3 is mediated by pancreatic serine proteases. In contrast, the proteases critical for reovirus replication in the lung are unknown. Neutrophil elastase (NE) is an acid-independent, inflammatory serine protease predominantly expressed by neutrophils. In addition to its normal role in microbial defense, aberrant expression of NE has been implicated in the pathology of acute respiratory distress syndrome (ARDS). Because reovirus replication in rodent lungs causes ARDS-like symptoms and induces an infiltration of neutrophils, we investigated the capacity of NE to promote reovirus virion uncoating. RESULTS: The human promonocyte cell line U937 expresses NE. Treatment of U937 cells with the broad-spectrum cysteine-protease inhibitor E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] and with agents that increase vesicular pH did not inhibit reovirus replication. Even when these inhibitors were used in combination, reovirus replicated to significant yields, indicating that an acid-independent non-cysteine protease was capable of mediating reovirus uncoating in U937 cell cultures. To identify the protease(s) responsible, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including NE and cathepsin (Cat) G. In the presence of E64, reovirus did not replicate efficiently in PMA-treated cells. To directly assess the role of NE in reovirus infection of U937 cells, we examined viral growth in the presence of N-Ala-Ala-Pro-Val chloromethylketone, a NE-specific inhibitor. Reovirus replication in the presence of E64 was significantly reduced by treatment of cells with the NE inhibitor. Incubation of virions with purified NE resulted in the generation of infectious subviron particles that did not require additional intracellular proteolysis. CONCLUSION: Our findings reveal that NE can facilitate reovirus infection. The fact that it does so in the presence of agents that raise vesicular pH supports a model in which the requirement for acidic pH during infection reflects the conditions required for optimal protease activity. The capacity of reovirus to exploit NE may impact viral replication in the lung and other tissues during natural infections.

Cell Differentiation↗
PubMed1999

Limited proteolysis of the IGF binding protein-2 (IGFBP-2) by a specific serine protease activity in early breast milk.

IGF in milk possibly promote maturation of the gastrointestinal tract in newborns. We studied the composition of milk samples derived from 99 healthy women at regular intervals during a period beginning 3 d and ending 6 mo after birth. The concentrations measured by RIA on d 3 were 10.7+/-0.4 ng/mL for IGF-II, 1.9+/-0.1 ng/mL for IGF-I, 100+/-5 ng/mL for IGF binding protein-3 (IGFBP-3), and 2163+/-108 ng/mL for IGFBP-2. All factor concentrations decreased by up to 70% in the course of the 6 mo. The most striking finding was an IGFBP-2-specific protease activity. Protease assays performed by incubation of 125I-IGFBP-2 with milk yielded fragments of 14, 16, 23, and 25 kD. 125I-IGFBP-3 was not cleaved. Proteolysis occurred only in milk from mothers until 4 wk postpartum and could be visualized by immunoblots. Since the affinity of the fragments to 125I-IGF-II was low, they were not demonstrable by ligand blot. Inhibitor studies and pH optimizing classified the IGFBP-2 protease as an Me2(+)-dependent serine protease with a pH optimum of 7 to 8. The proteolytic activity of further milk proteins, known as IGFBP proteases, was analyzed. Epidermal growth factor receptor peptide and prostate-specific antigen did not cleave IGFBP-2, although the protease activity correlated (r = 0.84, p < 0.00003) with the prostate-specific antigen concentration in milk. The y-nerve growth factor cleaved 125I-IGFBP-2, but in a completely different manner than the milk protease. In conclusion, the IGFBP-2 protease in milk is most probably a kallikrein. The specific proteolysis of IGF/IGFBP-2 complexes may increase the biologic availability of IGF in early milk. This mechanism may promote growth of the maternal breast epithelium and maturation of the gastrointestinal tract of newborns.

Female↗
PubMed1981

Hormonal regulation of epidermal growth factor and protease in the submandibular gland of the adult mouse.

The structure of the granular convoluted tubules of the mouse submandibular gland is influenced by androgens, adrenal steroids, and thyroid hormones. We wished to investigate the effects of variations in hormonal status on the quantitative and qualitative distribution of two secretory products of these tubules, epidermal growth factor (EGF) and protease. The effects of the thyroid and adrenal glands on EGF content and protease activity of the submandibular glands of adult female mice were studied by RIAs (EGF), enzyme assays (protease), and immunocytochemical methods. In animals rendered chronically hypothyroid by propylthiouracil (4 months) or in animals which were adrenalectomized and ovariectomized (3 weeks), protease activity and EGF levels were reduced by 81-97%. The administration of testosterone induced these polypeptides even in hypothyroid animals. Daily administration of L-T4 (T4; 1 micrograms/g BW) for 7 days increased EGF and protease activity 3.6-fold in intact mice and reversed the effect of hypothyroidism. EGF and protease were also induced by T4 in adrenalectomized and ovariectomized mice, although to a lesser degree than in intact animals. Immunocytochemical stainings of submandibular glands indicated that the number of granular convoluted tubule cells immunoreactive for EGF correlated with the levels of EGF determined by RIAs. With respect to immunostaining for protease, such a correlation was not observed. The data indicate multihormonal regulation of EGF and protease in the mouse submandibular gland.

Adrenal Glands↗
PubMed1982

Intracellular protease activity in glucocorticoid-mediated thymolysis.

The effect of dexamethasone on rat thymus protease activity was tested by following hydrolysis of 14C-labeled globin. Most of the proteolytic activity was located in the cytoplasmic fraction obtained from either whole thymus homogenates or isolated thymic lymphocytes. The protease showed an acid pH optimum and was inhibited by pepstatin and leupeptin. The particulate fractions exhibited only a negligible proteolytic activity throughout the pH range tested. The administration of dexamethasone (9 alpha-fluoro-11 beta, 17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3,20-dione; 1 mg/kg, ip) to adrenalectomized castrated rats caused a marked increase in the acid protease activity assayed in the cytosol of whole thymus or thymic lymphocytes, with no change in the particular enzyme activity. The sensitivity of the cytosolic enzyme to several protease inhibitors was unchanged after glucocorticoid treatment. Minimal effective dexamethasone doses for thymic involution and increases in protease activity were 0.01 and 0.1 mg/kg BW, respectively. The half-maximal thymolytic effect was obtained at 0.05 mg/kg dexamethasone, while the half-maximal effect on the protease was observed only at 0.30 mg/kg dexamethasone. In contrast, in vitro exposure of isolated thymic lymphocytes to 8.3 X 10(-6) M dexamethasone failed to affect the acid protease activity in the cytosol, but produced a marked time-dependent cytolytic response. These observations suggest that glucocorticoid-induced cytolysis in rat thymic lymphocytes is not mediated by a direct effect of the hormone on endogenous proteases.

Adrenalectomy↗
PubMed1985

Immunological localization and quantitation of the androgen-dependent secretory protease of the canine prostate.

We have recently reported on the isolation and characterization of the predominant protein of canine prostatic fluid and the subsequent identification of this protein as a proteolytic enzyme. In this report, we described the preparation and characterization of a rabbit antiserum against this secretory protease which was purified from canine prostatic fluid by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specificity of the antiserum has been established by immunoblot analysis and immunoprecipitation of the secretory protease present in canine prostatic fluid and tissue homogenates. The antiserum was used to localize (by immunofluorescence) and quantitate (by single radial immunodiffusion) the secretory protease in prostatic tissue obtained from intact and castrated dogs. These analyses can be summarized as follows: the secretory protease is strictly localized in the apical portions of the columnar epithelial cells in prostates of intact dogs, where it comprises almost 60% of the total Triton X-100-soluble protein; and the level of secretory protease decreases at least 500-fold in prostates of castrated dogs, demonstrating the androgen-dependent nature of this enzyme. Proteins immunologically related to the secretory protease were detected at low levels in the pancreas and salivary glands of the dog. Such proteins were not detectable in canine liver, kidney, vas deferens, testis, epididymis, lung, spleen, skin, small intestine, or sartorius muscle, suggesting a high degree of prostatic specificity for the secretory protease. These results demonstrate that the canine prostate is highly specialized with respect to its androgen-dependent synthesis and secretion of this secretory protease and that the antiserum specific for this protein can be used to assess canine prostatic function under varying hormonal conditions.

Animals↗
PubMed1998

A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions.

Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.

Animals↗
PubMed1994

Alteration in insulin-like growth factor-binding proteins (IGFBPs) and IGFBP-3 protease activity in serum and urine from acute and chronic renal failure.

The insulin-like growth factors, IGF-I and IGF-II, are proteins that promote cellular growth and differentiation of various organs, including the kidney. These peptides interact with high affinity cell surface receptors and bind to a family of IGF-binding proteins (IGFBPs). Altered serum and urinary IGFBP patterns in children with chronic renal failure have been previously described. In this study, we evaluated serum and urinary IGFBP profiles in acute renal failure patients (ARF; n = 10) and chronic renal failure patients (n = 10), using Western ligand blots. Most patients with acute or chronic renal failure showed decreased intact serum IGFBP-3 and increased serum IGFBP-2. Both groups displayed marked urinary IGFBP alterations, including increased urinary IGFBP-1 and totally absent urinary IGFBP-3, as detected by Western ligand blot. To evaluate altered IGFBP profiles, we performed IGFBP-3 protease assays with sera and urine from renal failure patients and normal controls. Although control urine had only minor protease activity (defined by the ability to degrade [125I]IGFBP-3), significant protease activity was found in urine from renal failure patients. The proteolytic pattern and susceptibility to protease inhibitors in most renal failure urine samples were the same as those seen in normal urine and with plasmin. Protease activity was completely inhibited by serine protease inhibitors. We speculate that urinary protease activity is mediated primarily by a serine protease(s), which may be involved in the modulation of renal IGF activity in health and disease.

Acute Kidney Injury↗
PubMed1992

Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221.

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus subtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a CM-glycinin digesting protease from soybean seeds.

A glycinin-digesting protease was purified from 4-h-imbibed soybean seeds to apparent homogeneity by a combination of DEAE-cellulose, gel filtration and Shodex IEC QA-824 chromatography. The purified soybean protease showed a single band on SDS-PAGE and degraded the carboxymethylated glycinin molecule at neutral or alkaline pH. The glycinin-digesting protease has an apparent molecular mass of 98 kilodaltons as estimated by SDS-PAGE under both reduced and non-reduced conditions. A peptidic substrate for the soybean protease was detected and purified from a tryptic digest of glycinin A5A4B3 complex; it corresponded to Ala(375)-Lys(381) of the B3 subunit. It was found that the protease could hydrolyze the peptide bond between Tyr(378) and Asn(389) of that peptide. The soybean protease was significantly inhibited by PMSF, indicating the endopeptidase is a serine protease. Moreover, the glycinin-digesting activity is also susceptible to EDTA and the inactivated enzyme could be restored by the addition of MnCl2, suggesting that Mn2+ is an important factor for the endopeptidase activity. To our knowledge, this is the first report of purification and characterization of a glycinin-digesting protease from soybean seeds.

Alanine↗