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)

In infectious and familial prion disorders, neurodegeneration is often seen without obvious deposits of the scrapie prion protein (PrP(Sc)), the principal cause of neuronal death in prion disorders. In such cases, neurotoxicity must be mediated by alternative pathways of cell death. One such pathway is through a transmembrane form of PrP. We have investigated the relationship between intracellular accumulation of prion protein aggregates and the consequent up-regulation of transmembrane prion protein in a cell model. Here, we report that exposure of neuroblastoma cells to the prion peptide 106-126 catalyzes the aggregation of cellular prion protein to a weakly proteinase K-resistant form and induces the synthesis of transmembrane prion protein, the proposed mediator of neurotoxicity in certain prion disorders. The N terminus of newly synthesized transmembrane prion protein is cleaved spontaneously on the cytosolic face of the endoplasmic reticulum, and the truncated C-terminal fragment accumulates on the cell surface. Our results suggest that neurotoxicity in prion disorders is mediated by a complex pathway involving transmembrane prion protein and not by deposits of aggregated and proteinase K-resistant PrP alone.
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PMID:Prion peptide 106-126 modulates the aggregation of cellular prion protein and induces the synthesis of potentially neurotoxic transmembrane PrP. 1168 69

A common T17A polymorphism in the signal peptide of the cytotoxic T-lymphocyte antigen 4 (CTLA-4), a T-cell receptor that negatively regulates immune responses, is associated with risk for autoimmune disease. Because the polymorphism is absent from the mature protein, we hypothesized that its biological effect must involve early stages of protein processing, prior to signal peptide cleavage. Constructs representing the two alleles were compared by in vitro translation, in the presence of endoplasmic reticulum membranes. We studied glycosylation by endoglycosidase H digestion and glycosylation mutant constructs, cleavage of peptide with inhibitors, and membrane integration by ultracentrifugation and proteinase K sensitivity. A major cleaved and glycosylated product was seen for both alleles of the protein but a band representing incomplete glycosylation was markedly more abundant in the predisposing Ala allele (32.7 +/- 1.0 versus 10.6% +/- 1.2 for Thr, p < 10(-9)). In addition, differential intracellular/surface partitioning was studied with co-transfection of the alleles fused to distinct fluorescent proteins in COS-1 cells. By quantitative confocal microscopy we found a higher ratio of cell surface/total CTLAThr(17) versus CTLAAla(17) (p = 0.01). Our findings corroborate observations, in other proteins, that the signal peptide can determine the efficiency of post-translational modifications other than cleavage and suggest inefficient processing of the autoimmunity predisposing Ala allele as the explanation for the genetic effect.
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PMID:A common autoimmunity predisposing signal peptide variant of the cytotoxic T-lymphocyte antigen 4 results in inefficient glycosylation of the susceptibility allele. 1224 7

We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ER in vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chains in vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.
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PMID:Protein unfolding is not a prerequisite for endoplasmic reticulum-to-cytosol dislocation. 1248 53

Signal peptides (SPs) direct nascent secretory and membrane proteins to the membrane of the endoplasmic reticulum. They are usually cleaved from the nascent polypeptide by signal peptidase and then further proteolytically processed. The SP of the pre-glycoprotein (pGP-C) of the lymphocytic choriomeningitis virus SPGP-C (signal peptide of pGP-C) shows different properties: 1) The SPGP-C is unusually long (58 amino acid residues) and contains two hydrophobic segments interrupted by a lysine residue. 2) The SPGP-C is cleaved only from a subset of pGP-C proteins. A substantial portion of pGP-C accumulates that still contains the SPGP-C.3)The cleaved SPGP-C is rather long-lived (t(1/2) of more than 6 h). 4) The cleaved SPGP-C resides in the membrane and is resistant to digestion with proteinase K even in the presence of detergents, suggesting a very compact structure. 5) SPGP-C accumulates in virus particles. These unusual features of the cleaved SPGP-C suggest that SPGP-C not only targets the nascent pGP-C to the endoplasmic reticulum membrane but also has additional functions in lymphocytic choriomeningitis virus life cycle.
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PMID:Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C. 1291 26

Recently, it was observed that reverse-translocated cytosolic PrP and PrP expressed in the cytosol induce rapid death in neurons (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). In this study, we investigated whether accumulation of prion protein (PrP) in the cytosol is toxic to human neurons in primary culture. We show that in these neurons, a single PrP isoform lacking signal peptide accumulates in the cytosol of neurons treated with epoxomicin, a specific proteasome inhibitor. Therefore, endogenously expressed PrP is subject to the endoplasmic reticulum-associated degradation (ERAD) pathway and is degraded by the proteasome in human primary neurons. In contrast to its toxicity in N2a cells, reverse-translocated PrP (ERAD-PrP) is not toxic even when neurons are microinjected with cDNA constructs to overexpress either wild-type PrP or mutant PrPD178N. We found that ERAD-PrP in human neurons remains detergent-soluble and proteinase K-sensitive, in contrast to its detergent-insoluble and proteinase K-resistant state in N2a cells. Furthermore, not only is microinjection of a cDNA construct expressing CyPrP not toxic, it protects these neurons against Bax-mediated cell death. We conclude that in human neurons, ERAD-PrP is not converted naturally into a form reminiscent of scrapie PrP and that PrP located in the cytosol retains its protective function against Bax. Thus, it is unlikely that simple accumulation of PrP in the cytosol can cause neurodegeneration in prion diseases.
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PMID:Cytosolic prion protein is not toxic and protects against Bax-mediated cell death in human primary neurons. 1291 44

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB-VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB-VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB-VPg polyprotein acts as a membrane-anchor for the replication complex.
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PMID:Topogenesis in membranes of the NTB-VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain. 1476 10

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat-treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two-step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K-digested ovalbumin, and the binding of calreticulin to proteinase K-digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide-binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen-like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein-scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.
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PMID:Interaction of C1q with the receptor calreticulin requires a conformational change in C1q. 1514 59

Sphingolipids are essential membrane components of eukaryotic cells. Their synthesis is initiated with the condensation of l-serine with palmitoyl-CoA, producing 3-ketodihydrosphingosine (KDS), followed by a reduction to dihydrosphingosine by KDS reductase. Until now, only yeast TSC10 has been identified as a KDS reductase gene. Here, we provide evidence that the human FVT-1 (hFVT-1) and mouse FVT-1 (mFVT-1) are functional mammalian KDS reductases. The forced expression of hFVT-1 or mFVT-1 in TSC10-null yeast cells suppressed growth defects, and hFVT-1 overproduced in cultured cells exhibited KDS reductase activity in vitro. Moreover, purified recombinant hFVT-1 protein exhibited NADPH-dependent KDS reductase activity. The identification of the FVT-1 genes enabled us to characterize the mammalian KDS reductase at the molecular level. Northern blot analyses demonstrated that both hFVT-1 and mFVT-1 mRNAs are ubiquitously expressed, suggesting that FVT-1 is a major KDS reductase. We also found the presence of hFVT-1 variants, which were differentially expressed among tissues. Immunofluorescence microscopic analysis revealed that hFVT-1 is localized at the endoplasmic reticulum. Moreover, a proteinase K digestion assay revealed that the large hydrophilic domain of hFVT-1, which contains putative active site residues, faces the cytosol. These results suggest that KDS is converted to dihydrosphingosine in the cytosolic side of the endoplasmic reticulum membrane. Moreover, the topology studies provide insight into the spatial organization of the sphingolipid biosynthetic pathway.
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PMID:FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that faces the cytosolic side of the endoplasmic reticulum membrane. 1532 38

High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11beta-hydroxysteroid dehydrogenase (11beta-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11beta-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.
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PMID:Elements in the N-terminal signaling sequence that determine cytosolic topology of short-chain dehydrogenases/reductases. Studies with retinol dehydrogenase type 1 and cis-retinol/androgen dehydrogenase type 1. 1535 69

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10-29, 101-123, and 127-149, and membrane-integration of residues 75-97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.
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PMID:Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation. 1571 79


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