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91.
92.
We investigated possible role(s) of N protein phosphorylation in the rabies virus replication process. A large amount of P proteins are associated with the viral nucleocapsid (NC) in the infected cell, the amount which was greatly decreased by phosphatase-treatment of the isolated NC, indicating that the phosphate group of N and/or P proteins is essential for their stable association with the NC. Immunoprecipitation studies were performed on the coexpressed normal N or phosphorylation deficient N(S389A) and P proteins, demonstrating that the P protein associated with phosphorylation-deficient NC-like structures was much less in amount than that associated with the wild type NC. Similar results were also obtained with a mutant P protein, PDeltaN19, which lacked the N-terminal 19 amino acids and was capable of binding to the NC-like structures but incapable of forming the RNA-free N-P complexes. Immunoprecipitation studies with mAb #402-13 further suggested that the NC-specific linear 402-13 epitope was exposed even on the P proteins which were associated with the phosphorylation-deficient NC-like structures, but such association was very weak as demonstrated by greatly decreased amounts of coprecipitated NC-like structures. From these results, we assume that the phosphorylation of N protein enhances the association between the 402-13 epitope-positive P protein and the NC probably by stabilizing such P-NC binding. 相似文献
93.
Henriques ES Fonseca N Ramos MJ 《Protein science : a publication of the Protein Society》2004,13(9):2355-2369
Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors. 相似文献
94.
Bhatt AN Khan MY Bhakuni V 《Protein science : a publication of the Protein Society》2004,13(8):2184-2195
The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway. 相似文献
95.
Using a phylogenomic approach with 10 fungi of very different virulence and habitat, we determined that there was substantial diversification of subtilase-type proteases early in ascomycete history (with subsequent loss in many lineages) but with no comparable diversification of trypsins. Patterns of intron loss and the degree of divergence between paralogues demonstrated that the proliferation of proteinase K subtilases and subtilisin type subtilases seen in pathogenic ascomycetes (Metarhizium anisopliae, Magnaporthe grisea, Fusarium graminearum) occurred after the basidiomycete/ascomycete split but predated radiation of ascomycete lineages. This suggests that the early ascomycetes had a lifestyle that selected for multiple proteases, whereas the current disparity in gene numbers between ascomycete lineages results from retention of genes in at least some pathogens that have been lost in other lineages (yeasts, Aspergillus nidulans, Neurospora crassa). A similar prevailing trend towards lineage specific gene loss of trypsins in saprophytes and some pathogens suggests that their phylogenetic breadth will have been much wider in early fungi than currently. 相似文献
96.
Hui Z Minamiguchi K Doi H Kinoshita N Kanouchi H Oka T 《In vitro cellular & developmental biology. Animal》2004,40(8-9):293-296
Summary An efficient Escherichia coli expression system for the production of mature-type alkaline serine protease II (mASP II) has been constructed. Complementary
deoxyribonucleic acid-encoding mASP II was inserted into the inducible bacterial expression vector pGE-30. After introduction
into E, coli, the plasmid was expressed by isopropyl-1-thio-β-d-galactopyranoside, and the recombinant product was purified using a Ni-nitrilotriacetic acid column The purified product
had the expected NH2-terminal sequence and showed a scrapie isoform of prion protein-degrading activity using hamster scrapie 263K prions as a
substrate. 相似文献
97.
98.
List K Szabo R Wertz PW Segre J Haudenschild CC Kim SY Bugge TH 《The Journal of cell biology》2003,163(4):901-910
Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation. 相似文献
99.
Early events of apoptosis following HSV-1 infection were investigated at the single-cell level using intensified fluorescence digital-imaging microscopy. The results provide evidence that infection of differentiated ND7 neuronlike cells by HSV-1 triggers detectable alterations indicative of physiological changes associated with the early stages of apoptosis. Less than 1 h after infection with HSV-1 (KOS strain) or K26GFP (GFP being fused to HSV-1 capsid protein VP26) we observed (i) moderate decrease in mitochondrial membrane potential (about 20%), (ii) exposure of phosphatidyl serine, (iii) morphological change in the mitochondria that became spherical instead of filamentous, and (iv) activation of caspase-8. Within 3 h changes reverted to normal, which indicated that apoptosis was counteracted very early following HSV-1 infection. Similar results were obtained with KOS-TK27GFP, lacking TK and UL24 proteins, suggesting that TK and UL24 play no role in apoptosis. In Vero cells mitochondrial changes characteristic of the apoptotic process were not observed following HSV-1 infection. The UV-inactivated K26GFP had the capacity to induce apoptosis in neuronlike cells. This real-time multiparametric analysis, in combination with relevant viral mutants, could be a useful approach for dissecting the roles of various viral genes in modulating apoptotic pathways during infection. 相似文献
100.
Oral treatment with vanadium of Zucker fatty rats activates muscle glycogen synthesis and insulin-stimulated protein phosphatase-1 activity 总被引:1,自引:0,他引:1
Since the glucose-lowering effects of vanadium could be related to increased muscle glycogen synthesis, we examined the in vivo effects of vanadium and insulin treatment on glycogen synthase (GS) activation in Zucker fatty rats. The GS fractional activity (GSFA), protein phosphatase-1 (PP1), and glycogen synthase kinase-3 (GSK-3) activity were determined in fatty and lean rats following treatment with bis(maltolato)oxovanadium(IV) (BMOV) for 3 weeks (0.2 mmol/kg/day) administered in drinking water. Skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). In both lean and fatty rats, muscle GSFA was significantly increased at 15 min following insulin stimulation. Vanadium treatment resulted in decreased insulin levels and improved insulin sensitivity in the fatty rats. Interestingly, this treatment stimulated muscle GSFA by 2-fold (p < 0.05) and increased insulin-stimulated PP1 activity by 77% (p < 0.05) in the fatty rats as compared to untreated rats. Insulin resistance, vanadium and insulin in vivo treatment did not affect muscle GSK-3 activity in either fatty or lean rats. Therefore, an impaired insulin sensitivity in the Zucker fatty rats was improved following vanadium treatment, resulting in an enhanced muscle glucose metabolism through increased GS and insulin-stimulated PP1 activity. 相似文献