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PubMed2002

The regulation of subtilisin-cleaved actin by tropomyosin/troponin.

Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.

Actins↗
PubMed2003

Cloning and biochemical characterization of blisterase, a subtilisin-like convertase from the filarial parasite, Onchocerca volvulus.

Blisterase is a subtilisin-like proprotein convertase of nematodes. The enzyme is named after the blistered cuticle found in Caenorhabditis elegans with the bli-4 e937 mutation. The critical role of the enzyme in cuticle production makes it a potential drug target for parasitic nematodes. We have cloned and expressed blisterase from the parasitic nematode Onchocerca volvulus, a major cause of blindness in Africa. The catalytic domain of the protease exhibits 84% identity with the corresponding domain of its closest homologue, C. elegans blisterase. O. volvulus blisterase expressed in insect cells has maximal activity in 1 mm calcium at neutral pH. The protease is inhibited by EDTA, the suicide substrate decanoyl-RVKR-chloromethylketone, alpha1-antitrypsin Portland and by its own propeptide. Substrate assays with fluorescent peptides show that O. volvulus blisterase requires a P4 arginine and a basic amino acid at P1 for cleavage. The kcat of blisterase on the peptide substrate, t-butyloxycarbonyl-RVRR-4-methylcoumaryl-7-amide was determined to be 0.018 s-1. In vitro cleavage studies with the nematode polyprotein antigen demonstrated that blisterase cleaved at tetrabasic (RRKR) but not at dibasic (KR) sites. This report describes the first biochemical characterization of the nematode specific protease, blisterase.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Conformational dynamics of subtilisin-chymotrypsin inhibitor 2 complex by coarse-grained simulations.

An off-lattice dynamic Monte Carlo (MC) method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2) and subtilisin in both free and complex forms over two time windows, referring to short and long time scales. The conformational dynamics of backbone bonds analysed from several independent trajectories reveal that: Both the inhibitor and the enzyme are restricted in their bond rotations, excluding a few bonds, upon binding; the effect being greatest for the loop regions, and for the inhibitor. A cooperativity in the near-neighbor bond rotations are observed on both time scales, whereas the cooperative rotations of the bonds far along the sequence appear only in the long time window, and the latter time window is where most of the interactions between the inhibitor and the enzyme are observed. Upon binding, the cooperatively rotating parts of the inhibitor and the enzyme are readjusted compared to their free forms, and new correlations appear. The binding loop, although it is the closest contact region, is not the only part of the inhibitor involved in the interactions with the enzyme. Loops 3 and 8 and the helices F and G in bound enzyme and the binding loop of the inhibitor contribute at the most to the collective motions of whole structure on the slow time scale and are apparently important for enzyme-inhibitor interactions and function. The results in general provide evidence for the contribution of the loops with cooperative motions to the extensive communication network of the complex.

Models, Molecular↗
PubMed2006

Characterization of an extracellular subtilisin protease of Rhizopus microsporus and evidence for its expression during invasive rhinoorbital mycosis.

An endoprotease Arp (alkaline Rhizopus protease) was identified and purified to virtual homogeneity from the culture supernatant of an isolate of Rhizopus microsporus var. rhizopodiformis recovered from a non-fatal case of rhinoorbital mucormycosis. N-terminal sequencing of the mature native enzyme was obtained for the first 20 amino acids and revealed high homology to serine proteases of the subtilisin subfamily. Arp migrated in SDS-PAGE with an estimated molecular mass of 33 kDa and had a pI determined to be at pH 8.8. Arp is proteolytically active against various substrates, including elastin, over a broad pH range between 6 and 12 with an optimum at pH 10.5. After invasive mucormycosis, specific antibodies against Arp were detected in stored serum samples taken from the patient from whom the R. microsporus strain of this study had been isolated. Furthermore, in search of factors involved in thrombosis as a typical complication of mucormycosis, a procoagulatory effect of the enzyme has recently been shown. Altogether, these data substantiate the expression of Arp during human rhinoorbital mucormycosis and suggest a role of the enzyme in pathogenesis.

Amino Acid Sequence↗
PubMed1990

Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin.

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.

Actin Cytoskeleton↗
PubMed1992

Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin.

Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low. In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.

Animals↗
PubMed1975

The hydrolysis of human IgG with subtilisin.

Small amounts of subtilisin (0.1 mg. per ml.) in the absence of cysteine will instantaneously hydrolyze "enzyme-sensitive" IgG globulins. This procedure permits the identification of antibodies in whole serum and myeloma globulins in the ultracentrifuge with simplicity and speed unmatched by other technics.

Bromelains↗
PubMed1999

A novel subtilisin-like protease gene from Arabidopsis thaliana is expressed at sites of lateral root emergence.

Differential screening of a cDNA library for mRNA species that specifically accumulate during auxin-induced lateral root formation in Arabidopsis thaliana led to the isolation of the AIR3 cDNA clone. The corresponding single copy gene consists of 10 exons which encode a protein that possesses all the characteristics of subtilisin-like proteases. The promoter of the AIR3 gene was fused to the gusA (beta-glucuronidase) reporter gene and introduced into Arabidopsis. Expression was almost completely restricted to the outer layers of the parental root at sites of lateral root emergence and could be observed even before protrusion of the newly formed root tip. In the presence of external auxin, GUS activity was visible throughout the parts of the root that are competent for lateral root formation. By digesting structural proteins in the extracellular matrix of cells located above sites of lateral root formation, AIR3 might weaken cell-to-cell connections and thus facilitate lateral root emergence.

Amino Acid Sequence↗
PubMed2002

Functional tolerance of Streptomyces subtilisin inhibitor toward conformational and stability changes caused by single-point mutations in the hydrophobic core.

Single amino acid mutations of Met103 in the hydrophobic core of a serine protease inhibitor, Streptomyces subtilisin inhibitor, caused little change in the inhibitory activity, as measured by the inhibitor constant, although some altered the thermodynamic stability of the protein considerably. (1)H NMR investigations showed that the conformational stress caused by the replacement of Met103 with Gly, Ala, Val, and Ile, namely, the effects of the cavities generated by replacements with smaller side-chains and of the steric distortions generated by beta-branched side-chains, caused considerable changes in the structural arrangement of the side-chains within the core. However, these structural changes were absorbed within the hydrophobic core, without distorting the structure of the reactive site essential for the protein function. These results provide an excellent example of the conformational flexibility of a protein core and the degree of its tolerance of an amino acid replacement. The results also reveal the crucially designed structural relationship between the core of the inhibitor and the enzyme-binding segment with the reactive site in a serine protease inhibitor.

Methionine↗
PubMed1999

Subtilisin-like proprotein convertases, PACE4 and PC8, as well as furin, are endogenous proalbumin convertases in HepG2 cells.

Serum albumin is synthesized as a larger precursor form, proalbumin, which undergoes proteolytic processing at a dibasic site by a hepatic proprotein convertase within the secretory pathway to generate the mature form. Although furin, a member of the subtilisin-like proprotein convertase (SPC) family, was thought to be the only candidate hepatic convertase for proalbumin, SPC family members other than furin were recently suggested to also be involved in proalbumin processing. This study was designed to identify the endogenous proprotein convertases involved in proalbumin processing. Since human hepatoma HepG2 cells are highly differentiated and produce major plasma proteins, this cell line was used as a model for hepatocytes. Northern blot analysis revealed that PACE4, furin and PC8 of the SPC family were expressed in HepG2 cells as well as in the liver. Ribonuclease protection assay showed that PACE4A-II mRNA is the major transcript in HepG2 cells among the PACE4 isoforms. The coexpression studies showed that furin, PACE4A-II and PC8 were all able to convert proalbumin to albumin correctly. To elucidate the roles of these endogenous SPC family members in proalbumin processing, the antisense RNA for PACE4, furin and PC8 was stably expressed in HepG2 cells, respectively. The expression of each antisense RNA resulted in approximately 30% inhibition of endogenous proalbumin processing. We therefore concluded that PACE4 and PC8, as well as furin, are involved in the processing of proalbumin in HepG2 cells, and that these SPC family members are functionally redundant in this processing.

Furin↗
PubMed1999

Secondary structures and structural fluctuation in a dimeric protein, Streptomyces subtilisin inhibitor.

Based on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C(alpha) signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the beta3 strand is completely missing and the alpha2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative. Higher protection against hydrogen exchange for residues in part of the beta4 strand implies that this region might serve as a folding core.

Amino Acid Sequence↗
PubMed1994

Amino acid sequences of Kunitz family subtilisin inhibitors from seeds of Canavalia lineata.

The amino acid sequences of two subtilisin inhibitors (CLSI-II and -III) from Canavalia lineata seeds were determined by manual Edman degradation using the DABITC/PITC double coupling method after enzymatic digestions and CNBr degradation. CLSI-II and -III consist of 190 and 183 amino acids, respectively, and have identical amino acid sequences except for in the C-terminal regions: an elongated sequence, Gly-Thr-Ile-Arg- Ser-Asp-Gly, was found at the C-terminus of CLSI-II. A short-chain analog protein without an N-terminal Asn residue was also detected in each inhibitor preparation. The inhibitors showed significant homology to Kunitz type inhibitors, but differed from them with respect to the half-cystine content. Among five half-cystine residues present in CLSI-III, two disulfide bonds link Cys44 to Cys88 and Cys142 to Cys149, Cys106 being present as a free cysteine residue. Phenylglyoxal treatment abolished the inhibitory activity of CLSI-III, indicating the participation of an Arg residue in the interaction with the enzyme. The reactive-site peptide bond was deduced to be Arg68-Gly69.

Amino Acid Sequence↗
PubMed1975

Temperature-jump studies on benzeneboronic acid-serine protease interactions. Subtilisin and alpha-chymotrypsin.

The interactions of benzeneboronic acid (BBA) as a transition state analog with subtilisin (EC 3.4.21.4) and with alpha-chymotrypsin (EC 3.4.21.1) were investigated kinetically by the temperature-jump method using pH indicators. For both enzymes, the concentration dependence of the relaxation time was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow, unimolecular process. The possibility of a trigonal-tetrahedral interconversion of BBA at the active site of the enzyme is discussed.

Benzene↗
PubMed1977

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor.

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.

Bacterial Proteins↗
PubMed1978

Crystal structure of a protein proteinase inhibitor, SSI (Streptomyces subtilisin inhibitor), at 4 A resolution.

The crystal structure of a protein proteinase inhibitor, SSI (Streptomyces subtilisin inhibitor), which strongly inhibits bacterial alkaline proteinases specifically, was determined at 4 A resolution using four heavy-atom derivatives. The SSI molecule can be described as an ellipsoid of about 30 X 40 X 65 A composed of two identical subunits each having dimensions of about 35 X 25 X 40 A and a molecular weight of 11,483. The subunit has an extensive beta-sheet structure, but no long alpha-helices are present. Based on the binding sites of platinum reagents known to form coordination complexes with methionine, it is speculated that the P1 residue, Met 73, of the reactive site is at the protruding edge of the subunit. At the subunit-subunit interface, a beta-sheet of one subunit is stacked on top of the corresponding beta-sheet of the other subunit.

Bacterial Proteins↗
PubMed1980

Acid denaturation steps of Streptomyces subtilisin inhibitor. A proton magnetic resonance study of individual histidine environment.

Resonance positions and intensities of the C(2) protons of histidyl residues (His 43 His 106) were followed in the NMR spectrum of a protein, Streptomyces subtilisin inhibitor (MW 23,000), in the pH range 2-9 at 30 degrees C, in order to clarify the microenvironment of the individual histidyl residues and the acid denaturation processes of the protein. The large difference in the 1H-2H exchange rate between the C(2) protons of the two histidyl residues indicates that, whereas His 106 is well exposed to the solvent, His 43 is highly shielded from the solvent. Protonation of His 106 occurs in the pH range 5-9, giving a pKa of 6.0 +/- 0.1. In contrast, His 43 can not be protonated in the native conformation, but is protonation occurs simultaneously with the acid denaturation of the protein in a narrow pH range 2.7-3.6, with pHmid of transition at 3.25. On the other hand, the denaturation of the His 106 environment starts at pH 5.0 and finishes at pH 2.7 with pHmid of transition at 3.7. The denaturation transitions are reversible with pH for both His 43 and His 106, but the rates of these transitions are slow (less than 25 s-1). The Hill coefficient for the His 106 transition was found to be 0.9 +/- 0.1 in the pH range 3.5-5.0, but to be distinctively larger than unity below pH 3.5. On the other hand, the Hill coefficient for the His 43 transition was found to be 4.4 +/- 0.6 for the whole range of transition (pH 2.7-3.6). These results combined with available information about the crystal structure lead us to conclude that the transition of His 106 in the pH range 3.5-5.0 represents a local denaturation caused by the protonation of one neighboring residue, probably Glu 102, whereas the transition of His 43 occurs as a result of cooperative protonation of several residues probably including Phe 113 and Asp 52, and is a denaturation step accompanied by the destruction of the major hydrophobic core.

Bacterial Proteins↗
PubMed1980

Solvent accessibility and microenvironment in a bacterial protein proteinase inhibitor SSI (Streptomyces subtilisin inhibitor).

Solvent accessibility (Lee, B. & Richards, F.M. (1971) J. Mol. Biol. 55, 379-400) was calculated for each atom of a bacterial protein proteinase inhibitor SSI (Streptomyces subtilisin inhibitor) based on crystallographic coordinates. Mainly based on this information, various chemical and spectroscopic (UV, Raman, NMR) observations made on the microenvironments of cystines, methionines, tryptophan, histidines, and tyrosines of SSI in solution were evaluated. Crystallographic data and the latter two sets of data were mainly at least qualitatively consistent with each other. These data include (1) the conformation of the two disulfide bridges, (2) the flexibility of the three methionyl side chains, (3) the extent of exposure of the indole ring of a tryptophan, (4) the environment of the two histidines, (5) the environment of the tyrosines, and (6) the hydrogen-deuterium exchangeability of peptide NH's. However, the extents of exposure of tyrosines deduced by solvent perturbation UV difference spectroscopy were significantly larger than those based on solvent accessibility calculations. Possible reasons for this discrepancy are discussed.

Bacterial Proteins↗
PubMed1981

Exposure of aromatic residues of Streptomyces subtilisin inhibitor. A photo-CIDNP study.

Exposure of aromatic residues, Tyr 7, Tyr 75, Tyr 93, His 43, His 106, and Trp 8, was studied by laser-induced photo-CIDNP in the 1H NMR spectrum of Streptomyces subtilisin inhibitor at 360 MHz. Only Tyr 7 and Tyr 75 gave strong CIDNP signals, whereas the rest of the aromatic residues gave no detectable signals in the temperature range 25-55 degrees C. From the temperature dependence data, it is concluded that Tyr 7 is well exposed at all temperatures, whereas the exposure of Tyr 75 increases with temperature, in agreement with the conclusion obtained by other methods. Agreements and discrepancies between the conclusions derived from the CIDNP data and the results so far obtained by other methods are compared for all the aforementioned aromatic residues.

Bacterial Proteins↗