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111.
Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation 总被引:1,自引:0,他引:1
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Monoubiquitylation is a well-characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin-binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded alpha-helices that bind ubiquitin independently, non-cooperatively and with modest affinity. The Vps27 N-terminal UIM engages the Leu8-Ile44-Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8-Ile44-Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin-binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome. 相似文献
112.
Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments. 相似文献
113.
Subunit composition of kainate receptors in hippocampal interneurons 总被引:16,自引:0,他引:16
Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function. 相似文献
114.
Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1 总被引:1,自引:0,他引:1
The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS. 相似文献
115.
Hotchkiss RS Tinsley KW Hui JJ Chang KC Swanson PE Drewry AM Buchman TG Karl IE 《Journal of immunology (Baltimore, Md. : 1950)》2000,164(7):3675-3680
Sepsis induces extensive apoptosis of lymphocytes, which may be responsible for the profound immune suppression of the disorder. Two potential pathways of sepsis-induced lymphocyte apoptosis, Fas and p53, were investigated. Lymphocyte apoptosis was evaluated 20-22 h after sepsis by annexin V or DNA nick-end labeling. Fas receptor-deficient mice had no protection against sepsis-induced apoptosis in thymocytes or splenocytes. p53 knockout mice (p53-/-) had complete protection against thymocyte apoptosis but, surprisingly, had no protection in splenocytes. p53-/- mice had no improvement in sepsis survival compared with appropriately matched control mice with sepsis. We conclude that both p53-dependent and p53-independent pathways of cell death exist in sepsis. This differential apoptotic response of thymocytes vs splenocytes in p53-/- mice suggests that either the cellular response or the death-inducing signal is cell-type specific in sepsis. The fact that p53-/- lymphocytes of an identical subtype (CD8-CD4+) were protected in thymi but not in spleens indicates that cell susceptibility to apoptosis differs depending upon other unidentified factors. 相似文献
116.
The structure of an insect chymotrypsin 总被引:3,自引:0,他引:3
Botos I Meyer E Nguyen M Swanson SM Koomen JM Russell DH Meyer EF 《Journal of molecular biology》2000,298(5):895-901
The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme. 相似文献
117.
Desai U Swanson R Bock SC Bjork I Olson ST 《The Journal of biological chemistry》2000,275(25):18976-18984
The contribution of Arg(129) of the serpin, antithrombin, to the mechanism of allosteric activation of the protein by heparin was determined from the effect of mutating this residue to either His or Gln. R129H and R129Q antithrombins bound pentasaccharide and full-length heparins containing the antithrombin recognition sequence with similar large reductions in affinity ranging from 400- to 2500-fold relative to the control serpin, corresponding to a loss of 28-35% of the binding free energy. The salt dependence of pentasaccharide binding showed that the binding defect of the mutant serpin resulted from the loss of approximately 2 ionic interactions, suggesting that Arg(129) binds the pentasaccharide cooperatively with other residues. Rapid kinetic studies showed that the mutation minimally affected the initial low affinity binding of heparin to antithrombin, but greatly affected the subsequent conformational activation of the serpin leading to high affinity heparin binding, although not enough to disfavor activation. Consistent with these findings, the mutant antithrombin was normally activated by heparin for accelerated inhibition of factor Xa and thrombin. These results support an important role for Arg(129) in an induced-fit mechanism of heparin activation of antithrombin wherein conformational activation of the serpin positions Arg(129) and other residues for cooperative interactions with the heparin pentasaccharide so as to lock the serpin in the activated state. 相似文献
118.
Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats 总被引:3,自引:0,他引:3
Morton H McKay DA Murphy RM Somodevilla-Torres MJ Swanson CE Cassady AI Summers KM Cavanagh AC 《Immunology and cell biology》2000,78(6):603-607
Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX-2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half-life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N-terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers. 相似文献
119.
120.
Ligation of proteins to ubiquitin requires activation of ubiquitin by E1, the ubiquitin-activating enzyme. Mutant alleles of E1 in mammalian cells have been crucial for dissecting the contribution of the ubiquitin system to cell function. Comparable mutants have been unavailable for Saccharomyces cerevisiae. Here we describe the isolation and characterization of a hypomorphic allele of S. cerevisiae E1. Protein modification by ubiquitin is strongly impaired in the mutant, inhibiting degradation of ubiquitin-proteasome pathway substrates as well as ubiquitin-dependent but proteasome-independent degradation of membrane receptors. This allele will be a useful tool for evaluating the ubiquitin-dependence of cellular processes in yeast, even those in which the proteasome is not involved. 相似文献