Phosphorylation of nonstructural 5A protein of hepatitis C virus: HCV group-specific hyperphosphorylation.
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We previously showed that two proteins with molecular weights of 56 and 58 kDa are produced from nonstructural protein 5A (NS5A) derived from hepatitis C virus (HCV)-1b genotype. The 56-kDa protein is phosphorylated at serine residues in NS5A, including those located in the C-terminal region of NS5A, while the 58-kDa protein, the hyperphosphorylated form of the 56-kDa protein, is phosphorylated at serine residues in the central region. This hyperphosphorylation is dependent on the presence of HCV NS4A protein. To clarify whether NS4A-dependent phosphorylation also occurs in other HCV genotypes, phosphorylation of NS5A was analyzed by two-dimensional gel electrophoresis. Here, we report that NS5A from the HCV-2a genotype was phosphorylated. However, hyperphosphorylation of NS5A occurs in the HCV-1b genotype but not in the -2a genotype. This result suggests that modification of NS5A phosphorylation reflects the virological features of HCV and that there are physiological differences in the roles of differently phosphorylated NS5A between HCV genotypes. (+info)
Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation.
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Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4]. (+info)
Self-aggregation of triadin in the sarcoplasmic reticulum of rabbit skeletal muscle.
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The 95 kDa transmembrane glycoprotein triadin is believed to be an essential component of excitation-contraction coupling in the junctional sarcoplasmic reticulum of skeletal muscle fibers. It is debatable whether triadin mediates intraluminal interactions between calsequestrin and the ryanodine receptor exclusively or whether this junctional protein provides also a cytoplasmic linkage between the Ca2+-release channel and the dihydropyridine receptor. Here, we could show that native triadin exists as disulfide-linked homo-polymers of above 3000 kDa. Under non-reducing conditions, protein bands representing the alpha1-dihydropyridine receptor and calsequestrin did not show an immunodecorative overlap with the extremely high-molecular-mass triadin clusters. Following chemical crosslinking, the ryanodine receptor and triadin exhibited a similarly decreased electrophoretic mobility. However, immunoblotting of diagonal non-reducing/reducing two-dimensional gels clearly demonstrated a lack of overlap between the immunodecorated bands representing triadin, the alpha1-dihydropyridine receptor, the ryanodine receptor and calsequestrin. Thus, in native membranes triadin appears to form large self-aggregates primarily. Although triadin exists in a close neighborhood relationship to the Ca2+-release channel tetramers, it does not seem to be directly linked to the other main triad components implicated in the regulation of the excitation-contraction-relaxation cycle and Ca2+-homeostasis. This agrees with a proposed role of triadin in the maintenance of overall triad architecture. (+info)
Autoantibodies in primary Sjogren's syndrome are directed against proteasomal subunits of the alpha and beta type.
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OBJECTIVE: The proteasome subunit HC9 (alpha3) has recently been identified as a major target of the humoral autoimmune response in patients with autoimmune myositis and systemic lupus erythematosus. Since B cell hyperreactivity is a common feature of systemic autoimmune diseases, patients with primary Sjogren's syndrome (SS) and other control groups were investigated to evaluate the significance of autoantibodies against the proteasome. METHODS: Analyses of autoantibodies directed against the 20S proteasome were performed using enzyme-linked immunosorbent assay, immunoblot, and 2-dimensional electrophoresis. Forty-three patients with primary SS, 47 patients with rheumatoid arthritis including 9 with secondary SS, 19 patients with gastrointestinal tumors, and 80 healthy controls were tested for antiproteasome antibodies. RESULTS: Antiproteasome antibodies were detected in 39% of patients (17 of 43) with primary SS. In contrast, only 1 of 47 patients with rheumatoid arthritis showed positive reactivity (P < 0.001). Serum samples from 19 tumor patients (P < 0.003) and 80 healthy controls (P < 0.001) were serologically negative. Moreover, immunoblotting and 2-dimensional analysis of the antiproteasome response revealed a polyspecific recognition pattern in 7 patients with primary SS. Different proteasomal subunits of the alpha and beta type, including subunits that carried the proteolytic active sites, were recognized by the patients' sera. CONCLUSION: The humoral antiproteasome response in primary SS, in contrast to its secondary form, is characterized by an extensive recognition pattern of several subunits, indicating a polyspecific B cell activation against the 20S proteasome. Moreover, proteolytically active beta-type subunits, which are important for the generation of major histocompatibility complex class I-restricted antigens, appear to be targets of the autoimmune response. The data indicate that the proteasome itself may stand on a cross point of pathways that links mechanisms of the immune defense with features of systemic autoimmunity. (+info)
Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.
(21/5851)
Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A. (+info)
Early activated replication origins within the cell cycle-regulated histone H4 genes in Physarum.
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It was previously shown that the two members of the cell cycle-regulated histone H4 gene family, H4-1 and H4-2, are replicated at the onset of S phase in the naturally synchronous plasmodium of Physarum polycephalum, suggesting that they are flanked by replication origins. It was further shown that a DNA fragment upstream of the H4-1 gene is able to confer autonomous replication of a plasmid in the budding yeast. In this paper, we re-investigated replication of the unlinked Physarum histone H4 genes by mapping the replication origin of these two loci using alkaline agarose gel and neutral/neutral 2-dimensional agarose gel electrophoreses. We showed that the two replicons containing the H4 genes are simultaneously activated at the onset of S phase and we mapped an efficient, bidirectional replication origin in the vicinity of each gene. Our data demonstrated that the Physarum sequence that functions as an ARS in yeast is not the site of replication initiation at the H4-1 locus. We also observed a stalling of the rightward moving replication fork downstream of the H4-1 gene, in a region where transient topoisomerase II sites were previously mapped. Our results further extend the concept of replication/transcription coupling in Physarum to cell cycle-regulated genes. (+info)
RpoS-dependent stress tolerance in Pseudomonas aeruginosa.
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Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans. The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species. To determine if the P. aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated. Disruption of the rpoS gene resulted in a two- to threefold increase in the rate of kill of stationary-phase cells. The rpoS mutant also survived less well than the parental strain during the initial phase of carbon or phosphate-carbon starvation. However, after 25 d starvation the remaining population of culturable cells was not significantly different. Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P. aeruginosa. Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation. (+info)
Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments.
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Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells. (+info)