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131.
Phosphorylation enhances mitochondrial targeting of GSTA4-4 through increased affinity for binding to cytoplasmic Hsp70 总被引:4,自引:0,他引:4
Robin MA Prabu SK Raza H Anandatheerthavarada HK Avadhani NG 《The Journal of biological chemistry》2003,278(21):18960-18970
Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4. 相似文献
132.
Qasim MA Lu W Lu SM Ranjbar M Yi Z Chiang YW Ryan K Anderson S Zhang W Qasim S Laskowski M 《Biochemistry》2003,42(21):6460-6466
The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm. 相似文献
133.
Turkey ovomucoid third domain (OMTKY3) is shown to exist at low pH as two distinctly folded, interconverting conformations. Activation parameters were determined for the transition, and these were of the type reported previously for cis/trans isomerizations of prolyl peptide bonds. Multidimensional, multinuclear NMR spectroscopy was used to determine the three-dimensional structure of each of the two states of P(5)-Pro(14)Asp OMTKY3 at pH 2.5 and 25 degrees C, under conditions where the two states have equal populations with interchange rates of 0.25 s(-1). The results showed that the two states differ by cis/trans isomerization of the P(8)-Tyr(11)-P(7)-Pro(12) peptide bond, which is cis in the conformer dominant at neutral pH and trans in the conformer appearing at low pH. The major structural differences were found to be in the region of the reactive site loop. The core of the protein, including the antiparallel beta-sheet and a alpha-helix, is preserved in both structures. The state with the cis peptide bond is similar to previously reported structures of OMTKY3 determined by NMR spectroscopy and X-ray crystallography. The cis-to-trans transition results in the relocation of the aromatic ring of P(8)-Tyr(11), disrupts many interactions between the alpha-helix and the reactive-site loop, and leads to more open spacing between this loop and the alpha-helix. In addition, the configurations of two of the three disulfide bonds, P(11)-Cys(8)- P(20)'-Cys(38), and P(3)-Cys(16)- P(17)'-Cys(35), are altered such that the C(alpha)-C(alpha) distances for each disulfide bridge are longer by approximately 1 A in the trans state than in the cis. Mutations at P(1)-Leu(18), P(6)-Lys(13), and P(5)-Pro(14) influence the position of the cis <= => trans equilibrium. In P(1)-Leu(18)Xxx OMTKY3 mutants, the trans state is more favored by P(1)-Gly(18) than by Ala(18) or Leu(18); in P(6)-Lys(13)Xxx OMTKY3 mutants, the trans state is more favored by P(6)-Glu(13) and P(6)-Asp(13) than Lys(13) or His(13). Stabilization of the trans state in P(5)-Pro(14)Xxx OMTKY3 mutants follows the series Xxx = Gly > Asp > Glu > Ala approximately equal His > Pro. In comparing the state with the trans peptide bond to that with the cis, the pK(a) values of P(12)-Asp(7) and P(1)'-Glu(19) are higher and those of P(9)-Glu(10) and P(25)'-Glu(43) are lower. The pK(a) values of other titrating groups in the molecule are similar in both conformational states. These pK(a) changes underlie the pH dependence of the conformational equilibrium and can be explained in part by observed structural differences. (15)N transverse relaxation results indicate that residues P(6)-Lys(13)-P(3)-Cys(16) in the trans state undergo a dynamic process on the microsecond-millisecond time scale not present in the cis state. 相似文献
134.
Genetics of quantitative traits in Arabidopsis thaliana 总被引:1,自引:0,他引:1
The genetic control of 22 quantitative traits, including developmental rates and sizes, was examined in generations of Arabidopsis thaliana derived from the cross between the ecotypes, Columbia (Col) and Landsberg erecta (Ler). The data were obtained from three sets of families raised in the same trial: the 16 basic generations, that is, parents, F(1)'s, F(2)'s, backcrosses, recombinant inbred lines (RILs) and a triple test cross (TTC), the latter produced by crossing the RILs to Col, Ler and their F(1). The data were analysed by two approaches. The first (approach A) involved traditional generation mean and variance component analysis and the second (B), based around the RILs and TTC families, involved marker-based QTL analysis.From (A), genetic differences between Col and Ler were detected for all traits with moderate heritabilities. Height at flowering was the only trait to show heterosis. Dominance was partial to complete for all height traits, and there was no overdominance but there was strong evidence for directional dominance. For most other traits, dominance was ambidirectional and incomplete, with average dominance ratios of around 80%. Epistasis, particularly of the duplicate type that opposes dominance, was a common feature of all traits. The presence of epistasis must imply multiple QTL for all traits.The QTL analysis located 38 significant effects in four regions of chromosomes I, II, IV and V, but not III. QTL affecting rosette size and leaf number were identified in all four regions, with days to maturity on chromosomes IV and V. The only QTL for height was located at the expected position of the erecta gene (chromosome II; 50 cM), but the additive and dominance effects of this single QTL did not adequately explain the generation means. The possible involvement of other interacting height QTL is discussed. 相似文献
135.
Alam SI Agarwal GS Kamboj DV Rai GP Singh L 《Indian journal of experimental biology》2003,41(2):177-180
A sensitive PCR based detection of Bacillus anthracis spores from environnment was standardized. Specific 1247bp amplicon could be detected with template concentration as low as 13 pg. Sensitivity was enhanced to 10 fold by nesting with second set of primers, forming 208bp amplicon. Extraction of DNA from spores purified from soil samples by aqueous polymer two-phase system followed by partial germination and freeze-thaw treatment yielded best results. Soil sample spiked with spores (8x10(2)/g of sample) could be detected with this method. 相似文献
136.
Parathyroid hormone regulation of type II sodium-phosphate cotransporters is dependent on an A kinase anchoring protein 总被引:1,自引:0,他引:1
Parathyroid hormone inhibits sodium-phosphate cotransport in proximal renal tubule cells through activation of several kinases. We tested the hypothesis that the activity of these kinases was coordinated by an A kinase anchoring protein (AKAP) by demonstrating that the type II sodium-phosphate cotransporter (NaPi-4) physically associated with an AKAP and that this association was necessary for regulation of phosphate transport by parathyroid hormone. Immunoprecipitation with anti-NaPi-4 antiserum and glutathione S-transferase pull-down with GST-NaPi-4 showed that NaPi-4 associated with AKAP79, protein kinase A catalytic and regulatory subunits, and the parathyroid hormone receptor in opossum kidney cells. When the regulatory subunit of protein kinase A was uncoupled from the AKAP by a competing peptide, parathyroid hormone lost the ability to inhibit phosphate transport. This result was confirmed by co-transfecting HEK293 cells with the sodium-phosphate cotransporter and wild type AKAP, a mutant AKAP79, or the empty vector. 8-Bromo-cAMP was able to inhibit phosphate transport in cells expressing the wild type AKAP79 but not empty vector or mutant AKAP79. We conclude that parathyroid hormone inhibits proximal renal tubule sodium-phosphate cotransport through a signaling complex dependent upon an AKAP. 相似文献
137.
A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity. 相似文献
138.
Qamar R Ayub Q Mohyuddin A Helgason A Mazhar K Mansoor A Zerjal T Tyler-Smith C Mehdi SQ 《American journal of human genetics》2002,70(5):1107-1124
Eighteen binary polymorphisms and 16 multiallelic, short-tandem-repeat (STR) loci from the nonrecombining portion of the human Y chromosome were typed in 718 male subjects belonging to 12 ethnic groups of Pakistan. These identified 11 stable haplogroups and 503 combination binary marker/STR haplotypes. Haplogroup frequencies were generally similar to those in neighboring geographical areas, and the Pakistani populations speaking a language isolate (the Burushos), a Dravidian language (the Brahui), or a Sino-Tibetan language (the Balti) resembled the Indo-European-speaking majority. Nevertheless, median-joining networks of haplotypes revealed considerable substructuring of Y variation within Pakistan, with many populations showing distinct clusters of haplotypes. These patterns can be accounted for by a common pool of Y lineages, with substantial isolation between populations and drift in the smaller ones. Few comparative genetic or historical data are available for most populations, but the results can be compared with oral traditions about origins. The Y data support the well-established origin of the Parsis in Iran, the suggested descent of the Hazaras from Genghis Khan's army, and the origin of the Negroid Makrani in Africa, but do not support traditions of Tibetan, Syrian, Greek, or Jewish origins for other populations. 相似文献
139.
The present investigation focuses on the synthesis and application of a cross-linking agent that is compatible with the solubility characteristics of chitosan. A water-soluble, blocked-diisocyanate was prepared as a bisulfite adduct to 1,6-hexamethylene diisocyanate, which proved to be stable for several weeks in aqueous, acidic chitosan solutions at room temperature. Thermal cross-linking of chitosan as cast, dried films was investigated by varying the NCO/NH(2) ratio from 0.0 to 1.2. Spectroscopic (IR), thermal (TGA), swelling, and structural (WAXD) studies indicated that chitosan was cross-linked in a concentration-dependent manner under mild thermal conditions: 60 degrees C for 24 h. Cross-linking inefficiency was concluded to be due to lack of mobility of the reacting species in the solid state. In a preliminary study, the enzymatic degradation with Chitinase (E. C. 3.2.1.14) from Streptomyces griseus was found to be the greatest for non-crosslinked chitosan, followed by chitin, and then by cross-linked samples. 相似文献
140.
CCR5 is a G-protein-coupled chemokine receptor that is used as a co-factor by macrophage-tropic (M-tropic) isolates of human immunodeficiency virus-1 (HIV-1) to gain entry into host cells. A 32-bp deletion in the CCR5 gene (CCR5-Delta32) leads to the production of an altered gene product that prevents HIV-1 from entering the host cell. This study was carried out to determine prevalence of CCR5-Delta32 allele frequency in a large Pakistani population sample (n = 821) representing 10 ethnic groups. No individual was homozygous for the mutant allele and the frequency of the CCR5-Delta32 allele ranged from 0.62% to 3.57%. The CCR5-Delta32 allele frequency was generally lower in populations from southern Pakistan. The overall frequency of the CCR5-Delta32 allele in Pakistan was 2.31%, which is much lower than that found in European populations and similar to that in the Middle East. This is consistent with the historical records and genetic data that indicate a close genetic affinity among these populations. This study demonstrates that the Pakistani population is highly susceptible to M-tropic isolates of HIV-1 and public health measures need to be enforced with urgency if Pakistan is to avoid an HIV epidemic. 相似文献