KLHL31促进C2C12细胞的肌原分化

人类KLHL31基因是本实验室已克隆的基因,其蛋白质含有保守的BTB和串连重复的Kelch结构域,已有报道表明其在人类成体骨骼肌和心肌组织中特异表达。RT-PCR分析表明在C2C12细胞肌原分化过程中过表达KLHL31能够提高肌原分化标志基因MyoD与Myogenin的转录水平;荧光报告系统分析发现过表达KLHL31能够增强肌原分化相关基因MCK启动子的活性,表明KLHL31能够促进C2C12细胞的肌原分化。     

可诱导型CRISPRi系统的构建及其初步应用

[目的]构建可诱导型CRISPRi系统以特异性地抑制靶基因的表达。[方法]采用PCR技术将人ZNF10基因的KRAB结构域编码序列分别克隆至d Cas9蛋白编码基因的5’端(KRAB-d Cas9)与3’端(d Cas9-KRAB)。设计并构建靶向荧光素酶报告基因起始密码子附近序列的4种gRNA表达载体,其表达受到含有Tet O调控元件的H1启动子调控。分析共表达d Cas9蛋白和gRNA对共转染的荧光素酶报告基因表达水平的影响。在此基础上,设计并构建靶向人KLHL21基因转录起始位点附近区域的3种gRNA表达载体,将其与KRAB-d Cas9融合表达载体分别共转染HEK293T细胞,24h后收集细胞并采用荧光实时定量PCR与蛋白质印迹技术分析KLHL21基因表达水平的变化。[结果]CRISPRi系统有效地抑制HEK293T细胞中荧光素酶报告基因与内源性的KLHL21基因的表达。共表达TetR抑制蛋白可阻断CRISPRi对荧光素酶报告基因表达的抑制作用,在细胞培养基中加入1μg/ml盐酸四环素可解除这种抑制作用。[结论]成功构建了可诱导型CRISPRi系统,可有效抑制真核细胞基因的表达。     

拟南芥DTX31基因表达研究

以模式植物拟南芥为材料,通过PCR和RT-PCR在DNA和RNA水平上筛选鉴定了DTX31基因对应的T-DNA插入突变体,对其表型变化进行了观察.通过半定量RT-PCR分析检测了DTX31基因在拟南芥不同器官及环境胁迫响应中的表达情况,结果发现:DTX31基因在根中表达最高,而在茎、叶、叶柄、花中的表达则较弱;盐和赤霉素使DTX31基因的表达迅速升高,盐胁迫2 h后的表达量达到最高峰,GA处理1 h时就达到最高峰,热激使DTX31基因的表达变化不明显.因此,推测该基因可能是盐和GA信号传导通路中的一个重要调控因子.     

人Collectrin同源基因的分离克隆及其在人肾集合管和远曲小管的特异表达

为探讨collectrin在人类疾病中的作用 ,利用人类collectrin同源性引物 ,经末端cDNA快速扩增法分离获得人collectrin基因全长序列并对collectrin进行生物信息学分析及定位表达研究 .结果发现 ,人类collectrin(GenBank登录号为AF2 2 9179)基因全长含 1345bp ,开放阅读框架编码 2 2 2个氨基酸 .在核苷酸和氨基酸水平 ,与小鼠collectrin序列分别有 86 9%和 87 4 %同源性 .生物信息学分析结果提示 ,collectrin为一个 2 5kD的具有一个信号肽和一个跨膜区的跨膜糖蛋白 .人类collectrin与人类血管紧张素转换酶相关的羧基肽酶 (ACE2 )具有 4 7 8%高度同源性 .人多组织Northern杂交结果显示 :collectrin基因为人类肾脏特异性表达基因 .原位杂交及免疫组化证实 ,与小鼠collectrin特异表达于集合管细胞不同 ,人collectrin基因mRNA及其蛋白产物除位于肾脏集合管细胞外 ,远曲肾小管细胞也有表达 .由此推论 ,人类collectrin基因为肾脏特异性表达基因 ,与人类血管紧张素转换酶相关的羧基肽酶具有高度同源性 ,可能为血管紧张素转换酶 (ACE)基因家族的新成员 .     

人类新基因Bzw2的克隆、抗体制备及表达分析

Bzw2( basic leucine zipper and W2 domains 2)基因是人类心脏发育候选基因,它由bzip结构域和W2结构域构成.为了研究该基因在心脏发育过程中的作用,利用生物信息学信息克隆了人类Bzw2基因全长,将所得的片段插入到原核表达载体pGEXT-4T-1载体中,利用BL21菌株表达该重组...     

家蚕miRNA Novel-31~*上调溶血素基因的表达

【目的】Novel-31*是在家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,Bm CPV)感染的家蚕中发现的一个差异表达miRNA。本研究旨在验证Novel-31*对其靶基因表达的调控作用,以便进一步研究miRNA及其靶基因在昆虫免疫调节中的作用。【方法】用生物信息学方法预测Novel-31*的靶基因,荧光定量PCR分析Novel-31*及其靶基因在家蚕感染Bm CPV后不同时间点的表达变化;构建miRNA慢病毒表达载体和靶基因慢病毒表达载体,转染293T细胞,同时合成Novel-31*mimics转染家蚕培养细胞Bm N,使用荧光定量PCR检测Novel-31*对靶基因表达的调控作用。【结果】生物信息学方法预测发现,溶血素基因是Novel-31*的靶基因,其结合位点位于溶血素基因的5'UTR区域。荧光定量PCR分析表明,Novel-31*及溶血素基因在感染Bm CPV的家蚕血淋巴细胞中呈现明显的上调表达。荧光定量PCR检测表明,在Novel-31*慢病毒表达载体和溶血素基因5'UTR慢病毒表达载体转染的293T细胞中和在转染Novel-31*mimics的家蚕Bm N细胞中,溶血素基因都上调表达。【结论】溶血素基因是miRNA Novel-31*的靶基因,Novel-31*与溶血素基因5'UTR结合,上调溶血素基因的表达。     

栽培草莓潜在印记基因FaTRG-31的克隆与特征分析北大核心CSCD

在前期通过筛选获得草莓候选印记基因FaTRG-31(turgor-responsive protein 31)的基础上,以八倍体栽培草莓‘红颜’(Fragaria×ananassa Duch.‘Benihoppe’)为材料,对FaTRG-31基因的编码序列进行克隆、生物信息学、组织表达、启动子和印记特性分析,以揭示该基因的作用机理,为草莓印记基因表达调控及生物学功能研究奠定基础。结果显示:(1)草莓FaTRG-31基因开放阅读框(ORF)全长999 bp,编码332个氨基酸,含有典型的Asn-Pro-Ala(NPA)基序和6个跨膜结构,定位于细胞质膜,属于典型的AQP蛋白(aquaporin)家族;系统进化树深入分析表明FaTRG-31为PIPs(plasma intrinsic proteins)亚家族1型蛋白。(2)草莓FaTRG-31在不同组织中均有表达,胚乳中的表达量最高,且在FaTRG-31基因上游克隆出的1989 bp启动子序列中发现含有与胚乳特异表达相关的作用元件,此外还有多种非生物胁迫响应元件和激素响应元件等。(3)草莓印记特性分析结果显示,FaTRG-31在胚乳中的SNP(single nucleotide polymorphism)位点处测序峰为单峰且是母本表达,即该基因在栽培草莓胚乳中为母本表达印记基因。(4)非生物胁迫处理后分析发现,草莓FaTRG-31基因能在不同程度上响应非生物胁迫。     

人类生精相关基因TSARG4的cDNA克隆

为了探索精子生成的分子机制 ,从人精子外部致密纤维蛋白相关基因SPAG4(spermantigen 4)和小鼠精母细胞中表达的AK0 0 62 2 5基因出发 ,找到两个人类EST ,BG72 0 5 64和AI70 0 45 4,其中BG72 0 5 64在人睾丸中表达。运用“间隙填充法”填平这两个EST之间的间隙 ,从人睾丸文库中快速克隆了同源于SPAG4和AK0 0 62 2 5基因的人类TSARG4基因 (testisandspermatogenesisrelatedgene 4) (GenBank登录号为AF40 13 5 0 ) ,并用RT PCR对该基因阅读框进行验证。TSARG4基因全长 12 5 2bp ,开放阅读框为 94～ 12 3 3bp ,定位于 2 0q11.2 ,推定编码 3 79个氨基酸 ,预计分子量为 43 0 81.45 ,等电点为 8.61,该基因与小鼠精母细胞基因AK0 0 62 2 5编码的氨基酸序列同源性 74% ,与人类SPAG4基因编码的氨基酸序列同源性 45 %。RT PCR表明人类TSARG4基因在多个组织中均有表达 ,而同源的小鼠AK0 0 62 2 5基因仅在睾丸中表达     

人类ZNF434基因的克隆、真核表达和组织表达谱分析

目的:克隆人类ZNF434基因并构建其真核表达质粒,鉴定ZNF434基因的组织表达谱。方法:实时定量PCR检测ZNF434基因在人体10种组织中的表达情况;克隆ZNF434基因的全长开放读码框区并连入真核表达载体pIRES2-EGFP,磷酸钙沉淀法转染HEK293T细胞,荧光显微镜和Western blot鉴定ZNF434蛋白的表达情况。结果:ZNF434基因在检测的人体组织中均有表达,其中骨髓和睾丸表达最高,成功克隆了ZNF434基因并构建了该基因的真核表达质粒ZNF434-pIRES2-EGFP,转染HEK293T细胞可观察到绿色荧光蛋白表达,Western blot可检测出分子量56kDa的Flag-ZNF434融合蛋白表达。结论:明确了人类ZNF434基因的组织表达谱,构建了该基因的真核表达载体,为该基因的进一步研究奠定了基础。     

在大肠杆菌中高效表达人类免疫缺陷病毒Ⅰ型(HIV-1)核心蛋白

将编码人类免疫缺陷病毒Ⅰ型(HIV-1)核心蛋白(Gag)P24的大部分基因片段,插入大肠杆菌表达载体pEX31A中,获得的表达产物MS2-Gag融合蛋白占菌体总蛋白的20％左右。Western blot结果证明,这种表达产物能被艾滋病(AIDS)患者血清中的特异性抗体所识别。这是国内首次报道在大肠杆菌中高效表达人类免疫缺陷病毒Ⅰ型核心蛋白。对表达的重组核心蛋白的意义及应用前景进行了讨论。     

A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells

Faithful cell-cycle progression is tightly controlled by the ubiquitin-proteasome system. Here we identify a human Cullin 3-based E3 ligase (Cul3) which is essential for mitotic division. In a complex with the substrate-specific adaptors KLHL9 and KLHL13, Cul3 is required for correct chromosome alignment in metaphase, proper midzone and midbody formation, and completion of cytokinesis. This Cul3-based E3 ligase removes components of the chromosomal passenger complex from mitotic chromosomes and allows their accumulation on the central spindle during anaphase. Aurora B directly binds to the substrate-recognition domain of KLHL9 and KLHL13 in vitro, and coimmunoprecipitates with the Cul3 complex during mitosis. Moreover, Aurora B is ubiquitylated in a Cul3-dependent manner in vivo, and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. We thus propose that the Cul3/KLHL9/KLHL13 E3 ligase controls the dynamic behavior of Aurora B on mitotic chromosomes, and thereby coordinates faithful mitotic progression and completion of cytokinesis.     

Update on the Kelch-like (KLHL) gene family

The Kelch-like (KLHL) gene family encodes a group of proteins that generally possess a BTB/POZ domain, a BACK domain, and five to six Kelch motifs. BTB domains facilitate protein binding and dimerization. The BACK domain has no known function yet is of functional importance since mutations in this domain are associated with disease. Kelch domains form a tertiary structure of β-propellers that have a role in extracellular functions, morphology, and binding to other proteins. Presently, 42 KLHL genes have been classified by the HUGO Gene Nomenclature Committee (HGNC), and they are found across multiple human chromosomes. The KLHL family is conserved throughout evolution. Phylogenetic analysis of KLHL family members suggests that it can be subdivided into three subgroups with KLHL11 as the oldest member and KLHL9 as the youngest. Several KLHL proteins bind to the E3 ligase cullin 3 and are known to be involved in ubiquitination. KLHL genes are responsible for several Mendelian diseases and have been associated with cancer. Further investigation of this family of proteins will likely provide valuable insights into basic biology and human disease.     

Decrease of WNK4 ubiquitination by disease-causing mutations of KLHL3 through different molecular mechanisms

Recently, we demonstrated that WNK4 is a substrate for KLHL3–Cullin3 (CUL3) E3 ubiquitin ligase complexes and that impaired WNK4 ubiquitination is a common mechanism for pseudohypoaldosteronism type II (PHAII) caused by WNK4, KLHL3, and CUL3 mutations. Among the various KLHL3 mutations that cause PHAII, we demonstrated that the R528H mutation in the Kelch domain decreased the binding to WNK4, thereby causing less ubiquitination and increased intracellular levels of WNK4. However, the pathogenic mechanisms of PHAII caused by other KLHL3 mutants remain to be determined. In this study, we examined the pathogenic effects of three PHAII-causing mutations in different KLHL3 domains; the protein levels of these mutants significantly differed when they were transiently expressed in HEK293T cells. In particular, S410L expression was low even with increased plasmid expression. The cycloheximide chase assay revealed that an S410L mutation in the Kelch domain significantly decreased the intracellular stability. Mutations in E85A in the BTB domain and C164F in the BACK domain decreased the binding to CUL3, and S410L as well as R528H demonstrated less binding to WNK4. In vitro and in vivo assays revealed that these mutants decreased the ubiquitination and increased the intracellular levels of WNK4 compared with wild-type KLHL3. Therefore, the KLHL3 mutants causing PHAII investigated in this study exhibited less ability to ubiquitinate WNK4 because of KLHL3’s low stability and/or decreased binding to CUL3 or WNK4.     

Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B., Sayen, M. R., Williams, S. D., Crow, M. T., and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors.     

KLHL38 involvement in non-small cell lung cancer progression via activation of the Akt signaling pathway

Lung cancer is the leading cause of cancer-related death worldwide. KLHL38 has been reported to be upregulated during diapause but downregulated after androgen treatment during the reversal of androgen-dependent skeletal muscle atrophy. This study aimed to clarify the role of KLHL38 in non-small cell lung cancer (NSCLC). KLHL38 expression was evaluated in tumor and adjacent normal tissues from 241 patients with NSCLC using immunohistochemistry and real-time PCR, and its association with clinicopathological parameters was analyzed. KLHL38 levels positively correlated with tumor size, lymph node metastasis, and pathological tumor-node-metastasis stage (all P < 0.001). In NSCLC cell lines, KLHL38 overexpression promoted PTEN ubiquitination, thereby activating Akt signaling. It also promoted cell proliferation, migration, and invasion by upregulating the expression of genes encoding cyclin D1, cyclin B, c-myc, RhoA, and MMP9, while downregulating the expression of p21 and E-cadherin. In vivo experiments in nude mice further confirmed that KLHL38 promotes NSCLC progression through Akt signaling pathway activation. Together, these results indicate that KLHL38 is a valuable candidate prognostic biomarker and potential therapeutic target for NSCLC.Subject terms: Lung cancer, Oncogenesis     

Molecular characterization of KLHL3, a human homologue of the Drosophila kelch gene

The Drosophila kelch protein is a structural component of ring canals and is required for oocyte maturation. Here, we report the cloning and genomic structure of a new human homologue of kelch, KLHL3. At the amino acid level, KLHL3 shares 77% similarity with Drosophila kelch and 89% similarity with Mayven (KLHL2), another human kelch homolog. The approximately 6.5-kb mRNA has a single open reading frame encoding a protein of 587 amino acids with a predicted molecular mass of 650 kDa. Like kelch and KLHL2, the KLHL3 protein contains a poxvirus and zinc finger domain at the N-terminus and six tandem repeats (kelch repeats) at the C-terminus. At least three isoforms, which differ in the length of the N-terminus, are produced and may be the result of alternative promoter usage. We also identified alternative polyadenylation sites and alternative splicing; thus, as many as 12 mRNA variants and six putative protein isoforms could be produced. The KLHL3 gene is mapped to human chromosome 5, band q31, contains 17 exons, and spans approximately 120 kb of genomic DNA. KLHL3 maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no inactivating mutations of KLHL3 in malignant myeloid disorders with loss of 5q.     

The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses

Death‐associated protein kinase (DAPK) was identified as a mediator of interferon (IFN)‐induced cell death. How IFN controls DAPK activation remains largely unknown. Here, we identify the BTB–Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin 3 (Cul3) via its Kelch‐repeat domain and BTB domain, respectively. The KLHL20–Cul3–ROC1 E3 ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20‐mediated DAPK ubiquitination is suppressed in cells receiving IFN‐α or IFN‐γ, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN‐induced autophagy. This study identifies KLHL20–Cul3–ROC1 as an E3 ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E3 ligase, in IFN‐induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy.     

Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.