ADAM家族研究进展

ＡＤＡＭ家族是近年来发现的一类锚定于细胞膜的细胞表面蛋白家族,该家族成员具有４个较为保守的潜在功能域,在细胞间粘连和融合等活动中有重要作用。迄今为止已有２５个成员被发现,它们在精卵结合、肌细胞融合、膜蛋白分子的加工等生理过程中发挥重要功能。     

辽宁省地区人乳头瘤病毒33型基因多态性分析

研究人乳头瘤病毒（Human papillomavirus,HPV）33型E6和E7基因在辽宁省地区的基因多态性分布情况,增加对HPV33基因多态性地理分布情况的了解,为HPV33的诊断,靶向药和疫苗的研发提供理论依据。使用巢式PCR扩增方法对从已确诊HPV33阳性患者宫颈细胞提取的DNA样本进行E6和E7基因的扩增并测序,随后应用MegaX软件将所得测序结果与参考序列比对确定突变位点并进行系统发育树的构建。应用PAML4.9软件中的Codeml程序找出突变中的正向选择位点。分别应用ABC Pred server,ProPred-I server和ProPred server寻找理想的B细胞免疫表位,人类白细胞抗原（Human leukocyte antigen,HLA）I类结合表位和人类白细胞抗原（HLA）II类结合表位。共得到HPV33E6和E7序列各136个。与HPV33参考序列（M12732.1）进行比对后,E6基因中观测到17个单核苷酸突变,同义突变7个,非同义突变10个。E7基因中观测到9个单核苷酸突变,其中同义突变3个,非同义突变6个。系统发育分析显示HPV33E6和E7...     

人FAM33A基因启动子的克隆与鉴定

目的鉴定人FAM33A基因的启动子,为进一步研究其转录调控机制奠定基础。方法采用5’RACE技术（5’端cDNA快速扩增）鉴定FAM33A的转录起始位点。采用PCR定向克隆、酶切亚克隆等策略,构建FAM33A启动子荧光素酶报告基因。采用Lipofeetamine^TM2000转染H1299细胞,并通过Dual-Lu-ciferase@ Reporter Assay System进行荧光素酶报告基因活性检测。结果确定了FAM33A的转录起始位点,构建了覆盖FAM33A 5’端ATG附近约2kb区域的一系列FAM33A启动子荧光素酶报告基因。启动子活性分析表明,这些重组体均具有较高的启动子活性,同时含有典型的GC盒以及Sp1、E2F和GATA-1等潜在的转录因子结合位点。结论FAM33A启动子区域主要定位于转录起始位点附近约590bp的区域内。     

成年大脑神经细胞特异性ADAM10基因敲除小鼠模型的建立与鉴定

去整合素和金属蛋白酶10(ADAM10)是一种能够水解30余种跨膜蛋白质的“脱落酶”(sheddase), 参与诸多生理过程和致病机制, 如胚胎发育、细胞粘附、信号转导、免疫反应、癌症和阿尔茨海默病。迄今, 已报道的ADAM10完全基因敲除小鼠和大脑神经前体细胞特异性ADAM10基因敲除小鼠分别于胚胎期或围产期死亡, 致使无法研究成年小鼠大脑神经细胞ADAM10基因的功能。文章利用本研究小组建立的CaMKIIα-Cre转基因小鼠与ADAM10^loxP/loxP转基因小鼠杂交, 获得了CaMKIIα-Cre/ADAM10^loxP/loxP小鼠, 并对其进行鉴定。利用PCR方法检测成年ADAM10 cKO小鼠大脑基因组DNA表明, ADAM10基因缺失主要发生在前脑皮层和海马中。荧光定量PCR检测结果显示, ADAM10 mRNA的表达水平在前脑皮层和海马中分别降低55.7%和60.8% ; 使用Western blotting方法研究发现, ADAM10成熟蛋白质的含量在前脑皮层和海马中分别减少63%和84.8% 。采用免疫组织化学方法检测表明, 成年ADAM10 cKO小鼠与野生型小鼠相比, 其大脑皮层和海马神经细胞的ADAM10免疫染色明显减弱, 而其它细胞如胶质细胞的免疫染色基本一致。总之, 文章成功制备了首个存活至成年的大脑神经细胞特异性ADAM10基因敲除(cKO)小鼠, 克服了小鼠因ADAM10缺失在胚胎期或围产期死亡的弊端, 为研究成年小鼠大脑神经细胞ADAM10基因的功能奠定了坚实的基础。     

人A33基因5′调控区的组织特异性表达元件

在对人A33基因 5′调控区初步研究的基础上 ,进一步采用体外足迹法、电泳迁移率变更分析(EMSA)和定点突变等实验对A33启动子的 - 10 4～ + 2 5bp区域进行了重点研究 .在A33启动子的- 10 4～ + 2 5bp区域内存在两个转录正调控元件 ,它们分别位于转录起始位点上游 - 86～ - 6 8区(A)和 - 40～ - 19区 (B) .通过对转录因子数据库的查找 ,发现A区与转录因子GKLF (gut enrichedKr櫣ｐｐｅｌ likefactor)的结合位点吻合 ,而B区则没有找到与之相应的转录因子 .EMSA实验表明 ,A区与核蛋白的结合存在组织特异性 ,而B区的结合则无组织特异性 .推测A区所包含的顺式调控元件很可能是决定A33基因组织特异性表达的关键元件 .根据B区所处的位置和富含AT来分析 ,它极有可能是和通用转录因子及RNA聚合酶结合的区域 .A和B两个区域的点突变都可使A33启动子的活性丧失 85 %以上     

CYC类基因在被子植物花发育中的研究进展

CYCLOIDEA(CYC)类基因属于TCP基因家族成员,在花发育过程中具有重要作用。CYC类基因在被子植物进化过程中发生基因复制事件,形成CYC1、CYC2和CYC3三大分支,其中CYC2分支成员在花对称性形成方面具有主要调控作用。CYC1和CYC3分支成员开展研究较少。综述了国内外CYC类基因的研究现状及其存在问题,并对CYC类基因的研究前景做了展望。     

人A33基因5′调控区的组织特异性表达元件

在对人A33基因 5′调控区初步研究的基础上 ,进一步采用体外足迹法、电泳迁移率变更分析(EMSA)和定点突变等实验对A33启动子的 - 10 4～ + 2 5bp区域进行了重点研究 .在A33启动子的- 10 4～ + 2 5bp区域内存在两个转录正调控元件 ,它们分别位于转录起始位点上游 - 86～ - 6 8区(A)和 - 40～ - 19区 (B) .通过对转录因子数据库的查找 ,发现A区与转录因子GKLF (gut enrichedKr櫣ｐｐｅｌ likefactor)的结合位点吻合 ,而B区则没有找到与之相应的转录因子 .EMSA实验表明 ,A区与核蛋白的结合存在组织特异性 ,而B区的结合则无组织特异性 .推测A区所包含的顺式调控元件很可能是决定A33基因组织特异性表达的关键元件 .根据B区所处的位置和富含AT来分析 ,它极有可能是和通用转录因子及RNA聚合酶结合的区域 .A和B两个区域的点突变都可使A33启动子的活性丧失 85 %以上     

ADAM17在恶性肿瘤中的研究及其进展

去整合素-金属蛋白酶17(adisintegrin and metalloproteinase 17,ADAM17)是近年来发现的金属蛋白酶解聚素(adisintegrin and metalloproteinase,ADAMs)家族成员之一,参与肿瘤发生发展的重要过程.去整合素-金属蛋白酶17(ADAM17)又称为肿瘤坏死因子转换酶(TACE),因此除了具有解聚素和金属蛋白酶的活性,还可以将没有活性的肿瘤坏死因子(TNF-α)从细胞膜上切割下来,并与其受体相结合,从而激活TNF-α下游的EGFR信号传导,此外还可以激活多条信号传导途径如Notch传导通路等,进而影响肿瘤细胞的粘附、凋亡、转移、增殖等生物学行为.纵观ADAM17的研究,在多种恶性肿瘤中呈高表达状态,且这种高表达状态与肿瘤侵润程度及转移情况相关.随着人们对ADAM17基础科学的研究不断深入,ADAM17的临床应用前景也正被不断开发,鉴于其在多种恶性肿瘤组织中高表达的情况,可将其作为许多肿瘤的诊断标志物、及判断其转移和预后情况.靶向药物的研究给恶性肿瘤患者带来了新的福音,利用EGFR为研究扳机点成功研制出许多靶向药物,在EGFR的配体释放环节,ADAM17尤为重要.本文总结了ADAM17在恶性肿瘤发展中的作用及其机制,对其在癌症治疗的应用前景进行展望.     

IL-33及其受体ST2的研究进展

IL-33是的IL-1家族的新成员,通过受体ST2活化Th2辅助性T细胞。近来在自身免疫性疾病、变态反应性疾病和心脏疾病的研究表明IL-33是炎症性因子。本文总结了IL-33和其受体ST2信号通路的研究进展。     

人肠组织特异抗原A33基因5′调控区的克隆及其功能分析

用基因组步行法 (genomewalker)克隆了人A33抗原基因的 5′调控区 94 0bp片段 ,并用PCR法鉴定了这一克隆的正确性 .将该序列提交GenBank ,登录号为AF2 0 0 6 2 6 .用引物延伸法 (primerex tension)确定了A33基因的转录起始位点 ,发现该位点位于一个TATA盒下游 10个碱基处 .以增强型绿色荧光蛋白为报告基因构建了不同长度的A33启动子 5′缺失载体 ,用脂质体介导的方法将这些载体转染LoVo、HeLa、2 93等细胞 ,比较了EGFP的表达水平 .研究发现 ,A33启动子上主要转录调控元件以及与组织特异性表达相关的转录调控元件位于A33启动子的 - 10 4～ + 2 5bp区域     

Crystal structure of the catalytic domain of human ADAM33

Adam33 is a putative asthma susceptibility gene encoding for a membrane-anchored metalloprotease belonging to the ADAM family. The ADAMs (a disintegrin and metalloprotease) are a family of glycoproteins implicated in cell-cell interactions, cell fusion, and cell signaling. We have determined the crystal structure of the Adam33 catalytic domain in complex with the inhibitor marimastat and the inhibitor-free form. The structures reveal the polypeptide fold and active site environment resembling that of other metalloproteases. The substrate-binding site contains unique features that allow the structure-based design of specific inhibitors of this enzyme.     

Human ADAM33: protein maturation and localization

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.     

Association of ADAM33 gene polymorphisms with adult concomitant allergic rhinitis and asthma in Chinese Han population

Allergic Rhinitis (AR) and allergic asthma (AS) are very common diseases involving genetic and environmental factors. Most patients with asthma also have rhinitis, which suggests the concept of ‘one airway, one disease’. A disintegrin and metalloproteinase 33 (ADAM33) was discovered as the first asthma-susceptible gene by positional cloning. To evaluate the potential influences of ADAM33 gene polymorphisms on concomitant allergic rhinitis and asthma (ARA), a case-control study was conducted in Han population of Northeast China. Six polymorphic sites (V4, T + 1, T2, T1, S1 and Q − 1) were genotyped in 135 ARA patients and 151 controls (CTR). Genotypes were determined by the polymerase chain restriction fragment length polymorphism (PCR-RFLP) method. Data was analyzed using the Chisquaretest and Haploview software. The SNPs (V4 G/C, T2 A/G, T1 G/A, and Q − 1A/G) of the ADAM33 gene may be the causal variants in ARA disease. Ximei Zhang and Dongju Su contributed equally to this work.     

Association of ADAM33 gene polymorphisms with asthma in Mongolian and Han groups in Inner Mongolia

Polymorphisms in the gene encoding for A disintegrin and metalloprotease 33 (ADAM33) are closely associated with the risk of bronchial asthma attacks in different populations. We collected blood samples from 248 asthma patients – 130 of the Han ethnic group and 118 of the Mongolian ethnic group – living in the Inner Mongolia region of China, and analyzed the single nuclear polymorphisms (SNPs) of the T1, T2 and V4 loci of the ADAM33 gene using PCR-RFLP (restriction fragment length polymorphism). In addition, we also tested 256 healthy controls (134 and 122 from the Han and Mongolian ethnic groups respectively) for the same SNPs. Three genotypes of the T1, T2 and V4 loci were predominantly detected: while polymorphisms in the T1 locus were significantly associated with asthma risk in both Mongolian and Han ethnicities (P?<?0.05, 1P?<?0.05), that in the V4 locus were relevant only in the Mongolian patients (P?<?0.05, 1P?>?0.05). In contrast, polymorphisms in the T2 locus showed no significant association with asthma risk in either ethnic group (P?>?0.05, 1P?>?0.05).     

Human fertilin β: Identification,characterization, and chromosomal mapping of an ADAM gene family member

Fertilin α/β (PH30 α/β) is a heterodimeric sperm surface protein containing binding and fusion domains with potential for interaction with integrin receptors on the oocyte. We report the cDNA cloning, deduced amino acid sequence, tissue specificity, and chromosomal mapping of human fertilin β. Encoded by a 2205 nucleotide open reading frame, the deduced amino acid sequence of human fertilin β contains pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like (EGF) repeat, transmembrane, and cytoplasmic domains. Due to this domain organization, human fertilin β has been identified as a member of the ADAM family, which is composed of membrane-anchored proteins having A Disintegrin And Metalloprotease domain. The amino acid sequence of human fertilin β shares 90%, 56%, and 55% identity, respectively, to monkey, guinea pig, and mouse fertilin β homologs. A phenylalanine-glutamate-glutamate (FEE) binding tripeptide within the disintegrin-like domain of human fertilin β, homologous to other fertilin β RGD-like (arginine-glycine-aspartic acid) tripeptides, could compete for recognition by integrins and other receptors. Northern analysis from 16 human tissues revealed human fertilin β's 2.9 kb message only in testis, which raises interest in possible clinical applications of this molecule as a contraceptive vaccinogen. Human fertilin β maps to chromosome 8, band p11.2, by fluorescence in situ hybridization and mouse/human somatic cell hybrid Southern hybridization. Mol. Reprod. Dev. 46:363–369, 1997. © 1997 Wiley-Liss, Inc.     

Protease domain of human ADAM33 produced by Drosophila S2 cells

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].     

Positive selection at reproductive ADAM genes with potential intercellular binding activity

Many genes with a role in reproduction, including those implicated in fertilization and spermatogenesis, have been shown to evolve at a faster rate relative to genes associated with other functions and tissues. These survey studies usually group a wide variety of genes with different characteristics and evolutionary histories as reproductive genes based on their site of expression or function. We have examined the molecular evolution of the ADAM (a disintegrin and metalloprotease) gene family, a structurally and functionally diverse group of genes expressed in reproductive and somatic tissue to test whether a variety of protein characteristics such as phylogenetic clusters, tissue of expression, and proteolytic and adhesive function can group fast evolving ADAM genes. We found that all genes were evolving under purifying selection (d(N)/d(S) < 1), although reproductive ADAMs, including those implicated in fertilization and spermatogenesis, evolved at the fastest rate. Genes with a role in binding to cell receptors in endogenous tissue appear to be evolving under purifying selection, regardless of the tissue of expression. In contrast, positive selection of codon sites in the disintegrin/cysteine-rich adhesion domains was detected exclusively in ADAMs 2 and 32, two genes expressed in the testis with a potential role in sperm-egg adhesion. Positive selection was detected in the transmembrane/cytosolic tail region of ADAM genes expressed in a variety of tissues.     

LGI1 and LGI4 bind to ADAM22, ADAM23 and ADAM11

The transmembrane protein ADAM22 is expressed at high levels in the brain. From its molecular structure, ADAM22 is thought to be an adhesion molecule or a receptor because it has functional disintegrin-like and cysteine-rich sequences in its ectodomain. The phenotypic analysis of ADAM22-deficient mice has indicated the important roles played by ADAM22 in proper neuronal function and peripheral nerve development, however, the precise molecular function of ADAM22 is still unknown. To understand the function of ADAM22 on a molecular basis, we identified ADAM22 binding proteins by using immunoprecipitation and mass spectrometric analysis. This analysis revealed that Leucine-rich glioma inactivated 1 (LGI1) is the most potent ADAM22 binding protein in mouse brain. By our quantitative cell-ELISA system, we demonstrated the specific binding of LGI1 with ADAM22. Furthermore, we showed that LGI4, a putative ADAM22 ligand, also bound to ADAM22. Characterization of the binding specificity of LGI1 and LGI4 suggested that ADAM22 is not a sole receptor, because ADAM11 and ADAM23 had a significant binding ability to LGI1 or LGI4. Therefore, LGI-ADAM system seems to be regulated not only by the affinity but also by the cell-type-specific expression of each protein. Our findings provide new clues to understand the functions of LGI1 and LGI4 as an ADAMs ligand.     

ADAM家族的结构特征与生物学功能

ADAM家族(A Disintegrin And Metalloproteinase domain)是一类包括去整合域和金属蛋白酶域的跨膜蛋白分子,具有发育、细胞间相互作用、精卵结合、肌管融合、神经发育和肿瘤发生等功能。本文对ADAM家族的结构和主要功能进行介绍。