Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cryptosporidium parvum is an enteric protozoan parasite of medical and veterinary importance. Dissemination of environmentally resistant oocysts in surface water plays an important role in the epidemiology of cryptospridiosis. Although the polymerase chain reaction (PCR) is a well-established technique and is widely used for detecting microorganisms, it is not routinely applied for monitoring waterborne C. parvum. In order to facilitate the application of PCR to the detection of waterborne C. parvum oocysts, a comparison of published PCR protocols was undertaken and different sample-preparation methods tested. The sensitivity of a one-step PCR method, consisting of 40 temperature cycles, was 10 purified oocysts or fewer than 100 oocysts spiked in raw lake water. The detection limit of two primer pairs, one targeting the ribosomal small subunit and another specific for a C. parvum sequence of unknown function, was approximately ten-fold lower than achieved with a primer pair targeting an oocyst shell protein gene. Three cycles of freezing/thawing were sufficient to expose oocyst DNA and resulted in higher sensitivity than proteinase K digestion, sonication or electroporation. Inhibition of PCR by surface water from different local sources was entirely associated with the soluble fraction of lake water. Membrane filtration was evaluated in bench-scale experiments as a means of removing lake water inhibitors and improving the detection limit of PCR. Using gel and membrane filtration, the molecular size of inhibitory solutes from lake water was estimated to less than 27 kDa.
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PMID:Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts. 935 75

Immunoglobin binding protein (BiP) molecules exist as both monomers and oligomers and phosphorylated BiP is restricted to the oligomeric pool. Modified BiP is not bound to proteins such as immunoglobulin heavy chain and consequently, may constitute an inactive form. Unlike earlier analysis of mammalian BiP isolated by two-dimensional gel electrophoresis, results here demonstrated that immunoprecipitated BiP displayed predominantly threonine phosphorylation with only a trace of detectable phosphoserine. Like other Hsp70 family members, BiP is comprised of three domains: an amino terminal domain which binds nucleotide, an 18 kilodalton domain which binds peptide, and a carboxyl terminal variable domain of unknown function. Cyanogen bromide cleavage and enzymatic digestion experiments mapped threonine phosphorylation to a site within a 47 amino acid sequence of the peptide binding domain which contains seven threonine residues. Partial proteinase K digestion in the presence of ATP independently verified that the in vivo phosphorylation site of mammalian (BiP) is located within the peptide binding domain. Furthermore, phosphorylation did not impede BiPs ATP-induced conformational change. Thus, the peptide binding domain of BiP is phosphorylated on threonine residue(s) mapping to not more than two tryptic fragments within the peptide binding domain. This location on the molecule could explain why phosphorylated BiP is not detected bound to proteins in vivo.
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PMID:In vivo threonine phosphorylation of immunoglobulin binding protein (BiP) maps to its protein binding domain. 949 82

The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP(C)) form, it is a soluble, alpha-helix-rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, "scrapie" form (PrP(Sc)), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in beta-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the alpha-helix-rich to the beta-sheet-rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is approximately 7000 times more efficient than dithiothreitol.
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PMID:Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form. 1116 30

MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.
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PMID:Characterization of the intracellular proteolytic cleavage of myocilin and identification of calpain II as a myocilin-processing protease. 1765 May 8

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.
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PMID:Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae. 1765 41

Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.
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PMID:Rv1698 of Mycobacterium tuberculosis represents a new class of channel-forming outer membrane proteins. 1843 14

The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis.
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PMID:Identification of outer membrane proteins of Mycobacterium tuberculosis. 1843 72

Fluid shear modulates many biological properties. How shear mechanosensing occurs in the extracellular matrix (ECM) and is transduced into cytoskeletal change remains unknown. Cochlin is an ECM protein of unknown function. Our investigation using a comprehensive spectrum of cutting-edge techniques has resulted in following major findings: (1) over-expression and down-regulation of cochlin increase and decrease intraocular pressure (IOP), respectively. The overexpression was achieved in DBA/2J-Gpnmb(+)/SjJ using lentiviral vectors, down-regulation was achieved in glaucomatous DBA/2J mice using targeted disruption (cochlin-null mice) and also using lentiviral vector mediated shRNA against cochlin coding region; (2) reintroduction of cochlin in cochlin-null mice increases IOP; (3) injection of exogenous cochlin also increased IOP; (4) increasing perfusion rates increased cochlin multimerization, which reduced the rate of cochlin proteolysis by trypsin and proteinase K; The cochlin multimerization in response to shear stress suggests its potential mechanosensing. Taken together with previous studies, we show cochlin is involved in regulation of intraocular pressure in DBA/2J potentially through mechanosensing of the shear stress.
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PMID:Cochlin, intraocular pressure regulation and mechanosensing. 2249 87

Flagella are multiprotein complexes necessary for swimming and swarming motility. In Salmonella enterica serovar Typhimurium, flagella-mediated motility is repressed by the PhoP/PhoQ regulatory system. We now report that Salmonella can move on 0.3% agarose media in a flagella-independent manner when experiencing the PhoP/PhoQ-inducing signal low Mg(2+). This motility requires the PhoP-activated mgtA, mgtC, and pagM genes, which specify a Mg(2+) transporter, an inhibitor of Salmonella's own F1Fo ATPase, and a small protein of unknown function, respectively. The MgtA and MgtC proteins are necessary for pagM expression because pagM mRNA levels were lower in mgtA and mgtC mutants than in wild-type Salmonella, and also because pagM expression from a heterologous promoter rescued motility in mgtA and mgtC mutants. PagM promotes group motility by a surface protein(s), as a pagM-expressing strain conferred motility upon a pagM null mutant, and proteinase K treatment eliminated motility. The pagM gene is rarely found outside subspecies I of S. enterica and often present in nonfunctional allelic forms in organisms lacking the identified motility. Deletion of the pagM gene reduced bacterial replication on 0.3% agarose low Mg(2+) media but not in low Mg(2+) liquid media. Our findings define a form of motility that allows Salmonella to scavenge nutrients and to escape toxic compounds in low Mg(2+) semisolid environments.
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PMID:Flagella-independent surface motility in Salmonella enterica serovar Typhimurium. 2562 75

Pathogenic Leptopira is the etiological agent of leptospirosis, the most widespread zoonotic infection in the world. The disease represents a major public health problem, especially in tropical countries. The present work focused on two hypothetical proteins of unknown function, encoded by the genes LIC13059 and LIC10879, and predicted to be surface-exposed proteins. The genes were cloned and the proteins expressed using E. coli as a host system. We report that the recombinant proteins interacted with extracellular matrix (ECM) laminin, in a dose-dependent fashion and are novel potential adhesins. The recombinant proteins were called Lsa25.6 (rLIC13059) and Lsa16 (rLIC10879), for Leptospiral surface adhesins, followed by the respective molecular masses. The proteins attached to plasminogen (PLG), generating plasmin, in the presence of PLG-activator uPA. Both proteins bind to fibrinogen (Fg), but only Lsa25.6 inhibited fibrin clotting by thrombin-catalyzed reaction. Moreover, Lsa16 interacts with the mammalian cell receptor E-cadherin, and could contribute to bacterial attachment to epithelial cells. The proteins were recognized by confirmed leptospirosis serum samples, suggesting that they are expressed during infection. The corresponding leptospiral proteins are surface exposed based on proteinase K accessibility assay, being LIC10879 most probably exposed in its dimer form. The data of this study extend the spectrum of surface-exposed proteins of L. interrogans and indicate a possible role of the originally annotated hypothetical proteins in infection processes.
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PMID:Multifunctional and Redundant Roles of Leptospira interrogans Proteins in Bacterial-Adhesion and fibrin clotting inhibition. 2860 Jan 23


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