Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.64 (proteinase K)
4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Lass (longevity-assurance homologue) family members, which are highly conserved among eukaryotes, function in ceramide synthesis. In the mouse, there are at least five Lass family members, Lass1, Lass2, Lass4, Lass5 and the hitherto uncharacterized Lass6. To investigate specific roles for each Lass member in ceramide synthesis, we cloned these five mouse proteins. Overproduction of any Lass protein in cultured cells resulted in an increase in cellular ceramide, but the ceramide species produced varied. Overproduction of Lass1 increased C18:0-ceramide levels preferentially, and overproduction of Lass2 and Lass4 increased levels of longer ceramides such as C22:0- and C24:0-ceramides. Lass5 and Lass6 produced shorter ceramide species (C14:0- and C16:0-ceramides); however, their substrate preferences towards saturated/unsaturated fatty acyl-CoA differed. In addition to differences in substrate preferences, we also demonstrated by Northern blotting that Lass family members are differentially expressed among tissues. Additionally, we found that Lass proteins differ with regard to glycosylation. Of the five members, only Lass2, Lass5 and Lass6 were N-glycosylated, each at their N-terminal Asn residue. The occurrence of N-glycosylation of some Lass proteins provides topological insight, indicating that the N-termini of Lass family members probably face the luminal side of the endoplasmic reticulum membrane. Furthermore, based on a proteinase K digestion assay, we demonstrated that the C-terminus of Lass6 faces the cytosolic side of the membrane. From these data we propose topology for the conserved Lag1 motif in Lass family members, namely that the N-terminal region faces the luminal side and the C-terminal region the cytosolic side of the endoplasmic reticulum membrane.
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PMID:Mammalian Lass6 and its related family members regulate synthesis of specific ceramides. 1582 95

Gangliosides have been shown to be plasma membrane receptors for both murine polyomavirus and SV40, while JC virus uses serotonin receptors. In contrast, little is known of the membrane receptor and entry pathway for BK virus (BKV), which can cause severe disease in immunosuppressed bone marrow and renal transplant patients. Using sucrose flotation assays, we investigated BKV binding to and interaction with human erythrocyte membranes and determined that this interaction was dependent on a neuraminidase-sensitive, proteinase K-resistant molecule. BKV was found to interact with the gangliosides GT1b and GD1b. The terminal alpha2-8-linked disialic acid motif, present in both of these gangliosides, is likely to be important for this interaction. We also determined that the addition of GD1b and GT1b to LNCaP cells, which are normally resistant to BKV infection, made them susceptible to the virus. In addition, BKV interacted with membranes extracted from the endoplasmic reticulum (ER) and infection was blocked by the addition of brefeldin A, which interferes with transport from the ER to the Golgi apparatus. These data demonstrate that BKV uses the gangliosides GT1b and GD1b as receptors and passes through the ER on the way to the nucleus.
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PMID:Identification of gangliosides GD1b and GT1b as receptors for BK virus. 1641 13

Inherited prion diseases are neurodegenerative pathologies related to genetic mutations in the prion protein (PrP) gene, which favour the conversion of PrP(C) into a conformationally altered pathogenic form, PrP(Sc). The molecular basis of PrP(C)/PrP(Sc) conversion, the intracellular compartment where it occurs and how this process leads to neurological dysfunction are not yet known. We have studied the intracellular synthesis, degradation and localization of a PrP mutant associated with a genetic form of Creutzfeldt-Jakob disease (CJD), PrPT182A, in transfected FRT cells. PrPT182A is retained in the endoplasmic reticulum (ER), is mainly associated with detergent-resistant microdomains (DRMs) and is partially resistant to proteinase K digestion. Although an untranslocated form of this mutant is polyubiquitylated and undergoes ER-associated degradation, the proteasome is not responsible for the degradation of its misfolded form, suggesting that it does not have a role in the pathogenesis of inherited diseases. On the contrary, impairment of PrPT182A association with DRMs by cholesterol depletion leads to its accumulation in the ER and substantially increases its misfolding. These data support the previous hypothesis that DRMs are important for the correct folding of PrP and suggest that they might have a protective role in pathological scrapie-like conversion of PrP mutants.
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PMID:Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum. 1644 48

The membrane-bound protein complex gamma-secretase is an intramembranous protease whose substrates are a number of type I transmembrane proteins including the beta-amyloid precursor protein (APP). A presenilin molecule is thought to be the catalytic unit of gamma-secretase and either of two presenilin homologues, PS1 or PS2, can play this role. Mutations in the presenilins, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer's disease. To look for possible molecular heterogeneity in presenilin/gamma-secretase we examined the ability of proteinase K (PK) to digest endogenously expressed presenilins in intact endoplasmic reticulum vesicles. We demonstrate the existence of two physically different forms of gamma-secretase-associated PS1, one that is relatively PK-sensitive and one that is significantly more PK-resistant. A similarly PK-resistant form of PS2 was not observed. We speculate that the structural heterogeneity we observe may underlie, at least in part, previous observations indicating the physical and functional heterogeneity of gamma-secretase. In particular, our results suggest that there are significant differences between gamma-secretase complexes incorporating PS1 and PS2. This difference may underlie the more dominant role of PS1 in the generation of beta-amyloid peptides and in familial Alzheimer's disease.
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PMID:Protease digestion indicates that endogenous presenilin 1 is present in at least two physical forms. 1675 46

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.
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PMID:Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis. 1680 41

Cotranslational translocation of apoB100 across the endoplasmic reticulum (ER) membrane is inefficient, resulting in exposure of nascent apoB on the cytosolic surface of the ER. This predisposes apoB100 to ubiquitinylation and targeting for proteasomal degradation. It has been suggested that pause transfer sequences (PTS) present throughout apoB cause inefficient translocation. On the other hand, our previous study demonstrated that the translocation efficiency of apoB100 is dependent on the presence of a beta-sheet domain between 29 and 34% of full-length apoB100 (Liang, J.-S., Wu, X., Jiang, H., Zhou, M., Yang, H., Angkeow, P., Huang, L.-S., Sturley, S. L., and Ginsberg, H. N. (1998) J. Biol. Chem. 273, 35216-35221); this region of apoB has no PTS. However, the effects of the beta-sheet domain may require the presence of PTS elsewhere in the N-terminal region of apoB100. To further investigate the roles of PTS and beta-sheet domains in the translocation of apoB100 across the ER, we transfected McArdle RH7777, HepG2, or Chinese hamster ovary cells with human albumin (ALB)/human apoB chimeric cDNA constructs: ALB/B12-17 (two PTS but no beta-sheet), ALB/B29-34 (beta-sheet but no PTS), ALB/B36-41 (two PTS and a beta-sheet), and ALB/B49-54 (neither PTS nor a beta-sheet). ALB/ALB1-40 served as a control. Compared with ALB/ALB1-40, secretion rates of ALB/B12-17, ALB/B29-34, and ALB/B36-41 were reduced. Secretion of ALB/B49-54 was similar to that of ALB/ALB1-40. However, only ALB/B29-34 and ALB/B36-41 had increased proteinase K sensitivity, ubiquitinylation, and increased physical interaction with Sec61alpha. These results indicate that the translocation efficiency of apoB100 is determined mainly by the presence of beta-sheet domains. PTS do not appear to affect translocation, but may affect secretion by other mechanisms.
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PMID:Translocation efficiency of apolipoprotein B is determined by the presence of beta-sheet domains, not pause transfer sequences. 1685 91

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

We identified and characterized a novel RING finger gene, Rines/RNF180, which is well conserved among vertebrates. Putative Rines gene product (Rines) contains a RING finger domain, a basic coiled-coil domain, a novel conserved domain (DSPRC) and a C-terminal hydrophobic region that is predicted to be a transmembrane domain. N-terminally epitope tagged-Rines (Nt-Rines) was detected in the endoplasmic reticulum membrane/nuclear envelope in cultured mammalian cells. Nt-Rines was not extracted by high salt or alkaline buffers and was degraded in intact endoplasmic reticulum treated with proteinase K, indicating that Nt-Rines is an integral membrane protein with most of its N-terminal regions in the cytoplasm. Rines was expressed in brain, kidney, testis and uterus of adult mice, and in developing lens and brain, particularly in the ventricular layer of the cerebral cortex at embryonic stages. In cultured cells, Nt-Rines can bind another protein and promoted its degradation. The degradation was inhibited by proteasomal inhibitors. In addition, Nt-Rines itself was heavily ubiquitinated and degraded by proteasome. The involvement of Rines in the ubiquitin-proteasome pathway was further supported by its binding to the UbcH6 ubiquitin-conjugating enzyme and by its trans-ubiquitination enhancing activities. These results suggest that Rines is a membrane-bound E3 ubiquitin ligase.
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PMID:Rines/RNF180, a novel RING finger gene-encoded product, is a membrane-bound ubiquitin ligase. 1836 70

Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.
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PMID:Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406. Structural importance of the crown domain. 1842 67

The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation.
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PMID:Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts. 1900 Jul 18


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