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

多发性内分泌肿瘤1型（multiple endocrine neoplasia type 1, MEN1）基因是由NIH合作小组（包括MDB成员）通过定位克隆鉴定的肿瘤抑制基因。在MEN1类受影响的受试者中发现了该基因的种系突变，并且在散发性内分泌和其他肿瘤中发现了该基因的体细胞突变。该基因编码一种名为menin的610残基蛋白，与其他已知蛋白没有同源性。我们已经开展了一系列旨在确定menin的结构、功能、亚细胞定位和表达范围的研究。我们已经产生了一系列的多克隆肽抗血清，已被证明在免疫印迹和免疫沉淀研究有用。此外，这些抗体检测重组形式menin的表达，用于结构和生化分析。我们在NHGRI的合作者对转染了编码menin的cDNA的293细胞进行了亚细胞分离和免疫印迹，并对gfp标记的menin进行了研究，结果表明menin主要定位于细胞核。酵母-双杂交筛选确定junD为主要的相互作用伙伴。对junD进行诱变以确定参与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蛋白被用于生化和结构（x射线晶体学）研究。三维结构的确定将为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提供见解。内源性menin的纯化也正在进行，以确定相关蛋白和可能的翻译后修饰。在与MEN1基因有条件敲除（由NHGRI合作者产生）的小鼠杂交后，在甲状旁腺等与MEN1相关的靶组织中产生了表达cre重组酶的转基因小鼠。对这类小鼠的研究将有助于深入了解脑膜蛋白在肿瘤形成中的作用。已从提交基因库的基因组序列中确定了menin的果蝇同源性。这使我们能够利用果蝇酵母-双杂交文库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，并利用果蝇遗传学（与Brian Oliver/NIDDK合作）研究menin及其相互作用蛋白的功能。

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.