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1.
Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 alpha chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients. 相似文献
2.
Pedersen CB Bross P Winter VS Corydon TJ Bolund L Bartlett K Vockley J Gregersen N 《The Journal of biological chemistry》2003,278(48):47449-47458
Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding, and preliminary experiments suggested that the variant protein displayed prolonged association with chaperonins and delayed formation of active enzyme. Accordingly, the molecular pathogenesis of SCAD deficiency may rely on intramitochondrial protein quality control mechanisms, including degradation and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate with mitochondrial hsp60 chaperonins; however, the variant SCAD proteins remained associated with hsp60 for prolonged periods of time. Biogenesis experiments at two temperatures revealed that some of the variant proteins (R22W, G68C, W153R, and R359C) caused severe misfolding, whereas others (R147W, G185S, and Q341H) exhibited a less severe temperature-sensitive folding defect. Based on the magnitude of in vitro defects, these SCAD proteins are characterized as folding-defective variants and mild folding variants, respectively. Pulse-chase experiments demonstrated that the variant SCAD proteins either triggered proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency. 相似文献
3.
Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli. 相似文献
4.
5.
Treatment of the W7MG1 mouse T lymphoma cell line with glucocorticoid stimulates directly or indirectly two observable steps in the processing of mouse mammary tumor virus (MMTV) envelope glycoprotein precursor Pr74: cleavage of Pr74 to yield the mature glycoprotein products gp52 and gp33, and processing of the N-linked oligosaccharides to endoglycosidase H (endo H)-resistant forms found on the mature products but not on the precursor. Therefore, the primary hormone-regulated event in this pathway must occur at or before the point where MMTV envelope proteins become endo H resistant. Pulse-chase analyses identified a novel endo H-resistant 80-kDa species (designated gp80) as a processing intermediate. Therefore, in contrast to conclusions drawn for the envelope proteins of several other retroviruses, proteolytic cleavage of MMTV envelope proteins occurs after acquisition of endo H resistance. Also, proteolytic cleavage cannot be the primary hormone-regulated step. Second, inhibition of mannosidase II by the drug swainsonine did not prevent Pr74 from being proteolytically processed, thus demonstrating that conversion of oligosaccharide chains from endo H-sensitive to -resistant forms was not a prerequisite for proteolytic cleavage. Therefore, the requisite hormone-regulated event in MMTV glycoprotein processing must precede both acquisition of endo H resistance and proteolytic cleavage. This places the regulated event in the endoplasmic reticulum or early Golgi. 相似文献
6.
Robert T. Sauer 《Protein science : a publication of the Protein Society》2013,22(11):1675-1687
Understanding the relationship between the amino‐acid sequence of a protein and its ability to fold and to function is one of the major challenges of protein science. Here, cases are reviewed in which mutagenesis, biochemistry, structure determination, protein engineering, and single‐molecule biophysics have illuminated the sequence determinants of folding, binding specificity, and biological function for DNA‐binding proteins and ATP‐fueled machines that forcibly unfold native proteins as a prelude to degradation. In addition to structure‐function relationships, these studies provide information about folding intermediates, mutations that accelerate folding, slow unfolding, and stabilize proteins against denaturation, show how new binding specificities and folds can evolve, and reveal strategies that proteolytic machines use to recognize, unfold, and degrade thousands of distinct substrates. 相似文献
7.
8.
Several proteolytic enzymes have been studied with regard to their ability to induce DNA synthesis and cell proliferation in resting chick embryo fibroblasts. Of the enzymes examined, thrombin, bromelin, and trypsin exhibit potent mitogenic activity, elastase has significant but less marked activity, whereas thermolysin, papain, and α-protease are inactive. The enzymes were also tested for their ability to induce morphological change or to remove two iodinatable proteins of 250,000 and 205,000 daltons. Although the larger protein is removed by some but not all of the proteases examined, every protease tested removed the smaller cell surface protein. The ability of proteases to stimulate cell growth could not be correlated directly with removal of either of these cell surface proteins; however, loss of the smaller protein does correlate with the reduction of both cytoplasmic spreading and cell-cell interactions observed after protease treatment. A secondary, later event of migration of cells into clumps is observed in those instances when protease treatment did not result in a loss of the 250K protein. A role for each of these proteins in the processes of cellular adhesion is discussed. 相似文献
9.
Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. 总被引:21,自引:16,他引:5
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To study the role of specific regions of the yellow fever virus NS2B protein in proteolytic processing and association with the NS3 proteinase domain, a series of mutations were created in the hydrophobic regions and in a central conserved hydrophilic region proposed as a domain important for NS2B function. The effects of these mutations on cis cleavage at the 2B/3 cleavage site and on processing at other consensus cleavage sites for the NS3 proteinase in the nonstructural region were then characterized by cell-free translation and transient expression in BHK cells. Association between NS2B and the NS3 proteinase domain and the effects of mutations on complex formation were investigated by nondenaturing immunoprecipitation of these proteins expressed in infected cells, by cell-free translation, or by recombinant vaccinia viruses. Mutations within the hydrophobic regions had subtle effects on proteolytic processing, whereas mutations within the conserved domain dramatically reduced cleavage efficiency or abolished all cleavages. The conserved domain of NS2B is also implicated in formation of an NS2B-NS3 complex on the basis of the ability of mutations in this region to eliminate both association of these two proteins and trans-cleavage activity. In addition, mutations which either eliminated proteolytic processing or had no apparent effect on processing were found to abolish recovery of infectious virus following RNA transfection. These results suggest that the conserved region of NS2B is a domain essential for the function of the NS3 proteinase. Hydrophobic regions of NS2B whose structural integrity may not be essential for proteolytic processing may have additional functions during viral replication. 相似文献
10.
Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain 总被引:19,自引:0,他引:19
Molecular genetic evidence indicates that there should be three different (Na+ + K+)-stimulated ATPase (Na,K-ATPase) alpha subunit isozymes in the brain where previously only two ("alpha" and "alpha(+)") were resolved as proteins. To detect and identify alpha 1, alpha 2, and alpha 3 isozymes, polypeptides made by cell-free translation (Schneider, J.W., Mercer, R.W., Gilmore-Hebert, M., Utset, M.F., Lai, C., Greene, A., and Benz, E.J., Jr. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 284-288) were analyzed by gel electrophoresis and proteolytic fingerprinting. Synthetic alpha 1 comigrated with tissue alpha 1, while alpha 2 and alpha 3 comigrated with the leading and trailing edges, respectively, of tissue "alpha(+)." Proteolytic fingerprints of newborn rat brain Na,K-ATPase labeled in vivo with L-[35S]methionine indicated the presence of alpha 1 and alpha 3, and a low level of alpha 2. Monoclonal antibodies were characterized by the electrophoretic mobility of their antigens and by their ability to recognize the Na,K-ATPases of kidney, brain, and skeletal muscle. The antibodies were used to assess isozyme expression in the brain. All three isozymes increased in abundance during development from the 18-day fetus to the adult. Small changes were seen in the relative level of expression of alpha 1 and alpha 3 at different developmental ages, while alpha 2 expression increased markedly between the neonate and adult. In adult brain, all three isozymes were found in all brain regions examined. We conclude that all three isozymes are expressed as proteins and that their expression and distribution must be under complex control. No single developmental age or macroscopic brain region provides an exclusive source of any of the isozymes. 相似文献
11.
The role of proteolysis in the beer chill-proofing action of the proteolytic enzyme papain (EC 3.4.22.2) has been investigated by comparing the chill-proofing ability of papain with that of a proteolytically inactive derivative, S-carboxymethylpapain. The latter was prepared by treating papain with bromoacetic acid to carboxymethylate selectively the single essential sulphydryl group of the enzyme, that of l-cysteine-25. Both papain and S-carboxymethylpapain were found to exhibit increasing chill-proofing ability with increasing concentration in beer; at a protein concentration in beer of 30 μg/ml the chill-proofing effect of each protein proved to be substantial. Papain, either in the presence or absence of sodium bisulphite, was, however, found to be more effective than S-carboxymethylpapain at all protein concentrations. It is concluded that the chill-proofing action of papain originates largely, but not wholly, from its proteolytic action. Similarly, the chill-proofing ability of the proteolytic enzyme chymotrypsin (EC 3.4.21.1) has been compared with that of its proteolytically inactive zymogen, chymotrypsinogen A. Both proteins were found to exhibit increasing chill-proofing ability with increasing concentration in beer. The chill-proofing effect of chymotrypsin was, however, found to be greater than that of chymotrypsinogen A at all protein concentrations in beer. On the basis of these results and the close similarities in the molecular structures of chymotrypsin and chymotrypsinogen A, it is concluded that the chill-proofing action of chymotrypsin also originates largely, but not wholly, in its proteolytic action. The results from this study collectively demonstrate that no straightforward correlation exists between the proteolytic activity added to beer and its resistance to chill-haze formation. 相似文献
12.
Allen Taylor Joseph J. Berger John Reddan Antoinette Zuliani 《In vitro cellular & developmental biology. Animal》1991,27(4):287-292
Summary Alterations in proteolytic capabilities have been associated with abnormalities in the aged eye lens, but in vivo tests of
this hypothesis have been difficult to pursue. To simulate aging, we cultured cells from an 8-yr-old rabbit to early (population-doubling
level 20 to 30) and late (population-doubling level > 125) passage. Long-lived (t1/2>10 h) and short-lived (t1/2<10 h) intracellular proteins were labeled with [3H]leucine, and the ability of the cells to mount a proteolytic response to the stress of serum withdrawal was determined.
For early passage cells, the average t1/2 of long-lived proteins in the presence and absence of serum was 62 and 39 h, respectively. For late-passage cells, the average
t1/2 of long-lived proteins in the presence and absence of serum was 58 and 43 h, respectively. The net increase in intracellular
proteolysis in the absence of serum was 59 and 35% for early and late-passage cells, respectively. Thus, in vitro-aged rabbit
lens epithelial cells mount only 60% the proteolytic response to serum removal shown in “younger” cells. The enhanced ability
of early passage cells to respond to serum removal seems to involve lower homeostatic levels of proteolysis in the presence
of serum and greater enhancement of proteolysis in the absence of serum. Less than 2% of the protein is in the pool of short-lived
proteins. Rates of proteolysis of short-lived proteins in the presence and absence of serum were indistinguishable. With respect
to basal proteolytic rates in the presence of serum and ability to mount a proteolytic response upon serum withdrawal, these
rabbit lens epithelial cells are similar to bovine lens epithelial cells and fibroblasts.
This work was supported in part by contract 53-3K06-5-10 U.S. Department of Agriculture, Washington, DC, Massachusetts Lions
Eye Research FUnd, Inc., the Daniel and Florence Guggenheim Foundation, and a grant EY00362 from the National Eye Institute,
Bethesda, MD. 相似文献
13.
Proteolytic Enzyme Preparation from Pseudomonas fragi: Its Action on Pig Muscle 总被引:2,自引:1,他引:1
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P. J. V. Tarrant Nadine Jenkins A. M. Pearson T. R. Dutson 《Applied microbiology》1973,25(6):996-1005
An extracellular preparation from Pseudomonas fragi with proteolytic enzyme activity was isolated, and its action on meat proteins and meat protein ultrastructure was studied. First, a suitable growth medium for proteolytic enzyme production was determined, and a method for partial purification of the proteolytically active fraction was developed. The enzyme preparation displayed optimal proteolytic activity at neutral pH and 35 C. Proteolytic activity was irreversibly lost by mild heat treatment. The enzyme preparation was tested for its ability to hydrolyze isolated pig muscle proteins. Myofibrillar protein was rapidly degraded, G-actin and myosin were broken down at a slower rate, and the sarcoplasmic proteins were least susceptible to hydrolysis. Electron micrographs of pork muscle showed that the proteolytic enzyme preparation caused a complete loss of dense material from the Z line. Similarities are discussed between the action of P. fragi extracellular proteolytic enzyme(s) on meat and normal bacterial spoilage of meat. 相似文献
14.
The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins. 总被引:5,自引:0,他引:5
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Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins. 相似文献
15.
Considerable salt-soluble protein degradation was observed in pork muscle inoculated with Pseudomonas fragi. During a 20-day incubation period at 10 C, the samples proceeded to rank spoilage or putrefaction. There was a large decrease in the salt-soluble protein fraction and a corresponding increase in nonprotein nitrogen. Disc gel electrophoretic patterns showed that breakdown of the salt-soluble proteins had occurred after incubation for 20 days. During incubation for 10 days at 10 C, P. fragi produced large amounts of extracellular proteolytic activity in ground pork. Most of the proteolytic activity appeared immediately after spoilage occurred. However, a significant increase in the ability to hydrolyze casein and a slight increase in the ability to hydrolyze denatured hemoglobin occurred prior to spoilage. 相似文献
16.
The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation. 相似文献
17.
Jeroen Hugenholtz Douwe van Sinderen Jan Kok Wil N. Konings 《Applied microbiology》1987,53(4):853-859
Two components of the proteolytic system, proteins A and B (J. Hugenholtz, F. Exterkate, and W. N. Konings, Appl. Environ. Microbiol. 48:1105-1110, 1984), have been studied in Streptococcus cremoris Wg2 by immunological methods. The components could not be separated by standard chromatography techniques because both proteins had almost identical molecular weights (about 140,000) and isoelectric points (pH 4.5). Specific antibodies were raised against proteins A and B by excision of the different immunoprecipitates from crossed immunoelectrophoresis gels. With these antibodies, protein A or B was removed from solutions containing both proteins. The purified proteins A and B possessed proteolytic activity and were inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Each of these proteins accounted for approximately 50% of the total proteolytic activity isolated from S. cremoris Wg2. The specific antibodies against the proteases were also used for immuno-gold labeling studies. The proteases were clearly seen to be located at the outside of the cell wall. The proteases had the same location when the genetic information coding for the proteases was cloned in Streptococcus lactis and Bacillus subtilis. 相似文献
18.
Brush border membranes isolated from rat small intestine were found to possess a cAMP-dependent protein kinase activity. Upon addition of cAMP, a rapid, time-dependent inactivation of this enzyme occurs, which was found to be due to a proteolytic activity identified in the membranes. This activity could not be assigned to previously known brush border proteases. The inactivation and the proteolytic degradation of the kinase could be reproduced also with the pure catalytic subunit of cAMP-dependent protein kinase (C) from rabbit skeletal muscle (M.W. 40000) which was cleaved by the membranal proteolytic activity with concomitant quantitative appearance of a degradation product (M.W. 30000) devoid of kinase activity. The membranal proteolytic activity appears to be specific for C since: (1) it does not degrade the other endogenous proteins in the membrane preparation; (2) it does not degrade any of six arbitrarily chosen proteins from other sources; (3) it catalyzes a limited proteolysis of C which could not be simulated by other proteolytic enzymes such as trypsin, clostripain, chymotrypsin and papain. The attack of C by the membranal protease is blocked by the presence of the nucleotide substrate of the kinase (MgATP). In addition, the undissociated and inactive form of the enzyme (R2C2) does not lose its potential enzymatic activity, and neither its catalytic nor its regulatory subunits are digested by the protease. The specific, restricted and limited action of the protease, together with the prevention of its action by the substrate and the regulatory protein (R) of the kinase raise the possibility that the membranal protease may have a distinct physiological (possibly regulatory) assignment. 相似文献
19.
Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively 总被引:1,自引:0,他引:1
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In previous reports it was demonstrated that the Nipah virus V and W proteins have interferon (IFN) antagonist activity due to their ability to block signaling from the IFN-alpha/beta receptor (J. J. Rodriguez, J. P. Parisien, and C. M. Horvath, J. Virol. 76:11476-11483, 2002; M. S. Park et al., J. Virol. 77:1501-1511, 2003). The V, W, and P proteins are all encoded by the same viral gene and share an identical 407-amino-acid N-terminal region but have distinct C-terminal sequences. We now show that the P protein also has anti-IFN function, confirming that the common N-terminal domain is responsible for the antagonist activity. Truncation of this N-terminal domain revealed that amino acids 50 to 150 retain the ability to block IFN and to bind STAT1, a key component of the IFN signaling pathway. Subcellular localization studies demonstrate that the V and P proteins are predominantly cytoplasmic whereas the W protein is localized to the nucleus. In all cases, STAT1 colocalizes with the corresponding Nipah virus protein. These interactions are sufficient to inhibit STAT1 activation, as demonstrated by the lack of STAT1 phosphorylation on tyrosine 701 in IFN-stimulated cells expressing P, V, or W. Therefore, despite their common STAT1-binding domain, the Nipah virus V and P proteins act by retaining STAT1 in the cytoplasm while the W protein sequesters STAT1 in the nucleus, creating both a cytoplasmic and a nuclear block for STAT1. We also show that the IFN antagonist activity of the P protein is not as strong as that of V or W, perhaps explaining why Nipah virus has evolved to express these two edited products. 相似文献
20.
Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins 总被引:4,自引:0,他引:4
Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation. 相似文献