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

多发性内分泌肿瘤1型(MEN1)基因是由包括MDB成员在内的NIH合作小组通过定位克隆确定的肿瘤抑制基因。在受影响的MEN1家族受试者中发现了该基因的胚系突变，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。该基因编码一个610残基的蛋白质，称为脑膜素，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，旨在确定薄荷素的结构、功能、亚细胞定位和表达范围。我们已经制备了一系列多克隆多肽抗血清，这些血清在免疫印迹和免疫沉淀研究中被证明是有用的。此外，这些抗体检测重组形式的薄荷素的表达，用于结构和生化分析。对293细胞的亚细胞分离和免疫印迹结合我们在NHGRI的合作者进行的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琼德是薄荷素相互作用的伙伴。对Jund进行突变，以确定参与薄荷素相互作用的特定残基。不与薄荷素相互作用的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薄荷素正被用于生化和结构(X射线结晶学)研究。三维结构的确定应该为蛋白质相互作用的功能域以及许多自然发生的错义突变导致功能丧失的机制提供洞察力。对内源性脑膜素的纯化也在进行，以确定相关蛋白和可能的脑膜素翻译后修饰。在甲状旁腺等与MEN1相关的靶组织中表达cre重组酶的转基因小鼠已经与MEN1基因条件敲除(由NHGRI合作者产生)的小鼠杂交。对这类小鼠的研究应该有助于深入了解脑膜素在肿瘤形成中的作用。-肿瘤抑制基因；多发性内分泌瘤

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. - tumor suppressor gene; multiple endocrine neoplasia