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911.
Dutkiewicz R Schilke B Cheng S Knieszner H Craig EA Marszalek J 《The Journal of biological chemistry》2004,279(28):29167-29174
Isu, the scaffold for assembly of Fe-S clusters in the yeast mitochondrial matrix, is a substrate protein for the Hsp70 Ssq1 and the J-protein Jac1 in vitro. As expected for an Hsp70-substrate interaction, the formation of a stable complex between Isu and Ssq1 requires Jac1 in the presence of ATP. Here we report that a conserved tripeptide, PVK, of Isu is critical for interaction with Ssq1 because amino acid substitutions in this tripeptide inhibit both the formation of the Isu-Ssq1 complex and the ability of Isu to stimulate the ATPase activity of Ssq1. These biochemical defects correlate well with the growth defects of cells expressing mutant Isu proteins. We conclude that the Ssq1-Isu substrate interaction is critical for Fe-S cluster biogenesis in vivo. The ability of Jac1 and mutant Isu proteins to cooperatively stimulate the ATPase activity of Ssq1 was also measured. Increasing the concentration of Jac1 and mutant Isu together but not individually partially overcame the effect of the reduced affinity of the Isu mutant proteins for Ssq1. These results, along with the observation that overexpression of Jac1 was able to suppress the growth defect of an ISU mutant, support the hypothesis that Isu is "targeted" to Ssq1 by Jac1, with a preformed Jac1-Isu complex interacting with Ssq1. 相似文献
912.
An alternative splicing product of the murine trpv1 gene dominant negatively modulates the activity of TRPV1 channels 总被引:3,自引:0,他引:3
Transient receptor potential vanilloid 1 (TRPV1), or vanilloid receptor 1, is the founding member of the vanilloid type of TRP superfamily of nonselective cation channels. TRPV1 is activated by noxious heat, acid, and alkaloid irritants as well as several endogenous ligands and is sensitized by inflammatory factors, thereby serving important functions in detecting noxious stimuli in the sensory system and pathological states in different parts of the body. Whereas numerous studies have been carried out using the rat and human TRPV1 cDNA, the mouse TRPV1 cDNA has not been characterized. Here, we report molecular cloning of two TRPV1 cDNA variants from dorsal root ganglia of C57BL/6 mice. The deduced proteins are designated TRPV1alpha and TRPV1beta and contain 839 and 829 amino acids, respectively. TRPV1beta arises from an alternative intron recognition signal within exon 7 of the trpv1 gene. We found a predominant expression of TRPV1alpha in many tissues and significant expression of TRPV1beta in dorsal root ganglia, skin, stomach, and tongue. When expressed in HEK 293 cells or Xenopus oocytes, TRPV1alpha formed a Ca(2+)-permeable channel activated by ligands known to stimulate TRPV1. TRPV1beta was not functional by itself but its co-expression inhibited the function of TRPV1alpha. Furthermore, although both isoforms were synthesized at a similar rate, less TRPV1beta than TRPV1alpha protein was found in cells and on the cell surface, indicating that the beta isoform is highly unstable. Our data suggest that TRPV1beta is a naturally occurring dominant-negative regulator of the responses of sensory neurons to noxious stimuli. 相似文献
913.
Roitelman J Masson D Avner R Ammon-Zufferey C Perez A Guyon-Gellin Y Bentzen CL Niesor EJ 《The Journal of biological chemistry》2004,279(8):6465-6473
Apomine, a novel 1,1-bisphosphonate ester, has been shown to lower plasma cholesterol concentration in several species. Here we show that Apomine reduced the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, both in rat liver and in cultured cells. Apomine resembles sterols such as 25-hydroxycholesterol in its ability to potently accelerate the rate of HMGR degradation by the ubiquitin-proteasome pathway, a process that depends on the transmembrane domain of the enzyme. The similarity between Apomine and sterols in promoting rapid HMGR degradation extends to its acute requirements for ongoing protein synthesis and mevalonate-derived non-sterol product(s) as a co-regulator. Yet, at suboptimal concentrations, sterols potentiated the effect of Apomine in stimulating HMGR degradation, indicating that these agents act via distinct modes. Furthermore, unlike sterols, Apomine inhibited the activity of acyl-CoA:cholesterol acyltransferase in intact cells but not in cell-free extracts. Apomine stimulated the cleavage of the precursor of sterol-regulatory element-binding protein-2 and increased the activity of low density lipoprotein receptor pathway. This Apomine-enhanced activation of sterol-regulatory element-binding protein-2 was prevented by sterols or mevalonate. Taken together, our results provide a molecular mechanism for the hypocholesterolemic activity of Apomine. 相似文献
914.
Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants
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Cammarato A Hatch V Saide J Craig R Sparrow JC Tobacman LS Lehman W 《Biophysical journal》2004,86(3):1618-1624
Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates. 相似文献
915.
916.
Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation. 相似文献
917.
Transcriptional profiling of Caulobacter crescentus during growth on complex and minimal media 总被引:2,自引:0,他引:2
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Hottes AK Meewan M Yang D Arana N Romero P McAdams HH Stephens C 《Journal of bacteriology》2004,186(5):1448-1461
Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation. 相似文献
918.
Vann WF Daines DA Murkin AS Tanner ME Chaffin DO Rubens CE Vionnet J Silver RP 《Journal of bacteriology》2004,186(3):706-712
The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent. 相似文献
919.
920.
c-Myc sensitization to oxygen deprivation-induced cell death is dependent on Bax/Bak, but is independent of p53 and hypoxia-inducible factor-1 总被引:8,自引:0,他引:8