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991.
Danielson SR Held JM Oo M Riley R Gibson BW Andersen JK 《The Journal of biological chemistry》2011,286(9):7601-7608
Differential cysteine oxidation within mitochondrial Complex I has been quantified in an in vivo oxidative stress model of Parkinson disease. We developed a strategy that incorporates rapid and efficient immunoaffinity purification of Complex I followed by differential alkylation and quantitative detection using sensitive mass spectrometry techniques. This method allowed us to quantify the reversible cysteine oxidation status of 34 distinct cysteine residues out of a total 130 present in murine Complex I. Six Complex I cysteine residues were found to display an increase in oxidation relative to controls in brains from mice undergoing in vivo glutathione depletion. Three of these residues were found to reside within iron-sulfur clusters of Complex I, suggesting that their redox state may affect electron transport function. 相似文献
992.
Nagashima K Shumway SD Sathyanarayanan S Chen AH Dolinski B Xu Y Keilhack H Nguyen T Wiznerowicz M Li L Lutterbach BA Chi A Paweletz C Allison T Yan Y Munshi SK Klippel A Kraus M Bobkova EV Deshmukh S Xu Z Mueller U Szewczak AA Pan BS Richon V Pollock R Blume-Jensen P Northrup A Andersen JN 《The Journal of biological chemistry》2011,286(8):6433-6448
Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems. 相似文献
993.
Liu X Hein J Richardson SC Basse PH Toptan T Moore PS Gjoerup OV Chang Y 《The Journal of biological chemistry》2011,286(19):17079-17090
Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. We identified the hVam6p cytoplasmic protein involved in lysosomal processing as a novel interactor with MCV LT but not SV40 LT. hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma binding site. MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering. MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication. 相似文献
994.
Kristoffersen SM Tourasse NJ Kolstø AB Økstad OA 《Molecular biology and evolution》2011,28(2):963-983
Many short (<400 bp) interspersed sequence repeats exist in bacteria, yet little is known about their origins, mode of generation, or possible function. Here, we present a comprehensive analysis of 18 different previously identified repeated DNA elements, bcr1-bcr18 (?kstad OA, Hegna I, Lindback T, Rishovd AL, Kolst? AB. 1999. Genome organization is not conserved between Bacillus cereus and Bacillus subtilis. Microbiology. 145:621-631.; Tourasse NJ, Helgason E, ?kstad OA, Hegna IK, Kolst? AB. 2006. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol. 101:579-593.), in 36 sequenced genomes from the Bacillus cereus group of bacteria. This group consists of genetically closely related species with variable pathogenic specificity toward different hosts and includes among others B. anthracis, B. cereus, and B. thuringiensis. The B. cereus group repeat elements could be classified into three categories with different properties: Group A elements (bcr1-bcr3) exhibited highly variable copy numbers ranging from 4 to 116 copies per strain, showed a nonconserved chromosomal distribution pattern between strains, and displayed several features characteristic of mobile elements. Group B repeats (bcr4-bcr6) were present in 0-10 copies per strain and were associated with strain-specific genes and disruptions of genome synteny, implying a possible contribution to genome rearrangements and/or horizontal gene transfer events. bcr5, in particular, was associated with large gene clusters showing resemblance to integrons. In agreement with their potentially mobile nature or involvement in horizontal transfers, the sequences of the repeats from Groups A and B (bcr1-bcr6) followed a phylogeny different from that of the host strains. Conversely, repeats from Group C (bcr7-bcr18) had a conserved chromosomal location and orthologous gene neighbors in the investigated B. cereus group genomes, and their phylogeny matched that of the host chromosome. Several of the group C repeats exhibited a conserved secondary structure or had parts of the structure conserved, possibly indicating functional RNAs. Accordingly, five of the repeats in group C overlapped regions encoding previously characterized riboswitches. Similarly, other group C repeats could represent novel riboswitches, encode small RNAs, and/or constitute other types of regulatory elements with specific biological functions. The current analysis suggests that the multitude of repeat elements identified in the B. cereus group promote genome dynamics and plasticity and could contribute to the flexible and adaptive life style of these bacteria. 相似文献
995.
996.
Uhlén M Björling E Agaton C Szigyarto CA Amini B Andersen E Andersson AC Angelidou P Asplund A Asplund C Berglund L Bergström K Brumer H Cerjan D Ekström M Elobeid A Eriksson C Fagerberg L Falk R Fall J Forsberg M Björklund MG Gumbel K Halimi A Hallin I Hamsten C Hansson M Hedhammar M Hercules G Kampf C Larsson K Lindskog M Lodewyckx W Lund J Lundeberg J Magnusson K Malm E Nilsson P Odling J Oksvold P Olsson I Oster E Ottosson J Paavilainen L Persson A Rimini R Rockberg J Runeson M Sivertsson A 《Molecular & cellular proteomics : MCP》2005,4(12):1920-1932
Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research. 相似文献
997.
Lee CC Wood MD Ng K Andersen CB Liu Y Luginbühl P Spraggon G Katagiri F 《Structure (London, England : 1993)》2004,12(3):487-494
AvrB is a Pseudomonas syringae type III effector protein that is translocated into host plant cells during attempted pathogenesis. Arabidopsis harboring the corresponding resistance protein RPM1 can detect AvrB and mount a rapid host defense response, thus avoiding active infection. In the plant cell, AvrB induces phosphorylation of RIN4, a key component in AvrB/RPM1 recognition. Although the AvrB/RPM1 system is among the best characterized of the numerous bacterial effector/plant resistance protein systems involved in plant disease resistance and pathogenesis, the details of the molecular recognition mechanism are still unclear. To gain further insights, the crystal structure of AvrB was determined. The 2.2 A structure exhibits a novel mixed alpha/beta bilobal fold. Aided by the structural information, we demonstrate that one lobe is the determinant of AvrB/RPM1 recognition specificity. This structural information and preliminary structure-function studies provide a framework for the future understanding of AvrB function on the molecular level. 相似文献
998.
999.
Christian Nyrop Albers Ole Stig Jacobsen Jens Aamand 《Applied microbiology and biotechnology》2014,98(2):957-967
Micropollutants in groundwater are given significant attention by water companies and authorities due to an increasing awareness that they might be present even above the legal threshold values. As part of our investigations of the possibility to remove the common groundwater pollutant 2,6-dichlorobenzamide (BAM) by introducing the efficient BAM degrader Aminobacter sp. MSH1 into biologically active sand filters, we investigated if the strain adheres to filters containing various filter materials and if the initial adherence and subsequent degradation of BAM could be optimized. We found that most of the inoculated MSH1 cells adhered fast and that parameters like pH and ionic strength had only a minor influence on the adhesion despite huge influence on cell surface hydrophobicity. At the given growth protocol, the MSH1 strain apparently developed a subpopulation that had lost its ability to adhere to the filter materials, which was supported by attempted reinoculation of non-adhered cells. Analysis by quantitative PCR showed that most cells adhered in the top of the filters and that some of these were lost from the filters during initial operation, while insignificant losses occurred after 1 day of operation. The inoculated filters were found to degrade 2.7 μg/L BAM to below 0.1 μg/L at a 1.1-h residence time with insignificant formation of known degradation products. In conclusion, most filter materials and water types should be feasible for inoculation with the MSH1 strain, while more research into degradation at low concentrations and temperatures is needed before this technology is ready for use at actual waterworks. 相似文献
1000.
Egon Persson Jesper J. Madsen Ole H. Olsen 《Protein science : a publication of the Protein Society》2014,23(12):1717-1727
Formation of the factor VIIa (FVIIa)‐tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure‐based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)‐like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ‐carboxyglutamic acid (Gla)‐containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly‐Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF‐mediated stabilization of the active conformation of FVIIa and abrogated TF‐induced activity enhancement. In addition, FVIIa variants with short linkers associated 80‐fold slower with soluble TF (sTF) as compared with wild‐type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca2+‐free FVIIa; stepwise truncation resulting in gradually higher activity with des(83–86)‐FVIIa reaching the level of Gla‐domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo‐FVIIa activity. 相似文献