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1.
Yue Ren Yang Li Yaojie Wang Tianlei Wen Xuhang Lu Shenghai Chang Xing Zhang Yuequan Shen Xue Yang 《The Journal of biological chemistry》2022,298(5)
Calcium homeostasis modulator 1 (CALHM1) is a voltage- and Ca2+-gated ATP channel that plays an important role in neuronal signaling. However, as the previously reported CALHM structures are all in the ATP-conducting state, the gating mechanism of ATP permeation is still elusive. Here, we report cryo-EM reconstructions of two Danio rerio CALHM1 heptamers with ordered or flexible long C-terminal helices at resolutions of 3.2 Å and 2.9 Å, respectively, and one D. rerio CALHM1 octamer with flexible long C-terminal helices at a resolution of 3.5 Å. Structural analysis shows that the heptameric CALHM1s are in an ATP-nonconducting state with a central pore diameter of approximately 6.6 Å. Compared with those inside the octameric CALHM1, the N-helix inside the heptameric CALHM1 is in the “down” position to avoid steric clashing with the adjacent TM1 helix. Molecular dynamics simulations show that as the N-helix moves from the “down” position to the “up” position, the pore size of ATP molecule permeation increases significantly. Our results provide important information for elucidating the mechanism of ATP molecule permeation in the CALHM1 channel. 相似文献
2.
Fangfang Zhong Hongyan Wang Tianlei Ying Zhong-Xian Huang Xiangshi Tan 《Amino acids》2010,39(2):399-408
Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway
of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a α-subunit (690 AA) and a heme-binding β-subunit (619
AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate
(cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation,
platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling
was made in this study. The recombinant human sGC β1 subunit (HsGCβ619) and its truncated N-terminal fragments (HsGCβ195 and
HsGCβ384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were
characterized by UV–vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and
the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl
species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism
was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient
expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC. 相似文献
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The receptor for human endothelial differentiation gene-1 protein (EDG-1) was C-terminally tagged with green fluorescent protein and expressed in the methylotrophic yeast, Pichia pastoris. EDG-1 expression was driven by the highly inducible alcohol oxidase 1 promoter. Expression of EDG-1 recombinant protein was detected by Western blot analysis and confocal microscopy. The recombinant EDG-1 receptor protein was located in the plasma membrane. Radioligand binding assays demonstrated that the␣EDG-1 receptors expressed in Pichia pastoris␣have specific and saturation binding of 32P-labeled sphingosine 1-phosphate. 相似文献
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Feiran Zhang Christy Hammack Sarah C. Ogden Yichen Cheng Emily M. Lee Zhexing Wen Xuyu Qian Ha Nam Nguyen Yujing Li Bing Yao Miao Xu Tianlei Xu Li Chen Zhiqin Wang Hao Feng Wei-Kai Huang Ki-jun Yoon Chao Shan Luoxiu Huang Zhaohui Qin Kimberly M. Christian Pei-Yong Shi Mingjiang Xu Menghang Xia Wei Zheng Hao Wu Hongjun Song Hengli Tang Guo-Li Ming Peng Jin 《Nucleic acids research》2016,44(18):8610-8620
7.
Tianlei Ying Rui Gong Tina W. Ju Ponraj Prabakaran Dimiter S. Dimitrov 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(11):1977-1982
Therapeutic monoclonal antibodies (mAbs) have been successful for the therapy of a number of diseases mostly cancer and immune disorders. However, the vast majority of mAbs approved for clinical use are full size, typically in IgG1 format. These mAbs may exhibit relatively poor tissue penetration and restricted epitope access due to their large size. A promising solution to this fundamental limitation is the engineering of smaller scaffolds based on the IgG1 Fc region. These scaffolds can be used for the generation of libraries of mutants from which high-affinity binders can be selected. Comprised of the CH2 and CH3 domains, the Fc region is important not only for the antibody effector function but also for its long half-life. This review focuses on engineered Fc based antibody fragments and domains including native (dimeric) Fc and monomeric Fc as well as CH2 and monomeric CH3, and their use as novel scaffolds and binders. The Fc based binders are promising candidate therapeutics with optimized half-life, enhanced tissue penetration and access to sterically restricted binding sites resulting in an increased therapeutic efficacy. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. 相似文献
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Tianlei Qiu Lili Liu Min Gao Lanhe Zhang Haireti Tursun Xuming Wang 《Biodegradation》2016,27(2-3):165-178
A solid-phase denitrification (SPD) reactor packed with poly (3-hydroxybutyrate-co-3-hydroxyvalerate) as a carbon source was incorporated into a recirculating aquaculture system (RAS) to remove accumulated nitrate. Bacterial community structures in different parts of the RAS, including biofilter unit, SPD reactor, and culture water, were analyzed using Illumina MiSeq sequencing technology. The data showed that nitrate levels decreased remarkably in the RAS connected with SPD reactor (RAS-DR). In contrast, nitrate levels increased continuously in the conventional RAS without SPD reactor (RAS-CK). Biofilter unit and culture water in RAS-DR developed lower species richness and higher bacterial community diversity than that in RAS-CK. The bacterial community structure of RAS was significantly affected by the SPD process and the changes included an increase in the proportion of Proteobacteria and Firmicutes and a decrease in Nitrospira abundance in RAS-DR. Firmicutes was the most abundant phylum (56.9 %) and mainly consisted of Clostridium sensu stricto (48.3 %) in SPD reactor. 相似文献
10.
Epithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N-glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E-cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N-glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N-glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin-catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with gamma-catenin and vinculin compared with the endogenous E-cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization. 相似文献