Structural and functional characterization of human SGT and its interaction with Vpu of the human immunodeficiency virus type 1

The small glutamine-rich tetratricopeptide repeat protein (SGT) belongs to a family of cochaperones that interacts with both Hsp70 and Hsp90 via the so-called TPR domain. Here, we present the crystal structure of the TPR domain of human SGT (SGT-TPR), which shows that it contains typical features found in the structures of other TPR domains. Previous studies show that full-length SGT can bind to both Vpu and Gag of human immunodeficiency virus type 1 (HIV-1) and the overexpression of SGT in cells reduces the efficiency of HIV-1 particle release. We show that SGT-TPR can bind Vpu and reduce the amount of HIV-1 p24, which is the viral capsid, secreted from cells transfected with the HIV-1 proviral construct, albeit at a lower efficiency than full-length SGT. This indicates that the TPR domain of SGT is sufficient for the inhibition of HIV-1 particle release but the N- and/or C-terminus also have some contributions. The SGT binding site in Vpu was also identified by using peptide array and confirmed by GST pull-down assay.     

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution,Obtained Using Small-Angle X-Ray Scattering

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed.     

Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates Arabidopsis immune responses

The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene-triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70-SGT1 association.     

嗜吞噬无形体Msp2蛋白与宿主互作靶蛋白筛选及生物信息分析

目的:筛选与嗜吞噬无形体Msp2蛋白互作的THP-1细胞靶蛋白,有助于理解病原体侵袭宿主的分子机理。方法:PCR获得无形体msp2基因克隆到pGBKT7载体上构建诱饵质粒并转化酵母菌株Y2HGold,营养缺陷培养基及蓝白斑实验验证诱饵质粒是否有自激活、毒性;抽提THP-1总RNA,经反转录、Long-distance PCR、同源重组等构建到pGADT7-Rec载体上并转化酵母菌株Y187,鉴定cDNA文库质量;酵母双杂交筛选与无形体Msp2互作的宿主细胞靶蛋白,生物信息学分析蛋白互作可能导致的信号通路变化。结果:成功构建诱饵质粒;cDNA文库容量达4×106克隆,插入片段大小100-3000 bp,且无污染;酵母双杂交获得宿主靶蛋白7个,分别是NADH脱氢酶(泛醌)1α亚体13(NDUFA13)、锌指蛋白36, C3H样2(ZFP36L2)、核糖体蛋白11(RPL11)、前胸腺素α(PTMA)、C19orf10、组织蛋白酶G(CTSG)、核糖体蛋白S25(RPS25);生物信息学分析,互作的宿主靶蛋白主要参与细胞增殖、细胞凋亡、溶酶体成熟及其它一些信号通路等生物学过程。结论:应用酵母双杂交系统初步筛选出与嗜吞噬细胞无形体表面蛋白Msp2互作的宿主细胞靶蛋白,并利用生物信息学初步分析了其参与的生物学过程,为进一步研究病原菌胞内生存分子机制奠定了基础。     

Structural and functional analysis of SGT1-HSP90 core complex required for innate immunity in plants

SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization.     

Characterization and expression of mammalian cyclin b3, a prepachytene meiotic cyclin

We report the identification and expression pattern of a full-length human cDNA and a partial mouse cDNA encoding cyclin B3. Cyclin B3 (CCNB3) is conserved from Caenorhabditis elegans to Homo sapiens and has an undefined meiotic function in female, but not male Drosophila melanogaster. We show that H. sapiens cyclin B3 interacts with cdk2, is localized to the nucleus, and is degraded during anaphase entry after the degradation of cyclin B1. Degradation is dependent on sequences conserved in a destruction box motif. Overexpression of nondegradable cyclin B3 blocks the mitotic cell cycle in late anaphase, and at higher doses it can interfere with progression through G(1) and entry into S phase. H. sapiens cyclin B3 mRNA and protein are detected readily in developing germ cells in the human testis and not in any other tissue. The mouse cDNA has allowed us to further localize cyclin B3 mRNA to leptotene and zygotene spermatocytes. The expression pattern of mammalian cyclin B3 suggests that it may be important for events occurring in early meiotic prophase I.     

Role of SGT1 in resistance protein accumulation in plant immunity

A highly conserved eukaryotic protein SGT1 binds specifically to the molecular chaperone, HSP90. In plants, SGT1 positively regulates disease resistance conferred by many Resistance (R) proteins and developmental responses to the phytohormone, auxin. We show that silencing of SGT1 in Nicotiana benthamiana causes a reduction in steady-state levels of the R protein, Rx. These data support a role of SGT1 in R protein accumulation, possibly at the level of complex assembly. In Arabidopsis, two SGT1 proteins, AtSGT1a and AtSGT1b, are functionally redundant early in development. AtSGT1a and AtSGT1b are induced in leaves upon infection and either protein can function in resistance once a certain level is attained, depending on the R protein tested. In unchallenged tissues, steady-state AtSGT1b levels are at least four times greater than AtSGT1a. While the respective tetratricopeptide repeat (TPR) domains of SGT1a and SGT1b control protein accumulation, they are dispensable for intrinsic functions of SGT1 in resistance and auxin responses.     

Structural and functional analysis of SGT1 reveals that its interaction with HSP90 is required for the accumulation of Rx, an R protein involved in plant immunity

SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1-HSP90 interaction by promoting ternary complex formation.     

Molecular cloning, characterization and expression analysis of B cell translocation gene 1 in grass carp Ctenopharyngodon idella

An expressed sequence tag (EST) of B cell translocation gene (BTG) 1 (gcbtg1) was obtained from a grass carp Ctenopharyngodon idellus intestinal complementary (c)DNA library and the full-length cDNA sequence was acquired by rapid amplification of cDNA ends (RACE) technology. The predicted Gcbtg1 protein contains the box A and box B motifs which characterized the BTG and transducer of ERBB2 (TOB) family. Multiple alignment analysis reveals that Gcbtg1 shares an overall identity of 65-94% with Gcbtg1s of other vertebrates. Real-time quantitative PCR analysis reveals that the highest expression level of gcbtg1 was detected in liver and the lowest in muscle. Western blotting analysis indicates that the immunological cross-reactivity occurs between C. idella and human Homo sapiens BTG1 protein. A 1008 bp 5'-flanking region sequence was cloned and analysed.     

人类新基因LACE1的克隆和特性初步分析

目的:构建人类新基因LACE1,同时对该基因进行初步的特性和功能研究.方法:通过生物信息学EST拼接技术,RT-PCR等技术,克隆出30个人类未知功能基因.利用RT-PCR技术对该基因的表达谱进行研究,同时结合绿色荧光蛋白与荧光显微镜对该蛋白的定位进行初步分析,并利用MTT初步分析该基因的功能.结果:成功克隆出30个未知功能的人类新基因,其中LACEI(Homo sapiens lactation elevated 1)是一个没有任何功能文献报道的新基因,通过生物信息学分析该基因NCBI:NM一145315.3,其cDNA全长为2 262bp,有13个外显子和12个内含子组成,主要定位于人6号染色体,该基因定位于细胞质中,MTT结果显示该基因能明显抑制细胞增值.结论:人类新基因LACEI是一个抑制细胞增值的相对保守的人类新基因.     

Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes

We describe the identification of a mutant in the Arabidopsis accession Columbia (Col-0) that exhibits enhanced downy mildew (edm1) susceptibility to several Peronospora parasitica isolates, including the RPP7-diagnostic isolate Hiks1. The mutation was mapped to chromosome IV and characterized physically as a 35-kb deletion spanning seven genes. One of these genes complemented the mutant to full wild-type resistance against all of the Peronospora isolates tested. This gene (AtSGT1b) encodes a predicted protein of 39.8 kD and is an Arabidopsis ortholog of yeast SGT1, which was described originally as a key regulatory protein in centromere function and ubiquitin-mediated proteolysis. AtSGT1b contains three tetratricopeptide repeats at the N terminus followed by a bipartite chord-containing SGT domain and an SGT-specific domain at the C terminus. We discuss the role of AtSGT1b in disease resistance and its possible involvement in ubiquitin-mediated proteolysis in plants.     

Entamoeba histolytica: inhibition of cellular functions by overexpression of EhGEF1, a novel Rho/Rac guanine nucleotide exchange factor

The molecular, biochemical, and cellular characterization of EhGEF1 protein is described. Complete cDNA sequence of 1890 bp revealed an open reading frame that encodes a protein of 69 kDa. EhGEF1 is constituted of Dbl homology domain, pleckstrin homology domain, and several putative regulation sites. Studies of guanine nucleotide exchange activity of EhGEF1 on several GTPases from Entamoeba histolytica and Homo sapiens showed preferential activation on EhRacG, suggesting that EhGEF1 protein could be involved in mechanisms related to actin cytoskeleton activation, cytokinesis, capping, and uroid formation in trophozoite. Confocal microscopy studies of pExEhNeo/HSV-tagged-EhGEF1-transfected cells showed that trophozoites stimulated with ConA, EhGEF1, and EhRacG were localized at plasma membrane. Cellular studies showed that F-actin content of pExEhNeo/HSV-tagged-EhGEF1-transfected trophozoites as well as cellular migration and cell damage capacity were significantly altered. The observations suggest that EhRacG was the principal target of EhGEF1 and that EhGEF1 may provide a link between F-actin dynamics and EhRacG signaling.     

Cloning and characterization of a human GDPD domain-containing protein GDPD5

Glycerophosphodiester phosphodiesterase (GDPD) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. GDPD5 has been reported in Mus musculus and Gallus gallus, but not in Homo sapiens. Here we report the cloning and characterization of a novel human GDPD domain-containing gene, GDPD5, isolated from human testis cDNA library, and mapped to 11q13.4-13.5 by searching the UCSC genomic database. The GDPD5 cDNA sequence of 3442 base pairs contains an open reading frame encoding 605 amino acids. The GDPD5 gene consists of 17 exons and encodes a putative protein with six transmembrane regions and a GDPD motif. Subcellular localization of GDPD5 demonstrated that the protein was localized in the cytoplasm when overexpressed in COS-7 cells. RT-PCR analysis showed that GDPD5 was widely expressed in human tissues and the expression levels in kidney and prostate were relatively low.     

Genome wide cloning of maize meiotic recombinase Dmc1 and its functional structure through molecular phylogeny

The development of meiotic division and associated genetic recombination paved the way for evolutionary changes. However, the secondary and tertiary structure and functional domains of many of the proteins involved in genetic recombination have not been studied in detail. We used the human Dmc1 gene product along with secondary and tertiary domain structures of Escherichia coli RecA protein to help determine the molecular structure and function of maize Dmc1, which is required for synaptonemal complex formation and cell cycle progression. The maize recombinase Dmc1 gene was cloned and characterized, using rice Dmc1 cDNA as an orthologue. The deduced amino acid sequence was used for elaborating its 3-D structure, and functional analysis was made with the CDD software, showing significant identity of the Dmc1 gene product in Zea mays with that of Homo sapiens. Based on these results, the domains and motives of WalkerA and WalkerB as ATP binding sites, a multimer site (BRC) interface, the putative ssDNA binding L1 and L2 loops, the putative dsDNA binding helix-hairpin-helix, a polymerization motif, the subunit rotation motif, and a small N-terminal domain were proposed for maize recombinase Dmc1.     

文昌鱼AmphiDC-like的全长cDNA克隆及其表达模式分析

本文旨在克隆文昌鱼树突样蛋白基因AmphiDC-like,采用实时 PCR法和原位分子杂交法对其表达模式进行分析.从文昌鱼神经胚cDNA文库测序得到的ESTs中筛选得到该基因片段,通过引物步移直接测序的方法,克隆得到其cDNA全长序列,对其推测的氨基酸序列进行同源性分析发现,该基因产物具有树突样细胞蛋白共有的保守区,并与多种生物的树突状细胞蛋白具有高度同源性,其中与脊椎动物的树突样细胞蛋白同源性较高. 实时 PCR结果显示, AmphiDC-like在文昌鱼受副溶血性弧菌(Vibrio parahemolyticus,Vp)感染后6 h到48 h表达均上调,并通过原位分子杂交技术观察到,该基因在文昌鱼鳃和消化道中有表达,为深入研究其功能奠定了基础.     

Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato

A cDNA encoding solanidine glucosyltransferase (SGT) was isolated from potato. The cDNA was selected from a yeast expression library using a positive selection based on the higher toxicity of steroidal alkaloid aglycons relative to their associated glycosylated forms. The cDNA contained an open reading frame encoding a 56 kDa polypeptide with regions of similarity to previously characterized UDP-glucosyltransferases. The enzyme activity and reaction products of recombinant SGT in yeast were consistent with those observed for the endogenous enzyme from potato. SGT mRNA and protein accumulated in tubers in response to wounding. The time course for SGT mRNA accumulation paralleled that of 3-hydroxy-3-methylglutaryl-coenzymeA isoform 1 ( hmg1 ) mRNA. Steady-state SGT mRNA levels also increased transiently upon wounding of leaves.     

SGT1 regulates Akt signaling by promoting beta-TrCP-dependent PHLPP1 degradation in gastric cancer cells

SGT1 (suppressor of G2 allele of Skp1) plays a role in various cellular processes including kinetochore assembly and protein ubiquitination by interacting with Skp1, a component of SCF E3 ligase complex. However, the function of SGT1 in cancer is largely unknown. Here, we showed that SGT1 was over-expressed in gastric cancer tissues and silencing of SGT1 by siRNAs significantly inhibited the growth and colony formation of gastric cancer cells. We further showed that SGT1 could regulate Akt signaling pathway by modulating Akt ser473 phosphorylation status. Moreover, we found that SGT1 was able to regulate the stability of PHLPP1, which is the direct phosphatase for Akt ser473 phosphorylation. Immunoprecipitation assay revealed that SGT1 could enhance the binding between PHLPP1 and beta-TrCP which has been documented to be able to target PHLPP1 for destruction. Decreased PHLPP1 in SGT1 over-expressed gastric cancer cells failed to dephosphorylate Akt and resulted in increased Akt ser473 phosphorylation and amplified downstream Akt signaling. Thus, our data revealed a previously uncovered role of SGT1 in gastric cancer development, and suggested that SGT1 could be a promising anti-cancer target to against gastric cancer.     

For security and stability: SGT1 in plant defense and development

SGT1 (suppressor of G-two allele of SKP1) is highly conserved among all eukaryotes. In plants, SGT1 interacts with various proteins, including molecular chaperones (HSP70 and HSP90) and certain SCF ubiquitin ligases, and hence SGT1 likely functions in protein folding and stability. Since these protein complexes are involved in many aspects of plant biology, plants with a defective SGT1 display a plethora of phenotypic alterations. In this mini-review we highlight the interaction between SGT1 with other protein complexes and summarize the function of SGT1 in plant defense responses and development, including the recent advancements in the understanding of the role of SGT1 in jasmonic acid (JA) biosynthesis and signaling.Key words: SGT1, HSP90, RAR1, immunity, development, jasmonate, coronatine, pathogen, herbivore     

.用酵母双杂交系统筛选CENP-E相互作用蛋白

纺锤体检验点(spindle checkpoint)是一个重要的细胞分裂生化调节通路, 可监督染色体正确分离和传代．着丝粒相关蛋白E (centromere-associated protein E, CENP-E)是一个分子量为312 kD的微管马达驱动蛋白,可以衔接纺锤体微管与动点并参与纺锤体检验点调控．为研究CENP-E的作用机理,以其动点结合区域为诱饵蛋白,用酵母双杂交技术从人HeLa细胞 cDNA 文库中筛选出了Nuf2蛋白．体外的pull-down实验和体内的免疫共沉淀实验表明, Nuf2蛋白通过其卷曲螺旋(coiled-coil) 功能域特异结合CENP-E的 C 末端区域,间接免疫荧光显示Nuf2与CENP-E共定位于细胞有丝分裂期染色体的动点．由此推论, CENP-E 通过Nuf2的直接作用参与构筑动点-微管界面,进而参与细胞有丝分裂纺锤体检验点信号转导通路,为染色体正确分离发挥调控作用．