Transcriptional regulation of molybdoenzyme synthesis in Escherichia coli in response to molybdenum: ModE-molybdate, a repressor of the modABCD (molybdate transport) operon is a secondary transcriptional activator for the hyc and nar operons.
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Escherichia coli growing under anaerobic conditions produces several molybdoenzymes, such as formate hydrogenlyase (formate to H2 and CO2; hyc and fdhF genes) and nitrate reductase (narGHJI genes). Synthesis of these molybdoenzymes, even in the presence of the cognate transcriptional activators and effectors, requires molybdate in the medium. Besides the need for molybdopterin cofactor synthesis, molybdate is also required for transcription of the genes encoding these molybdoenzymes. In E. coli, ModE was previously identified as a repressor controlling transcription of the operon encoding molybdate transport components (modABCD). In this work, the ModE protein was also found to be a required component in the activation of hyc-lacZ to an optimum level, but only in the presence of molybdate. Mutant ModE proteins which are molybdate-independent for repression of modA-lacZ also restored hyc-lacZ expression to the wild-type level even in the absence of molybdate. Nitrate-dependent enhancement of transcription of narX-lacZ was completely abolished in a modE mutant. Nitrate-response by narG-lacZ and narK-lacZ was reduced by about 50% in a modE mutant. DNase I footprinting experiments revealed that the ModE protein binds the hyc promoter DNA in the presence of molybdate. ModE-molybdate also protected DNA in the intergenic region between narXL and narK from DNase I hydrolysis. DNA sequences (5' TAYAT 3' and 5' GTTA 3') found in ModE-molybdate-protected modABCD operator DNA were also found in the ModE-molybdate-protected region of hyc promoter DNA (5' GTTA-7 bp-CATAT 3') and narX-narK intergenic region (5' GTTA-7 bp-TACAT 3'). Based on these results, a working model is proposed in which ModE-molybdate serves as a secondary transcriptional activator of both the hyc and narXL operons which are activated primarily by the transcriptional activators, FhlA and NarL, respectively. (+info)
The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein.
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This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center. (+info)
4-Hydroxynonenal prevents NF-kappaB activation and tumor necrosis factor expression by inhibiting IkappaB phosphorylation and subsequent proteolysis.
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Extensively oxidized low density lipoprotein (ox-LDL), a modulator of atherogenesis, down-regulates the lipopolysaccharide (LPS)-induced activation of transcription factor NF-kappaB. We investigated whether 4-hydroxynonenal (HNE), a prominent aldehyde component of ox-LDL, represents one of the inhibitory substances. NF-kappaB activation by stimuli such as LPS, interleukin (IL)-1beta, and phorbol ester, but not tumor necrosis factor (TNF), was reversibly inhibited by HNE in a dose-dependent manner in human monocytic cells, whereas AP-1 binding was unaffected. Using similar HNE concentrations, LPS-induced kappaB- and TNF or IL-8 promoter-dependent transcription was prevented. Furthermore, pretreatment with HNE suppressed TNF production but not lactate dehydrogenase levels. Under these conditions the binding of LPS to monocytic cells was not significantly affected. However, induced proteolysis of the inhibitory proteins IkappaB-alpha, IkappaB-beta, and, at a later time point, IkappaB-epsilon was prevented. This is not due to inhibition of the proteasome, the major proteolytic activities of which remain unaffected, but rather to a specific prevention of the activation-dependent phosphorylation of IkappaB-alpha. This is the first report which demonstrates that HNE specifically inhibits the NF-kappaB/Rel system. Down-modulation of NF-kappaB-regulated gene expression may contribute at certain stages of atherosclerosis to low levels of chronic inflammation and may also be involved in other inflammatory/degenerative diseases. (+info)
Ubiquitination and degradation of ATF2 are dimerization dependent.
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Ubiquitination and proteasome-dependent degradation are key determinants of the half-lives of many transcription factors. Homo- or heterodimerization of basic region-leucine zipper (bZIP) transcription factors is required for their transcriptional activities. Here we show that activating transcription factor 2 (ATF2) heterodimerization with specific bZIP proteins is an important determinant of the ubiquitination and proteasome-dependent degradation of ATF2. Depletion of c-Jun as one of the ATF2 heterodimer partners from the targeting proteins decreased the efficiency of ATF2 ubiquitination in vitro, whereas the addition of exogenously purified c-Jun restored it. Similarly, overexpression of c-Jun in 293T human embryo kidney cells increased ATF2 ubiquitination in vivo and reduced its half-life in a dose-dependent manner. Mutations of ATF2 that disrupt its dimerization inhibited ATF2 ubiquitination in vitro and in vivo. Conversely, removal of residues 150 to 248, as in a constitutively active ATF2 spliced form, enhanced ATF2 dimerization and transactivation, which coincided with increased ubiquitination and decreased stability. Our findings indicate the increased sensitivity of transcriptionally active dimers of ATF2 to ubiquitination and proteasome-dependent degradation. Based on these observations, we conclude that increased targeting of a transcriptionally active ATF2 form indicates the mechanism by which the magnitude and the duration of the cellular stress response are regulated. (+info)
Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system.
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BACKGROUND: Angiotensin II activates NAD(P)H-dependent oxidases via AT1-receptor stimulation, the most important vascular source of superoxide (O2*-). The AT1 receptor is upregulated in vitro by low-density lipoprotein. The present study was designed to test whether hypercholesterolemia is associated with increased NAD(P)H-dependent vascular O2*- production and whether AT1-receptor blockade may inhibit this oxidase and in parallel improve endothelial dysfunction. METHODS AND RESULTS: Vascular responses were determined by isometric tension studies, and relative rates of vascular O2*- production were determined by use of chemiluminescence with lucigenin, a cypridina luciferin analogue, and electron spin resonance studies. AT1-receptor mRNA was quantified by Northern analysis, and AT1-receptor density was measured by radioligand binding assays. Hypercholesterolemia was associated with impaired endothelium-dependent vasodilation and increased O2*- production in intact vessels. In vessel homogenates, we found a significant activation of NADH-driven O2*- production in both models of hyperlipidemia. Treatment of cholesterol-fed animals with the AT1-receptor antagonist Bay 10-6734 improved endothelial dysfunction, normalized vascular O2*- and NADH-oxidase activity, decreased macrophage infiltration, and reduced early plaque formation. In the setting of hypercholesterolemia, the aortic AT1 receptor mRNA was upregulated to 166+/-11%, accompanied by a comparable increase in AT1-receptor density. CONCLUSIONS: Hypercholesterolemia is associated with AT1-receptor upregulation, endothelial dysfunction, and increased NADH-dependent vascular O2*- production. The improvement of endothelial dysfunction, inhibition of the oxidase, and reduction of early plaque formation by an AT1-receptor antagonist suggests a crucial role of angiotensin II-mediated O2*- production in the early stage of atherosclerosis. (+info)
Crotonobetaine reductase from Escherichia coli consists of two proteins.
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Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)). CI has been purified to electrophoretic homogeneity from a cell-free extract of E. coli O44 K74. The purified protein shows l(-)-carnitine dehydratase activity and its N-terminal amino acid sequence is identical to the caiB gene product from E. coli O44 K74. The relative molecular mass of CI has been determined to be 86100. It is composed of two identical subunits with a molecular mass of 42600. The isoelectric point of CI was found to be 4.3. CII was purified from an overexpression strain in one step by ion exchange chromatography on Fractogel EMD TMAE 650(S). The N-terminal amino acid sequence of CII shows absolute identity with the N-terminal sequence of the caiA gene product, i.e. of the postulated crotonobetaine reductase. The relative molecular mass of the protein is 164400 and it is composed of four identical subunits of molecular mass 41500. The isoelectric point of CII is 5.6. CII contains non-covalently bound FAD in a molar ratio of 1:1. In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII. A still unknown low-molecular-mass effector described for the l(-)-carnitine dehydratase is also necessary for crotonobetaine reductase activity. Monoclonal antibodies were raised against the two components of crotonobetaine reductase. (+info)
Interaction of thioredoxins with target proteins: role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2-oxoacid dehydrogenase complexes.
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The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides. (+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)