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Molecular architecture of human properdin, a positive regulator of the alternative pathway of complement 总被引:7,自引:0,他引:7
C A Smith M K Pangburn C W Vogel H J Müller-Eberhard 《The Journal of biological chemistry》1984,259(7):4582-4588
The structure of the human properdin molecule was investigated by hydrodynamic, spectroscopic, and transmission electron microscope studies. Sucrose density gradient ultracentrifugation of purified, functionally active properdin showed a single component sedimenting at 5.5 S. Electron microscopic examination by two different methods, however, revealed polydispersity of the protein which consisted of cyclic dimers, trimers, tetramers, pentamers, and higher cyclic oligomers. Approximately 80% of the oligomers were dimers, trimers, and tetramers. Monomers could not be detected. These polymers could be partially separated by gel filtration on Sephacryl S-300 and all fractions were active in terms of binding to C3b. The specific activity increased with oligomer size. When reexamined after incubation at 37 degrees C for 4 h or at 4 degrees C for 2 weeks, the chromatographic behavior of the oligomers and their electron microscopic appearance were unchanged, suggesting that in vitro no rapid equilibration occurred. The protomer is clearly visualized within polymers as a flexible, rod-like structure 26.0 nm in length and 2.5 nm in diameter, with pronounced thickening at each end. The monomer is bivalent with respect to binding to other properdin monomers and the binding sites are localized to the ends of the structure. A model could be devised which is consistent with the distinct geometry of the intersubunit contacts observed in micrographs. The circular dichroism spectrum of properdin suggests the presence of little alpha helix or beta structure and shows positive ellipticity at 231 nm. In contrast to previous investigators, we conclude that isolated human properdin is polydisperse and consists of a set of cyclic polymers constructed from a single highly asymmetric and flexible protomer. 相似文献
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J Hofsteenge M Blommers D Hess A Furmanek O Miroshnichenko 《The Journal of biological chemistry》1999,274(46):32786-32794
C-Mannosylation is a unique form of protein glycosylation, involving the C-glycosidic attachment of a mannosyl residue to the indole moiety of Trp. In the two examples found so far, human RNase 2 and interleukin-12, only the first Trp in the recognition motif WXXW is specifically C-mannosylated. To establish the generality of protein C-mannosylation, and to learn more about its mechanism, the terminal components of the human complement system (C6, C7, C8,and C9), which contain multiple and complex recognition motifs, were examined. Together with C5b they form the cytolytic agent, the membrane attack complex. These are the first proteins that are C-mannosylated on more than one Trp residue as follows: six in C6, four in C7, C8alpha, and C8beta, and two in C9. Thus, from the 113 Trp residues in the complete membrane attack complex, 50 were found to undergo C-mannosylation. The other important finding is that in C6, C7, C8, and C9 Trp residues without a second Trp (or another aromatic residue) at the +3 position can be C-mannosylated. This shows that they must contain an additional C-mannosylation signal. Whether this is encoded in the primary or tertiary structure is presently unknown. Finally, all modified Trp residues are part of the highly conserved core of the thrombospondin type 1 repeats present in these proteins. Since this module has been found in a large number of other proteins, the results suggest further candidates for C-mannosylation. 相似文献
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Osteoclasts are hematopoietic lineage derived-multinucleated cells that resorb bone. Their activity in balance with that of osteoblast is essential for bone homeostasis. Receptor activator of NF-κB ligand (RANKL) is known as an essential cytokine for the osteoclastogenesis, and c-Jun signaling in cooperation with NFAT family is crucial for RANKL-regulated osteoclastogenesis. We show here that schlafen2 (Slfn2), a member of a new family of growth regulatory genes involved in thymocyte development, is critical for osteoclastogenesis. RANKL selectively induces Slfn2 expression in osteoclast precursors via Rac1 signaling pathway. Targeted inhibition of Slfn2 by small interfering RNAs (siRNAs) markedly inhibits the formation of osteoclasts by diminishing the activation of c-Jun and the expression of c-Jun and NFATc1. In contrast, the overexpression of Slfn2 markedly increased phosphorylation and transactivation of c-Jun by RANKL. Together, these results indicate that Slfn2 has an essential role in osteoclastogenesis, functioning upstream of c-Jun and NFATc1. 相似文献
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Gupta-Rossi N Ortica S Meas-Yedid V Heuss S Moretti J Olivo-Marin JC Israël A 《The Journal of biological chemistry》2011,286(21):18720-18730
The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates. 相似文献
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<正>Dear Editor,With the inevitable trend of global warming, it is urgent to understand how plants sense and respond to temperature increases for designing new crop varieties that can tolerate high ambient temperature. In Arabidopsis thaliana, high ambient temperature promotes hypocotyl elongation in seedlings and stimulates petiole elongation and hyponasty in rosette leaves. These changes in architecture are collectively 相似文献
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Hasseine LK Murdaca J Suavet F Longnus S Giorgetti-Peraldi S Van Obberghen E 《Experimental cell research》2007,313(9):1927-1942
Both VEGF and insulin are implicated in the pathogenesis of diabetic retinopathy. While it has been established for many years that the number of cell surface receptors impacts upon VEGF and insulin action, little is known about the precise machinery and proteins driving VEGF-R2 and IR degradation. Here, we investigate the role of Hepatocyte growth factor-Regulated tyrosine kinase Substrate (Hrs), a regulator of RTK trafficking, in VEGF and insulin signaling. We report that ectopic expression of Hrs increases VEGF-R2 and IR number and tyrosine phosphorylation, leading to amplification of their downstream signaling. The UIM (Ubiquitin Interacting Motif) domain of Hrs is required for Hrs-induced increases in VEGF-R2, but not in IR. Furthermore, Hrs is tyrosine-phosphorylated in response to VEGF and insulin. We show that the UIM domain is required for Hrs phosphorylation in response to VEGF, but not to insulin. Importantly, Hrs co-localizes with both VEGF-R2 and IR and co-immunoprecipitates with both in a manner independent of the Hrs-UIM domain. Finally, we demonstrate that Hrs inhibits Nedd4-mediated VEGF-R2 degradation and acts additively with Grb10. We conclude that Hrs is a positive regulator of VEGF-R2 and IR signaling and that ectopic expression of Hrs protects both VEGF-R2 and IR from degradation. 相似文献
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Kimura H Kwan KM Zhang Z Deng JM Darnay BG Behringer RR Nakamura T de Crombrugghe B Akiyama H 《PloS one》2008,3(9):e3174
Background
Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.Methodology/Principal Findings
We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.Conclusions/Significance
Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis. 相似文献10.
Csortos C Czikora I Bogatcheva NV Adyshev DM Poirier C Olah G Verin AD 《American journal of physiology. Lung cellular and molecular physiology》2008,295(3):L440-L450
TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection. 相似文献
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The beta-chain of human interleukin 12 (IL-12) contains at position 319-322, the sequence Trp-x-x-Trp. In human RNase 2 this is the recognition motif for a new, recently discovered posttranslational modification, i.e., the C-glycosidic attachment of a mannosyl residue to the side chain of tryptophan. Analysis of C-terminal peptides of recombinant IL-12 (rHuIL-12) by mass spectrometry and NMR spectroscopy revealed that Trp-319beta is (partially) C-mannosylated. This finding was extended by in vitro mannosylation experiments, using a synthetic peptide derived from the same region of the protein as an acceptor. Furthermore, human B-lymphoblastoid cells, which secrete IL-12, were found to contain an enzyme that carries out the C-mannosylation reaction. This shows that nonrecombinant IL-12 is potentially C-mannosylated as well. This is only the second report on a C-mannosylated protein. However, the occurrence of the C-mannosyltransferase activity in a variety of cells and tissues, and the presence of the recognition motif in many proteins indicate that more C-mannosylated proteins may be found. 相似文献
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Lyudmila Krassikova Boxi Zhang Divya Nagarajan Andr Lima Queiroz Merve Kacal Evangelos Samakidis Helin Vakifahmetoglu-Norberg Erik Norberg 《Cell death and differentiation》2021,28(3):1091
Cancer cells undergo complex metabolic alterations. The mechanisms underlying the tuning of cancer metabolism are under active investigation. Here, we identify the uncharacterized deubiquitinase JOSD2 as a positive regulator of cancer cell proliferation by displaying comprehensive effects on glucose catabolism. We found that JOSD2 directly controls a metabolic enzyme complex that includes Aldolase A, Phosphofructokinase-1 and Phosphoglycerate dehydrogenase, in vitro and in vivo. Further, JOSD2 expression, but not a catalytically inactive mutant, deubiquitinates and stabilizes the enzyme complex, thereby enhancing their activities and the glycolytic rate. This represents a selective JOSD2 feature that is not shared among other Machado–Joseph disease DUBs or observed in nontransformed cells. JOSD2 deficiency displays cytostatic effects and reduces glycolysis in a broad spectrum of tumor cells of distinct origin and its expression correlates with poor prognosis in non-small cell lung cancer. Overall, our study provides evidence for a previously unknown biological mechanism in which JOSD2 integrates glucose and serine metabolism with potential therapeutic implications.Subject terms: Cancer metabolism, Lung cancer 相似文献
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SarA is an essential positive regulator of Staphylococcus epidermidis biofilm development 总被引:6,自引:0,他引:6
Tormo MA Martí M Valle J Manna AC Cheung AL Lasa I Penadés JR 《Journal of bacteriology》2005,187(7):2348-2356
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