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901.
HspBP1, an Hsp70 cochaperone,has two structural domains and is capable of altering the conformation of the Hsp70 ATPase domain 总被引:3,自引:0,他引:3
We present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding. 相似文献
902.
Ibarra CA Chowdhury P Petrich JW Atkins WM 《The Journal of biological chemistry》2003,278(21):19257-19265
The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9. 相似文献
903.
Acid-sensing ion channels in malignant gliomas 总被引:6,自引:0,他引:6
Berdiev BK Xia J McLean LA Markert JM Gillespie GY Mapstone TB Naren AP Jovov B Bubien JK Ji HL Fuller CM Kirk KL Benos DJ 《The Journal of biological chemistry》2003,278(17):15023-15034
High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells. 相似文献
904.
Teena L. Browning David A. Taggart Catherine Rummery Robert L. Close Mark D.B. Eldridge 《Conservation Genetics》2001,2(2):145-156
The use of molecular genetic techniques can aidwildlife managers in setting priorities anddevising management strategies for scatteredpopulations of threatened taxa. In this study,six remnant populations of the criticallyendangered brush-tailed rock-wallaby (Petrogale penicillata) in Victoria, Australia,were examined using karyotypic, microsatellite(11 loci) and mitochondrial DNA (mtDNA) controlregion sequence analysis. Each remnantpopulation was found to be genetically distinct(unique microsatellite alleles and controlregion haplotypes), but had low geneticdiversity. This distribution of geneticdiversity between, rather than withinpopulations, is most likely a consequence ofrecent severe reductions in population size anddispersal that have occurred since Europeansettlement. The six mtDNA control regionhaplotypes identified in the Victorianpopulations were all closely related (average1.3% sequence divergence), and only 2%divergence separated haplotypes from EastGippsland and the Grampians (550 km to thewest). In contrast there was considerablesequence divergence (7.7%) between theVictorian haplotypes and those found in P.penicillata from elsewhere in the speciesrange. In comparison, 8.8% divergenceseparates P. penicillata from the closelyrelated P. herberti. The Victorianhaplotypes also formed a distinct and wellsupported monophyletic group that excludedhaplotypes from other P. penicillata andP. herberti. In light of these data, werecommend that the remnant Victorianpopulations of P. penicillata be managedseparately from remaining populations in NewSouth Wales and Queensland; and thatindividuals be regularly exchanged amongst theVictorian populations to increase theirdiversity and reduce the likelihood ofinbreeding depression. 相似文献
905.
Minas Papadopoulos Berthold Nock Theodosia Maina Ioannis Pirmettis Catherine Raptopoulou Anastasios Tasiopoulos Anastasios Troganis Themistoklis Kabanos Aris Terzis Efstratios Chiotellis 《Journal of biological inorganic chemistry》2001,6(2):159-165
Novel oxorhenium and oxotechnetium complexes based on the tetradentate 1-(2-hydroxybenzamido)-2-(pyridinecarboxamido)benzene, H3L, ligand have been synthesized and characterized herein. Thus, by reacting equimolar quantities of the triply deprotonated ligand L3- with the suitable MO3+ precursor, the following neutral MOL complexes could be easily produced following similar synthetic routes: M = Re (1), M = 99gTc (2), and M = 99mTc (3). Complexes 1 and 2, prepared in macroscopic amounts, were chemically characterized and their structure determined by single-crystal X-ray analysis. They are isostructural metal chelates, adopting a distorted square pyramidal geometry around the metal. The N3O donor atom set of the tetradentate ligand defines the basal plane and the oxygen atom of the M = O core occupies the apex of the pyramid. Complex 3 forms quantitatively at tracer level by mixing the H3L ligand with Na99mTcO4 generator eluate in aqueous alkaline media and using tin chloride as reductant in the presence of citrate. Its structure was established by chromatographic comparison with prototypic complexes 1 and 2 using high-performance liquid chromatographic techniques. When challenged with excess glutathione in vitro, complex 3 is rapidly converted to hydrophilic unidentified metal species. Tissue distribution data after administration of complex 3 in vivo revealed a significant uptake and retention of this compound in brain tissue. 相似文献
906.
Adamy C Oliviero P Eddahibi S Rappaport L Samuel JL Teiger E Chassagne C 《American journal of physiology. Heart and circulatory physiology》2002,283(2):H733-H740
Right ventricular myocardial hypertrophy during hypoxic pulmonary hypertension is associated with local renin-angiotensin system activation. The expression of angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptors in this setting has never been investigated. We have therefore examined the chronic hypoxia pattern of AT(1) and AT(2) expression in the right and left cardiac ventricles, using in situ binding and RT-PCR assays. Hypoxia produced right, but not left, ventricular hypertrophy after 7, 14, and 21 days, respectively. Hypoxia for 2 days was associated in each ventricle with a simultaneous and transient increase (P < 0.05) in AT(1) binding and AT(1) mRNA levels in the absence of any significant change in AT(2) expression level. Only after 14 days of hypoxia, AT(2) binding increased (P < 0.05) in the two ventricles, concomitantly with a right ventricular decrease (P < 0.05) in AT(2) mRNA. Along these data, AT(1) and AT(2) binding remained unchanged in both the left and hypertrophied right ventricles from rats treated with monocrotaline for 30 days. These results indicate that chronic hypoxia induces modulations of AT(1) and AT(2) receptors in both cardiac ventricles probably through direct and indirect mechanisms, respectively, which modulations may participate in myogenic (at the level of smooth or striated myocytes) rather than in the growth response of the heart to hypoxia. 相似文献
907.
Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes 总被引:1,自引:0,他引:1
Amadou A Nawrocki A Best-Belpomme M Pavoine C Pecker F 《American journal of physiology. Cell physiology》2002,282(6):C1339-C1347
Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A2 (PLA2) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca2+ concentration([Ca2+]i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca2+]i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca2+]i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca2+]i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA2.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA2/AA pathway in mediating thecontractile effects of TNF-. 相似文献
908.
Translated Alu sequence determines nuclear localization of a novel catalytic subunit of casein kinase 2 总被引:3,自引:0,他引:3
Hilgard P Huang T Wolkoff AW Stockert RJ 《American journal of physiology. Cell physiology》2002,283(2):C472-C483
Casein kinase 2 (CK2) is atetrameric enzyme constitutively expressed in all eukaryotic tissues.The two known isoforms of the catalytic subunit, CK2 and CK2',have been reported to have distinct tissue-dependent subcellulardistributions. We recently described a third isoform of the catalyticsubunit, designated CK2", which is highly expressed in liver.Immunoblot analysis of HuH-7 human hepatoma cell fractions as well asimmunofluorescent microscopy revealed that CK2" was exclusivelylocalized to the nucleus and preferentially associated with the nuclearmatrix. CK2 and CK2' were found in nuclear, membrane, andcytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2" sequence resulted in release of the truncated green fluorescent protein fusion protein from the nuclear matrix and redistribution to both the nucleus and the cytoplasm. Demonstration that the carboxy terminus is necessary but not sufficient for nuclearretention indicates that the underlying mechanism of CK2" nuclearlocalization is dependent on the secondary structure of the holoenzymedirected by the carboxy-terminal sequence. 相似文献
909.
Lyford GL Strege PR Shepard A Ou Y Ermilov L Miller SM Gibbons SJ Rae JL Szurszewski JH Farrugia G 《American journal of physiology. Cell physiology》2002,283(3):C1001-C1008
Smooth muscle exhibitsmechanosensitivity independent of neural input, suggesting thatmechanosensitive pathways reside within smooth muscle cells. The nativeL-type calcium current recorded from human intestinal smooth muscle ismodulated by stretch. To define mechanosensitive mechanisms involved inthe regulation of smooth muscle calcium entry, we cloned the1C L-type calcium channel subunit (CaV1.2)from human intestinal smooth muscle and expressed the channel in aheterologous system. This channel subunit retained mechanosensitivitywhen expressed alone or coexpressed with a 2 calciumchannel subunit in HEK-293 or Chinese hamster ovary cells. Theheterologously expressed human cardiac 1C splice formalso demonstrated mechanosensitivity. Inhibition of kinase signalingdid not affect mechanosensitivity of the native channel. Truncation of the 1C COOH terminus, which containsan inhibitory domain and a proline-rich domain thought to mediatemechanosensitive signaling from integrins, did not disruptmechanosensitivity of the expressed channel. These data demonstratemechanical regulation of calcium entry through molecularly identifiedL-type calcium channels in mammalian cells and suggest that themechanosensitivity resides within the pore forming1C-subunit. 相似文献
910.
Shaw AC Larsen MR Roepstorff P Christiansen G Birkelund S 《FEMS microbiology letters》2002,212(2):193-202
The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'. 相似文献