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1.
Bauer DE Jackson JG Genda EN Montoya MM Yudkoff M Robinson MB 《Neurochemistry international》2012,61(4):566-574
GLAST is the predominant glutamate transporter in the cerebellum and contributes substantially to glutamate transport in forebrain. This astroglial glutamate transporter quickly binds and clears synaptically released glutamate and is principally responsible for ensuring that synaptic glutamate concentrations remain low. This process is associated with a significant energetic cost. Compartmentalization of GLAST with mitochondria and proteins involved in energy metabolism could provide energetic support for glutamate transport. Therefore, we performed immunoprecipitation and co-localization experiments to determine if GLAST might co-compartmentalize with proteins involved in energy metabolism. GLAST was immunoprecipitated from rat cerebellum and subunits of the Na(+)/K(+) ATPase, glycolytic enzymes, and mitochondrial proteins were detected. GLAST co-localized with mitochondria in cerebellar tissue. GLAST also co-localized with mitochondria in fine processes of astrocytes in organotypic hippocampal slice cultures. From these data, we hypothesized that mitochondria participate in a macromolecular complex with GLAST to support oxidative metabolism of transported glutamate. To determine the functional metabolic role of this complex, we measured CO(2) production from radiolabeled glutamate in cultured astrocytes and compared it to overall glutamate uptake. Within 15min, 9% of transported glutamate was converted to CO(2). This CO(2) production was blocked by inhibitors of glutamate transport and glutamate dehydrogenase, but not by an inhibitor of glutamine synthetase. Our data support a model in which GLAST exists in a macromolecular complex that allows transported glutamate to be metabolized in mitochondria to support energy production. 相似文献
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K R Shelton P M Egle J M Todd 《Biochemical and biophysical research communications》1986,134(2):492-498
A step in the induction of a 30- to 35-kD stress protein may be the reaction of chemical inducers with glutathione. Effective inducers are sulfhydryl reagents. Further, a comparison of three reagents, 1-chloro-2,4-dinitrobenzene, diethylmaleate, and N-ethylmaleimide, indicates that the first two, which have considerable selectivity for glutathione, are strong inducers of the stress protein but the third, which is much more reactive with protein sulfhydryls, is either a poor or ineffective inducer. A decrease in cellular glutathione does not appear to be inductive, however. An increase in modified glutathione remains as a possible signal for the induction of this stress protein. 相似文献
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Auxin affects many aspects of plant growth and development. We previously used chemical genetics to dissect auxin-signaling mechanisms and identified a small molecule, sirtinol, that constitutively activated auxin signaling (Y. Zhao et al. [2003], Science 301: 1107-1110). Here we describe the isolation, characterization, and cloning of an Arabidopsis mutant Atcand1-1 that emerged from a genetic screen for mutants insensitive to sirtinol. Loss-of-function mutants of AtCAND1 were resistant to sirtinol and auxin, but not to gibberellins or brassinolide. Atcand1 displayed developmental phenotypes similar to those of axr1, namely, short petioles, downwardly curling leaves, short inflorescence, and reduced fertility. AtCAND1 is homologous to human CAND1, a protein that is composed almost entirely of HEAT-repeat units and has been implicated in regulating the assembly and disassembly of the SCF protein degradation machinery. Taken together with previous biochemical studies, this work helps to elucidate the roles of AtCAND1 in protein degradation and auxin signaling. 相似文献
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《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2022,1869(5):119212
Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex.SummaryIn this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function. 相似文献
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Harold C. Smith Stanley G. Harris Martin Zillmann Susan M. Berget 《Experimental cell research》1989,182(2):521-533
The role of nuclear matrix proteins in premessenger RNA splicing has been investigated using antibodies raised against isolated rat liver nuclear matrix and cross-reactive with a 65-kDa HeLa cell nuclear matrix protein (IGA-65). IGA-65 is an internal nuclear matrix component which can be solubilized as a component of nuclear splicing extracts, by the action of endogenous ribonucleases, EDTA, and DTT during extract preparation. Preincubation of splicing extract with antibodies against IGA-65 (anti-IGA-65) inhibited in vitro splicing of exogenous adenovirus precursor RNA. Furthermore, assembly of precursor RNA into active spliceosome complexes was inhibited by pretreatment of extracts with anti-IGA-65, suggesting a role for IGA-65 during early spliceosome assembly. The IGA-65 present in splicing extracts was distinguishable from known U-snRNP and hnRNP proteins on protein gels. Furthermore, electrophoresis of splicing extract on native gels indicated that IGA-65 was present in protein complexes different from those containing U-snRNPs or hnRNP C protein. The data support identification of complexes containing IGA-65 as nuclear factors involved in pre-mRNA splicing and, by extension, suggest a role for the nuclear matrix during processing in vivo. 相似文献
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Brock DA Hatton RD Giurgiutiu DV Scott B Jang W Ammann R Gomer RH 《Eukaryotic cell》2003,2(4):788-797
Developing Dictyostelium cells aggregate to form fruiting bodies containing typically 2 × 104 cells. To prevent the formation of an excessively large fruiting body, streams of aggregating cells break up into groups if there are too many cells. The breakup is regulated by a secreted complex of polypeptides called counting factor (CF). Countin and CF50 are two of the components of CF. Disrupting the expression of either of these proteins results in cells secreting very little detectable CF activity, and as a result, aggregation streams remain intact and form large fruiting bodies, which invariably collapse. We find that disrupting the gene encoding a third protein present in crude CF, CF45-1, also results in the formation of large groups when cells are grown with bacteria on agar plates and then starve. However, unlike countin− and cf50− cells, cf45-1− cells sometimes form smaller groups than wild-type cells when the cells are starved on filter pads. The predicted amino acid sequence of CF45-1 has some similarity to that of lysozyme, but recombinant CF45-1 has no detectable lysozyme activity. In the exudates from starved cells, CF45-1 is present in a ~450-kDa fraction that also contains countin and CF50, suggesting that it is part of a complex. Recombinant CF45-1 decreases group size in colonies of cf45-1− cells with a 50% effective concentration (EC50) of ~8 ng/ml and in colonies of wild-type and cf50− cells with an EC50 of ~40 ng/ml. Like countin− and cf50− cells, cf45-1− cells have high levels of cytosolic glucose, high cell-cell adhesion, and low cell motility. Together, the data suggest that CF45-1 participates in group size regulation in Dictyostelium. 相似文献
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A Sm-like protein complex that participates in mRNA degradation 总被引:22,自引:0,他引:22
In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p-Lsm8p) are associated with U6 snRNA. Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA. Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p. Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p-Lsm7p, forms a new seven-subunit complex. We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry. This confirmed the association of the Lsm2p-Lsm8p complex with U6 snRNA. In contrast, the Lsm1p-Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation. Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs. Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step. These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming. 相似文献
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Ubiquitin-like (UBL)-ubiquitin-associated (UBA) proteins such as Rad23 and Dsk2 mediate the delivery of polyubiquitinated proteins to the proteasome in the ubiquitin-proteasome pathway. We show here that budding yeast peptidyl-tRNA hydrolase 2 (Pth2), which was previously recognized as a peptidyl-tRNA hydrolase, is a UBL domain-binding protein that participates in the ubiquitin-proteasome pathway. Pth2 bound to the UBL domain of both Rad23 and Dsk2. Pth2 also interacted with polyubiquitinated proteins through the UBA domains of Rad23 and Dsk2. Pth2 overexpression caused an accumulation of polyubiquitinated proteins and inhibited the growth of yeast. Ubiquitin-dependent degradation was accelerated in the pth2Delta mutant and was retarded by overexpression of Pth2. Pth2 inhibited the interaction of Rad23 and Dsk2 with the polyubiquitin receptors Rpn1 and Rpn10 on the proteasome. Furthermore, Pth2 function involving UBL-UBA proteins was independent of its peptidyl-tRNA hydrolase activity. These results suggest that Pth2 negatively regulates the UBL-UBA protein-mediated shuttling pathway in the ubiquitin-proteasome system. 相似文献
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Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity
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O Staub F Verrey T R Kleyman D J Benos B C Rossier J P Kraehenbuhl 《The Journal of cell biology》1992,119(6):1497-1506
High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function. 相似文献
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Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends.
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VP39 is a bifunctional vaccinia virus protein that acts as both a cap- dependent 2'-O-Methyltransferase and a poly(A) polymerase processivity factor. An analysis of C-terminal truncation mutants of a GST-VP39 fusion protein indicated the presence of a protease-sensitive C-terminal "tail" 36-43 amino acids in length that is non-essential for VP39 function. Fourteen new VP39 pointmutants, containing either single or multiple-clustered amino acid substitutions, were expressed in Escherichia coli. Of the eight that retained either one or both of the activities of VP39, seven were specifically methyltransferase-defective. None was specifically defective in adenylyltransferase stimulation. The nature of the methyltransferase defects in 10 of the methyltransferase-specific defectives, identified both herein and in a previous study (Schnierle BS, Gershon PD, Moss B, 1994, J biol Chem 269:20700-20706), was investigated using two novel substrate-binding assays. Three of the mutants (and possible a fourth), whose lesions were juxtaposed and centrally located within VP39, exhibited anomalous S-adenosyl-(L)-methionine (AdoMet) binding behavior, identifying residues important for AdoMet binding and possible also for catalysis. A surface plasmon resonance-based assay measured the interaction of VP39 with uncapped and 5'-cap 0-terminated oligo(A). A cap 0- dependent association-rate enhancement was observed for wild-type VP39 and 4 of the 10 mutant proteins. Two others were identified as defective in cap binding, and a third as partially defective. The lesions within the latter three mutants were closely apposed, and located toward the N-terminus of VP39. We have thus identified regions of VP39 important for interaction with its two substrates for cap-dependent methyltransferase activity: AdoMet and cap 0. 相似文献
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Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C. Protein S-binding protein 总被引:5,自引:0,他引:5
F J Walker 《The Journal of biological chemistry》1986,261(23):10941-10944
The apparent molecular weight of functional protein S in citrated plasma was observed to be between 115,000 and 130,000 as measured by sedimentation equilibrium in the air-driven ultracentrifuge. The molecular weight of the functional protein decreased to approximately 62,000 when copper ions were added to the plasma. This suggested the presence of a protein S-binding protein in plasma, which was confirmed by gel filtration experiments. Frontal analysis of plasma indicated that functional protein S could exist in as many as three forms. Addition of copper ions to plasma reduced the number of forms to one. In order to isolate the binding protein, plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in a 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A protein, eluted in the 0.6 M NaCl wash, was observed to bind to protein S in gel filtration experiments. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S. 相似文献
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《Cellular signalling》2014,26(9):1870-1877
Mitogen-inducible gene-6 (Mig-6) is a cytosolic multiadaptor protein that is best known for its role as a negative feedback regulator of epidermal growth factor receptor (EGFR) mediated signalling. Alternative roles of Mig-6 are becoming increasingly recognised. Consistently with this, Mig-6 was demonstrated to be involved in a broad spectrum of cellular events including tumour suppression which may include the induction of cellular senescence. Here, we investigated the mechanisms of Mig-6 induced premature cell senescence. Endogenous Mig-6 is poorly expressed in young fibroblasts, whilst its expression rises in cells presenting with typical features of senescence. Overexpression of Mig-6 is sufficient to trigger premature cellular senescence of early passage diploid lung fibroblasts (WI-38). Interestingly, Mig-6 overexpression reduced retinoblastoma protein (pRb) phosphorylation at the inactivating Ser249/Thr252 sites. We also found that phosphorylation of these sites in pRb is increased in the presence of the B-Raf V600E oncogenic mutation. We further show that Mig-6 overexpression reduces B-Raf V600E mediated pRb inactivation and preserves pRb function. 相似文献
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Receptor protein tyrosine phosphatase alpha participates in the m1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity. 总被引:3,自引:0,他引:3
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The phosphorylation state of a given tyrosine residue is determined by both protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activities. However, little is known about the functional interaction of these opposing activities at the level of an identified effector molecule. G protein-coupled receptors (GPCRs), including the m1 muscarinic acetylcholine receptor (mAChR), regulate a tyrosine kinase activity that phosphorylates and suppresses current generated by the Kv1.2 potassium channel. We examined the possibility that PTPs also participate in this signaling pathway since the tyrosine phosphatase inhibitor vanadate increases the extent of both Kv1.2 phosphorylation and suppression. We show that an endogenous transmembrane tyrosine phosphatase, receptor tyrosine phosphatase alpha (RPTPalpha), becomes tyrosine phosphorylated and co-immunoprecipitates with Kv1.2 in a manner dependent on m1 receptor activation. The N- and C-termini of Kv1.2 are shown to bind RPTPalpha in vitro. Overexpression of RPTPalpha in Xenopus oocytes increases resting Kv1.2 current. Biochemical and electrophysiological analysis reveals that recruiting RPTPalpha to Kv1.2 functionally reverses the tyrosine kinase-induced phosphorylation and suppression of Kv1.2 current in mammalian cells. Taken together, these results identify RPTPalpha as a new target of m1 mAChR signaling and reveal a novel regulatory mechanism whereby GPCR-mediated suppression of a potassium channel depends on the coordinate and parallel regulation of PTK and PTP activities. 相似文献
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The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site. 相似文献
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