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121.
Adenovirus invades host cells by first binding to host receptors through a trimeric fiber, which contains three domains: a receptor-binding knob domain, a long flexible shaft domain, and a penton base-attachment tail domain. Although the structure of the knob domain associated with a portion of the shaft has been solved by X-ray crystallography, the in situ structure of the fiber in the virion is not known; thus, it remains a mystery how the trimeric fiber attaches to its underlying pentameric penton base. By high-resolution cryo-electron microscopy, we have determined the structure of the human adenovirus type 5 (Ad5) to 3.6-Å resolution and have reported the full atomic models for its capsid proteins, but not for the fiber whose density cannot be directly interpreted due to symmetry mismatch with the penton base. Here, we report the determination of the Ad5 fiber structure and its mode of attachment to the pentameric penton base by using an integrative approach of multi-resolution filtering, homology modeling, computational simulation of mismatched symmetries, and fitting of atomic models into cryo-electron microscopy density maps. Our structure reveals that the interactions between the trimeric fiber and the pentameric penton base are mediated by a hydrophobic ring on the top surface of the penton base and three flexible tails inserted into three of the five available grooves formed by neighboring subunits of penton base. These interaction sites provide the molecular basis for the symmetry mismatch and can be targeted for optimizing adenovirus for gene therapy applications. 相似文献
122.
Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina 总被引:4,自引:0,他引:4
Clearance of synaptic glutamate by glial cells is required for the normal function of excitatory synapses and for prevention of neurotoxicity. Although the regulatory role of glial glutamate transporters in glutamate clearance is well established, little is known about the influence of glial glutamate metabolism on this process. This study examines whether glutamine synthetase (GS), a glial-specific enzyme that amidates glutamate to glutamine, affects the uptake of glutamate. Retinal explants were incubated in the presence of [(14)C]glutamate and glutamate uptake was assessed by measurement of the amount of radioactively labeled molecules within the cells and the amount of [(14)C]glutamine released to the medium. An increase in GS expression in Müller glial cells, caused by induction of the endogenous gene, did not affect the amount of glutamate accumulated within the cells, but led to a dramatic increase in the amount of glutamine released. This increase, which was directly correlated with the level of GS expression, was dependent on the presence of external sodium ions, and could be completely abolished by methionine sulfoximine, a specific inhibitor of GS activity. Our results demonstrate that GS activity significantly influences the uptake of glutamate by the neural retina and suggest that this enzyme may represent an important target for neuroprotective strategies. 相似文献
123.
Nicolas Collin Regis Gomes Clarissa Teixeira Lily Cheng Andre Laughinghouse Jerrold M. Ward Dia-Eldin Elnaiem Laurent Fischer Jesus G. Valenzuela Shaden Kamhawi 《PLoS pathogens》2009,5(5)
Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-γ at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG2 antibody levels and significant IFN-γ production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-γ and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response. 相似文献
124.
125.
Gray NE Lam LN Yang K Zhou AY Koliwad S Wang JC 《The Journal of biological chemistry》2012,287(11):8444-8456
Intracellular triacylglycerol (TG) hydrolysis and fatty acid release by the white adipose tissue (WAT) during a fast is stimulated by counter-regulatory factors acting in concert, although how adipocytes integrate these lipolytic inputs is unknown. We tested the role of angiopoietin-like 4 (Angptl4), a secreted protein induced by fasting or glucocorticoid treatment, in modulating intracellular adipocyte lipolysis. Glucocorticoid receptor blockade prevented fasting-induced tissue Angptl4 expression and WAT TG hydrolysis in mice, and TG hydrolysis induced by fasts of 6 or 24 h was greatly reduced in mice lacking Angptl4 (Angptl4(-/-)). Glucocorticoid treatment mimicked the lipolytic effects of fasting, although with slower kinetics, and this too required Angptl4. Thus, fasting-induced WAT TG hydrolysis requires glucocorticoid action and Angptl4. Both fasting and glucocorticoid treatment also increased WAT cAMP levels and downstream phosphorylation of lipolytic enzymes. Angptl4 deficiency markedly reduced these effects, suggesting that Angptl4 may stimulate lipolysis by modulating cAMP-dependent signaling. In support of this, cAMP levels and TG hydrolysis were reduced in primary Angptl4(-/-) murine adipocytes treated with catecholamines, which stimulate cAMP-dependent signaling to promote lipolysis, and was restored by treatment with purified human ANGPTL4. Remarkably, human ANGPTL4 treatment alone increased cAMP levels and induced lipolysis in these cells. Pharmacologic agents revealed that Angptl4 modulation of cAMP-dependent signaling occurs upstream of adenylate cyclase and downstream of receptor activation. We show that Angptl4 is a glucocorticoid-responsive mediator of fasting-induced intracellular lipolysis and stimulates cAMP signaling in adipocytes. Such a role is relevant to diseases of aberrant lipolysis, such as insulin resistance. 相似文献
126.
Ilani T Fishburn CS Levavi-Sivan B Carmon S Raveh L Fuchs S 《Cellular and molecular neurobiology》2002,22(1):47-56
D2 and D3 dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D2 receptor couples only to inhibitory G proteins, D3 receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D2 and D3 receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D2 receptor couple to Gi protein in a pattern characteristic of D2 receptor. On the other hand chimeras containing a third cytoplasmic loop of D3 receptor have coupling characteristics like those of D3 receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D2 and D3 to G proteins. 相似文献
127.
128.
Liu G Kennedy R Greenshields DL Peng G Forseille L Selvaraj G Wei Y 《Molecular plant-microbe interactions : MPMI》2007,20(10):1308-1319
The agriculturally important genus Colletotrichum is an emerging model pathogen for studying defense in Arabidopsis. During the process of screening for novel pathogenic Colletotrichum isolates on Arabidopsis, we found significant differences in defense responses between detached and attached leaf assays. A near-adapted isolate Colletotrichum linicola A1 could launch a typical infection only on detached, but not attached, Arabidopsis leaves. Remarkably, resistance gene-like locus RCH1-mediated resistance in intact plants also was compromised in detached leaves during the attacks with the virulent reference isolate C. higginsianum. The differences in symptom development between the detached leaf and intact plant assays were further confirmed on defense-defective mutants following inoculation with C. higginsianum, where the greatest inconsistency occurred on ethylene-insensitive mutants. In intact Arabidopsis plants, both the salicylic acid- and ethylene-dependent pathways were required for resistance to C. higginsianum and were associated with induced expression of pathogenesis-related genes PR1 and PDF1.2. In contrast, disease symptom development in detached leaves appeared to be uncoupled from these defense pathways and more closely associated with senescence: an observation substantiated by coordinated gene expression analysis and disease symptom development, and chemically and genetically mimicking senescence. 相似文献
129.
Isolation and characterization of a novel thermostable neopullulanase-like enzyme from a hot spring in Thailand 总被引:1,自引:0,他引:1
Tang K Kobayashi RS Champreda V Eurwilaichitr L Tanapongpipat S 《Bioscience, biotechnology, and biochemistry》2008,72(6):1448-1456
A gene encoding a thermostable pullulan-hydrolyzing enzyme was isolated from environmental genomic DNA extracted from soil sediments of Bor Khleung hot spring in Thailand. Sequence comparison with related enzymes suggested that the isolated enzyme, designated Env Npu193A, was most likely a neopullulanase-like enzyme. Env Npu193A was expressed in Pichia pastoris as a monomeric recombinant protein. The purified Env Npu193A exhibited pH stability ranging from 3 to 9. More than 60% of enzyme activity was retained after incubation at 60 degrees C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and gamma-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75 degrees C with K(m) and V(max) toward pullulan of 1.22+/-0.3% and 23.24+/-1.7 U/mg respectively. Env Npu193A exhibited distinct biochemical characteristics as compared with the previously isolated enzyme from the same source. Thus, a culture-independent approach with sequence-basing was found to be an effective way to discover novel enzymes displaying unique substrate specificity and high thermostability from natural bioresources. 相似文献
130.
The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders. 相似文献