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Cytosolic Proteins (cytosolic + protein)
Selected AbstractsThe first two-dimensional reference map of the fission yeast, Schizosaccharomyces pombe proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2005Namkyu Sun Abstract Cytosolic proteins of Schizosaccharomyces pombe were separated by two-dimensional (2-D) gel electrophoresis, to construct the first 2-D reference map. In the pI range 4,7, more than 500,spots were detected by silver staining, and 70 different proteins corresponding to 111,spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry, where necessary. In the pI range 6,9, approximately 330,spots were detected, and 31,proteins corresponding to 38,spots were identified by mass spectrometry. More than 50% of the identified proteins were involved in amino acid, carbohydrate or nucleotide metabolism, and energy production. A second large group of identified proteins comprises heat shock and other stress related proteins and chaperones. [source] Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-terminiFEBS JOURNAL, Issue 5 2006Ulrike Ziechner Human ether à go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674,683, BD-C2: 711,721) and one in the N-terminus (BD-N: 151,165). Binding of GST-fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD-N and BD-C2 provided dissociation constants in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM-EF-hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed. [source] The role of mitochondria, cytochrome c and caspase-9 in embryonic lens fibre cell denucleationJOURNAL OF ANATOMY, Issue 2 2002E. J. Sanders Abstract During the differentiation of secondary lens fibre cells from the lens epithelium, the fibre cells lose all of their cytoplasmic organelles as well as their nuclei. The fibre cells, containing crystallins, which confer optical clarity, then persist in the adult lens. The process of denucleation of these cells has been likened to an apoptotic event which is not followed by the plasma membrane changes that are characteristic of apoptosis. We have examined the expression and subcellular translocation of molecules of the apoptotic cascade in differentiating lens epithelial cells in culture. In this culture system, the epithelial cells differentiate into lentoids composed of lens fibre cells. We find that caspase-9, which is expressed and activated before embryonic day 12 in intact lenses, is localized in the cytosol outside mitochondria in non-differentiating cultured cells. In lentoid cells, caspase-9 migrates into mitochondria after the latter undergo a membrane permeability transition that is characteristic of apoptotic cells. At the same time, caspase-9 co-localizes with cytochrome c in the cytosol. The cytochrome c is apparently released from the mitochondria in lentoid cells after the mitochondrial membrane permeability transition and during the period of nuclear shrinkage. Also during this time, the mitochondria aggregate around the degenerating nuclei. Cytochrome c disappears rapidly, while mitochondrial breakdown occurs approximately coincident with the disappearance of the nuclei, but mitochondrial remnants persist together with cytochrome c oxidase, which is a mitochondrial marker protein. Apaf-1, another cytosolic protein of the apoptotic cascade, also migrates to the permeabilized mitochondria and also co-localizes with caspase-9 and cytochrome c in the cytosol or mitochondria of denucleating cells, thus providing evidence for the formation of an ,apoptosome' in these cells, as in apoptotic cells. At no time did we observe the translocation of molecules between cytoplasmic compartments and the nucleus in differentiating lentoid cells. We suggest that the uncoupling of nuclear and membrane apoptotic events in these cells may be due to the early permeability changes in the mitochondria, resulting in the loss of mitochondrial signalling molecules, or to the failure of molecules to migrate to the nucleus in these cells, thus failing to activate nuclear-plasma membrane signalling pathways. [source] Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14,Toll-like receptor,nuclear factor ,B pathwayJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2003Jun-ichi Kido Objectives:, Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor ,B (NF-,B). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14,TLR,NF-,B pathway and the cellular mechanism of calprotectin release in human neutrophils. Material and methods:, Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-,B, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-,B binding activity using electrophoretic mobility shift assays. Results:, P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-,B inhibitors suppressed P-LPS-induced NF-,B binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. Conclusion:, These results suggest that calprotectin release is induced by P-LPS via the CD14,TLR2,NF-,B signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems. [source] Staphylococcal NreB: an O2 -sensing histidine protein kinase with an O2 -labile iron,sulphur cluster of the FNR typeMOLECULAR MICROBIOLOGY, Issue 3 2004Annegret Kamps Summary The nreABC (nitrogen regulation) operon encodes a new staphylococcal two-component regulatory system that controls dissimilatory nitrate/nitrite reduction in response to oxygen. Unlike other two-component sensors NreB is a cytosolic protein with four N-terminal cysteine residues. It was shown that both the NreB,cysteine cluster and Fe ions are required for function. Isolated NreB was converted to the active form by incubation with cysteine desulphurase, ferrous ions and cysteine. This activation is typical for FeS-containing proteins and was reversed by oxygen. During reconstitution an absorption band at 420 nm and a yellow-brownish colour (typical for an FNR-type iron,sulphur cluster formation) developed. After alkylation of thiol groups in NreB and in the cysteine mutant NreB(C62S) almost no iron,sulphur cluster was incorporated; both findings corroborated the importance of the cysteine residues. Comparison of the kinase activity of (i) the reconstituted (ii) the unreconstituted, and (iii) the unreconstituted and deferrated NreB,His indicated that NreB kinase activity depended on iron availability and was greatly enhanced by reconstitution. NreB is the first direct oxygen-sensing protein described in staphylococci so far. Reconstituted NreB contains 4,8 acid-labile Fe and sulphide ions per NreB which is in agreement with the presence of 1,2 iron,sulphur [4Fe-4S]2+ clusters of the FNR-type. Unlike FNR, NreB does not act directly as transcriptional activator, but transfers the phosphoryl group to the response regulator NreC. [source] Intratesticular localization of the organic solute carrier protein, OSCP1, in spermatogenic cells in miceMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2008Kazuyuki Hiratsuka Abstract Organic solute carrier protein 1 (OSCP1) is a recently described human gene that facilitates the transport of various organic solutes into the cell, when expressed in frog eggs. In this study, we cloned a mouse ortholog of OSCP1 encoding 379 amino acid protein, with 94% homology to the human counterpart. The mouse OSCP1 mRNA was predominantly expressed in the testis, in which it was attributed to the spermatogenic cells, except the spermatogonia. Immunohistochemistry confirmed that OSCP1 protein is continuously expressed during spermatogenesis in a stage- and cell type-specific manner, in the leptotene spermatocytes at stage IX through step 15 spermatids. Subcellular fractionation of mouse testis homogenates indicated that OSCP1 is a 45-kDa cytosolic protein. Moreover, when green fluorescent protein-OSCP1 fusion constructs were transfected into cultured cells, the fluorescence localized evenly in the cytoplasm. These results suggest that mouse testis OSCP1 may indirectly mediate substrate uptake into meiotic and spermiogenic germ cells, within the cytosol. Mol. Reprod. Dev. 75: 1495,1504 © 2008 Wiley-Liss, Inc. [source] Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plantsPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2008Alessandra Barbante Summary The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions. [source] Redox regulation of cyclophilin A by glutathionylationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Pietro Ghezzi Abstract Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin,A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin,A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo. Using MALDI-MS we identified Cys52 and Cys62 as targets of glutathionylation in T,lymphocytes, and, using bioinformatic tools, we defined the reasons for the susceptibility of these residues to the modification. In addition, we found by circular dichroism spectroscopy that glutathionylation has an important impact on the secondary structure of CypA. Finally, we suggest that glutathionylation of CypA may have biological implications and that CypA may play a key role in redox regulation of immunity. [source] Structure of the thioredoxin-like domain of yeast glutaredoxin 3ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2008Lydia M. Gibson Yeast glutaredoxin 3 (Grx3) is a cytosolic protein that regulates the activity of the iron-responsive transcriptional activator Aft1. This member of the monothiol glutaredoxin family contains a thioredoxin-like domain and a glutaredoxin-like domain, which both possess a monothiol active site. The crystal structure of the thioredoxin-like domain has been determined at 1.5,Å resolution and represents the first published structure of this domain for the monothiol glutaredoxin family. The loop containing the signature motif WAxxC is partially disordered, indicating a greater degree of flexibility in this region compared with classical dithiol thioredoxins with a WCGPC active-site motif. [source] Regulation of Mg2+ -independent Ca2+ -ATPase by a low molecular mass protein purified from bovine brainBIOFACTORS, Issue 4 2006Srabasti Ghoshal Abstract The goat sperm microsomal membranes have been found to contain an Mg2+ -independent Ca2+ -ATPase, a low affinity but highly active enzyme sharing similarities with the SERCA family of ATPases. The present study reports the identification and characterization of a 14 kilodalton cytosolic protein from bovine brain which can act as an endogenous stimulator of the enzyme with an S50 (concentration producing 50% stimulation) of 0.8 , molar. Kinetic analysis suggests that the stimulation is noncompetitive with respect to the substrate, and the binding site(s) of the stimulator and substrate are distinct. Binding of the stimulator to the enzyme is reversible. The stimulator increases the affinity of the enzyme for calcium as evident from a decrease in K0.5 of the enzyme for calcium in presence of the stimulator. Radioactive labeling of the enzyme with [,;- 32P]-ATP suggests that the stimulator enhances the rate of dephosphorylation of the phosphoenzyme intermediate without altering the phosphorylation reaction step. The stimulatory effect of the protein has been observed only for the Mg2+ -independent form of the enzyme, the Mg2+ -dependent form being unaffected. [source] Enhanced sensitivity to cis -diamminedichloro-platinum(II) of a human carcinoma cell line with mutated p53 gene by cyclin-dependent kinase inhibitor p21WAF1 expressionCANCER SCIENCE, Issue 3 2003Mamoru Satou p53-mediated induction of p21WAF1, a cyclin-dependent protein kinase inhibitor, is known to protect cancer cells from the cytotoxic effects of anti-cancer drugs or ,-irradiation. Since the p53 gene is frequently inactivated in cancer cells, we examined whether p21WAF1 expression may alter the sensitivity of cancer cells with mutated p53 gene to anti-cancer drugs. Cells of a colon cancer cell line DLD-1 were transfected with p21WAF1 expression vector controlled by a tetracycline-repressable promoter and transfec-tants were cloned (Dp21,1). p21WAF1 expression induced by removal of tetracycline from culture media repressed cell proliferation and resulted in altered cell shape, suggesting induction of differentiation. Dp21,1 cells with p21WAF1 expression were more sensitive to cis -diamminedichloroplatinum(II) (CDDP) (IC50 value, 10 ,M) than those without p21WAF1 expression (IC50, 22 ,M). Sensitivity to doxorubicin was not different between Dp21,1 cells with and without p21WAF1 expression. DNA ladder formation was observed in Dp21,1 cells treated with CDDP, indicating that the enhanced sensitivity to CDDP involves apoptosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic protein revealed that subunit protein bands with Mr 55 kDa and 44 kDa were markedly increased in cells with p21WAF1 expression. By im-munoblotting, these proteins were identified as c-Jun N-terminal kinase (JNK) 2 and p38 mitogen-activated protein kinase (MAPK) 8, respectively, both of which are believed to be involved in apoptosis induction by CDDP. These results suggest that p21WAF1 may enhance the sensitivity of colon cancer cells with mutated p53 gene to CDDP, possibly through the JNK and p38 MAPK pathways. (Cancer Sci 2003; 94: 286,291) [source] Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2ELECTROPHORESIS, Issue 14 2006Richard C. Barry Abstract A proteomic map of Sulfolobus solfataricus,P2, an archaeon that grows optimally at 80°C and pH,3.2, was developed using high-resolution 2-DE and peptide mass fingerprinting. A total of 867,protein spots (659,aqueous Tris-soluble spots and 208,aqueous Tris-insoluble) were mapped over IPG,3,10, 4,7, and 6,11, with second-dimensional gels made of 8,18%,polyacrylamide. Three hundred and twenty-four different gene products were represented by the 867,spots, with 274,gene products being identified in the Tris-soluble fractions and 100,gene products in the Tris-insoluble portion. Fifty gene products were found on gels from both fractions. Additionally, an average of 1.50 ± 0.12 isoforms/protein was identified. This mapping study confirmed the expression of proteins involved in numerous metabolic, transport, energy production, nucleic acid replication, translation, and transcription pathways. Of particular interest, phosphoenolpyruvate carboxykinase,(SSO2537) was detected even though the pathway for gluconeogenesis is unknown for this archaeon. Tris-soluble fractions contained many cytosolic proteins while Tris-insoluble fractions contained many membrane-associated proteins, including ABC transporters and an ATP synthase. This study provides an optimized 2-DE approach for investigating the biochemical pathways and post-translational modifications employed by Sulfolobus to survive in its extreme environment. [source] New concepts in bilirubin encephalopathyEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2003J. D. Ostrow Abstract Revised concepts of bilirubin encephalopathy have been revealed by studies of bilirubin toxicity in cultured CNS cells and in congenitally jaundiced Gunn rats. Bilirubin neurotoxicity is related to the unbound (free) fraction of unconjugated bilirubin (Bf), of which the dominant species at physiological pH is the protonated diacid, which can passively diffuse across cell membranes. As the binding affinity of plasma albumin for bilirubin decreases strikingly as albumin concentration increases, previously reported Bf values were underestimated. Newer diagnostic tests can detect reversible neurotoxicity before permanent damage occurs from precipitation of bilirubin (kernicterus). Early toxicity can occur at Bf only modestly above aqueous saturation and affects astrocytes and neurons, causing mitochondrial damage, resulting in impaired energy metabolism and apoptosis, plus cell-membrane perturbation, which causes enzyme leakage and hampers transport of neurotransmitters. The concentrations of unbound bilirubin in the cerebro-spinal fluid and CNS cells are probably limited mainly by active export of bilirubin back into plasma, mediated by ABC transporters present in the brain capillary endothelium and choroid plexus epithelium. Intracellular bilirubin levels may be diminished also by oxidation, conjugation and binding to cytosolic proteins. These new concepts may explain the varied susceptibility of neonates to develop encephalopathy at any given plasma bilirubin level and the selective distribution of CNS lesions in bilirubin encephalopathy. They also can suggest better strategies for predicting, preventing and treating this syndrome. [source] Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycleFEBS JOURNAL, Issue 17 2001Shin-ichi Yokota The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of ,, , and ,-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the , and , subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower , and , subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the , and , subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of , and , subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells. [source] Detection of a homotetrameric structure and protein,protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insightsFEMS YEAST RESEARCH, Issue 1 2010Clayton Luiz Borges Abstract Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role in fungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was overexpressed in bacteria and a polyclonal antibody to this protein was produced. We identified a 180-kDa protein species reactive to the antibody produced in mice against the P. brasiliensis recombinant purified formamidase of 45 kDa. The 180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide mass fingerprinting using MS. The identical mass spectra generated by the 180 and the 45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formamidase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights into formamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism. [source] Chronic Ethanol Consumption Induces Global Hepatic Protein HyperacetylationALCOHOLISM, Issue 2 2010Blythe D. Shepard Background:, Although the clinical manifestations of alcoholic liver disease are well described, little is known about the molecular basis for liver injury. Recent studies have indicated that chronic alcohol consumption leads to the lysine-hyperacetylation of several hepatic proteins, and this list is growing quickly. Methods:, To identify other hyperacetylated proteins in ethanol-fed livers, we chose a proteomics approach. Cytosolic and membrane proteins (excluding nuclei) were separated on 2D gels, transferred to PVDF and immunoblotted with antibodies specific for acetylated lysine residues. Hyperacetylated proteins were selected for trypsin digestion and mass spectrometric analysis. Results:, In all, 40 proteins were identified, 11 of which are known acetylated proteins. Remarkably, the vast majority of hyperacetylated membrane proteins were mitochondrial residents. Hyperacetylated cytosolic proteins ranged in function from metabolism to cytoskeletal support. Notably, 3 key anti-oxidant proteins were identified whose activities are impaired in ethanol-treated cells. We confirmed that the anti-oxidant enzyme, glutathione peroxidase 1, actin and cortactin are hyperacetylated in ethanol-treated livers. Conclusions:, Alcohol-induced hyperacetylation of multiple proteins may contribute to the development of liver injury. The abundance of acetylated mitochondrial proteins further suggests that this modification is important in regulating liver metabolism and when perturbed, may contribute to the progression of a variety of metabolic diseases. [source] Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteriaMOLECULAR MICROBIOLOGY, Issue 6 2007Luciano F. Huergo Summary Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the PII family. In this study we have characterized in vitro complex formation between the AmtB and PII proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB,PII complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg2+ and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB,PII complex defines a new regulatory role for Amt proteins in Prokaryotes. [source] Trigger Factor and DnaK possess overlapping substrate pools and binding specificitiesMOLECULAR MICROBIOLOGY, Issue 5 2003Elke Deuerling Summary Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, ,tig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37°C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted ,tig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities. [source] Proteomic profiling of exosomes: Current perspectivesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2008Richard J. Simpson Professor Abstract Exosomes are 40,100,nm membrane vesicles of endocytic origin secreted by most cell types in vitro. Recent studies have shown that exosomes are also found in vivo in body fluids such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk. While the biological function of exosomes is still unclear, they can mediate communication between cells, facilitating processes such as antigen presentation and in trans signaling to neighboring cells. Exosome-like vesicles identified in Drosophila (referred to as argosomes) may be potential vehicles for the spread of morphogens in epithelia. The advent of current MS-based proteomic technologies has contributed significantly to our understanding of the molecular composition of exosomes. In addition to a common set of membrane and cytosolic proteins, it is becoming increasingly apparent that exosomes harbor distinct subsets of proteins that may be linked to cell-type associated functions. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses such as HIV suggest their participation in pathological situations. Interestingly, the recent observation that exosomes contain both mRNA and microRNA, which can be transferred to another cell, and be functional in that new environment, is an exciting new development in the unraveling exosome saga. The present review aims to summarize the physical properties that define exosomes as specific cell-type secreted membrane vesicles. [source] Sequential detergent fractionation of primary neurons for proteomics studiesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2008Simonetta Bernocco Dr. Abstract Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent-based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment-specific manner. The sequential use of detergent-containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane-bound or enclosed, nuclear, and cytoskeletal proteins. Fractionation of neurons was validated by comparison of the distribution of known subcellular marker proteins in the four extracts using Western blotting. Comparison of extracts by DIGE showed a clear difference in protein composition demonstrating significant variations with a fold change (FC) of at least 1.20 for 82% of the detected spots. Using proteins identified in these spots that could be assigned a subcellular localization based on descriptions in the Uniprot database, an extraction efficiency of 85% was calculated for cytosolic proteins in extract 1, 90% for membrane-bound and membrane-enclosed proteins in extract 2, 82% for nuclear proteins in extract 3 and 38% for cytoskeletal and RAFT proteins in extract 4. [source] BTB and TAZ domain scaffold proteins perform a crucial function in Arabidopsis developmentTHE PLANT JOURNAL, Issue 1 2009Hélène S. Robert Summary In Arabidopsis, bric-a-brac, tramtrack and broad (BTB) domain scaffold proteins form a family of 80 proteins that have involvement in various signaling pathways. The five members of the subfamily of BTB AND TAZ DOMAIN proteins (BT1,BT5) have a typical domain structure that is only observed in land plants. Here, we present a functional analysis of the BT family, of which at least four members are encoded by auxin-responsive genes. BT1 is a short-lived protein that is characteristically targeted for degradation by the 26S proteasome. Expression pattern, gene structure and sequence analyses indicate that BT1 and BT2 are closely related. They both localize to the nucleus and the cytosol, whereas the remaining BT proteins were determined as cytosolic proteins. Detailed molecular and phenotypic analysis of plants segregating for null mutations in the BT family revealed substantial redundancy among the BT members, and highlighted that BT proteins perform crucial roles in both male and female gametophyte development. BT2 seems to be the predominant gene in this process, in which it is functionally replaced by BT3 and BT1 through reciprocal transcription regulation. Compensational expression alters the steady-state mRNA levels among the remaining BT family members when other BT members are lost, and this contributes towards functional redundancy. Our data provide a surprising example of functional redundancy among genes required during gametophyte development, something that could not be detected in the current screens for gametophyte mutants. [source] Synthetic dimer of indole-3-carbinol: Second generation diet derived anti-cancer agent in hormone sensitive prostate cancerTHE PROSTATE, Issue 5 2006Venkata P.S. Garikapaty Abstract BACKGROUND Cruciferous vegetables have been found to have anti-prostate cancer effects. The active compounds mediating these effects include indoles such as indole-3-carbinol (I3C) and isothiocyanates. I3C is unstable having tissue tropic effects and clinical utility has been partly addressed by the synthesis of a more stable dimer diindolylmethane (DIM). METHODS Anti-proliferative activity was measured by XTT assay and cytosolic proteins quantitated by Western blot analysis. RESULTS DIM (IC50 50 µM) is a better anti-proliferative agent than I3C (IC50 150 µM) in androgen dependent LNCaP cells, inhibits DNA synthesis, and growth of R1881 stimulated LNCaP cells. Androgen receptor (AR), cyclin D1, and cdk4, induced by R1881, are downregulated by DIM. DIM downregulates phosphorylated Akt and phosphatidyl inositol 3-kinase and downstream inhibition of cyclin D1 and cdk4. CONCLUSION These studies provide evidence that DIM is a second-generation chemopreventive agent with a viable cellular target and has clinical potential as an anti-prostate cancer chemopreventive. © 2005 Wiley-Liss, Inc. [source] Crystallographic characterization of the PDZ1 domain of the human Na+/H+ exchanger regulatory factorACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2001Gordon Webster The Na+/H+ exchanger regulatory factor (NHERF) contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. The human NHERF PDZ1 domain, which spans residues 11,99, interacts specifically with carboxy-terminal residues of the ,2 adrenergic receptor and the cystic fibrosis transmembrane conductance regulator. The NHERF PDZ1 was expressed in Escherichia coli as a soluble protein, purified and crystallized in the unbound form using the vapor-diffusion method with 2,M ammonium sulfate as the precipitant. Diffraction data were collected to 1.5,Å resolution using synchrotron radiation. The crystals belong to space group P3121 or P3221, with unit-cell parameters a = b = 51.6, c = 58.9,Å, and one molecule in the asymmetric unit. [source] Use of thallium to identify monovalent cation binding sites in GroELACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Philip D. Kiser GroEL is a bacterial chaperone protein that assembles into a homotetradecameric complex exhibiting D7 symmetry and utilizes the co-chaperone protein GroES and ATP hydrolysis to assist in the proper folding of a variety of cytosolic proteins. GroEL utilizes two metal cofactors, Mg2+ and K+, to bind and hydrolyze ATP. A K+ -binding site has been proposed to be located next to the nucleotide-binding site, but the available structural data do not firmly support this conclusion. Moreover, more than one functionally significant K+ -binding site may exist within GroEL. Because K+ has important and complex effects on GroEL activity and is involved in both positive (intra-ring) and negative (inter-ring) cooperativity for ATP hydrolysis, it is important to determine the exact location of these cation-binding site(s) within GroEL. In this study, the K+ mimetic Tl+ was incorporated into GroEL crystals, a moderately redundant 3.94,Å resolution X-ray diffraction data set was collected from a single crystal and the strong anomalous scattering signal from the thallium ion was used to identify monovalent cation-binding sites. The results confirmed the previously proposed placement of K+ next to the nucleotide-binding site and also identified additional binding sites that may be important for GroEL function and cooperativity. These findings also demonstrate the general usefulness of Tl+ for the identification of monovalent cation-binding sites in protein crystal structures, even when the quality and resolution of the diffraction data are relatively low. [source] Proteome mapping of overexpressed membrane-enriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovaniBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 4 2010Awanish Kumar WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Over 60% of patients with visceral leishmaniasis (VL) in India and Sudan have become unresponsive to treatment with pentavalent antimonials, the first line of drugs for over 60 years. The drug resistance mechanism, studied so far in in vitro selected laboratory strains, has been attributed to various biochemical parameters. The resistance to Sb (V) in Leishmania field isolates is still unexplored. WHAT THIS STUDY ADDS In order to elucidate for the first time the mechanism of drug resistance in field isolates, this study was done in those clinically relevant field isolates which were either responsive or non responsive to SAG. A comparison of proteome profiles of membrane-enriched as well as cytosolic protein fractions of these isolates has pinpointed the multiple overexpressed proteins in resistant isolates. This study has indicated their possible essential role in antimony resistance of the parasite and provides a vast field to be exploited to find much needed novel treatment strategies against VL. AIMS This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism. METHODS The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF. RESULTS Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine,leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL. CONCLUSION This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins. [source] | ||||||||||||||||