STRUCTURE AND FUNCTION OF THE MEN1 GENE AND ITS PROTEIN PRODUCT, MENIN

Men1 基因及其蛋白质产物 Menin 的结构和功能

• 批准号: 6432134
• 负责人: ALLEN M. SPIEGEL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432134
• 关键词: antiserum; gene expression; human genetic material tag; immunoprecipitation; multiple endocrine neoplasia; neoplasm /cancer genetics; oncoproteins; point mutation; protein structure function; transfection; tumor suppressor genes; tumor suppressor proteins; western blottings

The multiple endocrine neoplasia type 1 (MEN1) gene is a tumor suppressor gene identified by positional cloning by an NIH collaborative group including members of MDB. Germline mutations in the gene are found in affected subjects of MEN1 kindreds, and somatic mutations in the gene have been identified in sporadic endocrine and other tumors. The gene encodes a 610 residue protein termed menin without homology to other known proteins. We have initiated a series of studies aimed at defining the structure, function, subcellular localization, and range of expression of menin. We have generated a series of polyclonal peptide antisera that have proved useful in immunoblot and immunoprecipitation studies. Furthermore,these antibodies detect the expression of recombinant forms of menin to be used for structural and biochemical analyses. Subcellular fractionation and immunoblotting of 293 cells transfected with the cDNA encoding menin, in conjunction with studies of GFP-tagged menin conducted by our collaborators in NHGRI have shown that menin is primarily localized to the nucleus. A yeast-two-hybrid screen identified junD as a menin interacting partner. Mutagenesis of junD was done to identify specific residues involved in menin interaction. The biologic properties of junD mutants that do not interact with menin are being studied. Purified recombinant menin expressed in E coli is being used for biochemical and structural (X-ray crystallography) studies. Determination of 3-D structure should offer insights into functional domains for protein interaction and the mechanism whereby many naturally occurring missense mutations cause loss of function. Purification of endogenous menin is also being pursued to identify associated proteins and possible post-translational modifications of menin. Transgenic mice expressing cre recombinase in target tissues relevant to MEN1 such as parathyroid have been generated for crosses with mice with a conditional knockout (generated by NHGRI collaborators) of the MEN1 gene. Study of such mice should offer insights into the role of menin in tumor formation.The drosophila ortholog of menin has been identified from genomic sequence submitted to Genebank. This has allowed us to search for potential interacting proteins using drosophila yeast-two-hybrid libraries, to test for interaction of candidate proteins with drosophila menin, and to employ drosophila genetics (collaboration with Brian Oliver/NIDDK) to study the function of menin and its interacting proteins.

多发性内分泌瘤1型（MEN 1）基因是由包括MDB成员在内的NIH协作组通过定位克隆鉴定的肿瘤抑制基因。在MEN 1激酶受影响的受试者中发现了该基因的生殖系突变，并且在散发性内分泌肿瘤和其他肿瘤中发现了该基因的体细胞突变。该基因编码一个610个残基的蛋白质，称为menin，与其他已知蛋白质没有同源性。我们已经开始了一系列的研究，旨在确定的结构，功能，亚细胞定位，和表达的范围的menin。我们已经产生了一系列的多克隆肽抗血清，已被证明是有用的免疫印迹和免疫沉淀研究。此外，这些抗体检测重组形式的menin的表达，用于结构和生化分析。亚细胞分级分离和免疫印迹293细胞转染的cDNA编码的menin，与我们的合作者在NHGRI进行的GFP标记的menin的研究表明，menin主要定位于细胞核。酵母双杂交筛选确定junD为menin相互作用伴侣。对junD进行诱变以鉴定参与menin相互作用的特定残基。不与menin相互作用的junD突变体的生物学特性正在研究中。在大肠杆菌中表达的纯化重组menin正用于生化和结构（X射线晶体学）研究。三维结构的确定应该提供深入了解蛋白质相互作用的功能域和许多自然发生的错义突变导致功能丧失的机制。内源性menin的纯化也正在进行，以确定相关的蛋白质和可能的翻译后修饰menin。已经产生了在与MEN 1相关的靶组织如甲状旁腺中表达cre重组酶的转基因小鼠，用于与具有MEN 1基因的条件性敲除（由NHGRI合作者产生）的小鼠杂交。通过对这些小鼠的研究，我们可以深入了解menin在肿瘤形成中的作用。这使我们能够使用果蝇酵母双杂交文库来寻找潜在的相互作用蛋白，测试候选蛋白与果蝇menin的相互作用，并采用果蝇遗传学（与Brian奥利弗/NIDDK合作）来研究menin及其相互作用蛋白的功能。