Strukturelle und funktionelle Analysen zum humanen LMBD1-Mangel und Aufklärung der molekularen Ursache einer neuen Cbl-Komplementationsgruppe

人类 LMBD1 缺陷的结构和功能分析以及新 Cbl 互补组分子原因的阐明

• 批准号: 182906966
• 负责人: Professor Dr. Frank Rutsch
• 金额: --
• 依托单位: Klinik für Kinder- und Jugendmedizin - Allgemeine Pädiatrie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/182906966?language=en
• 关键词: Strukturelle; und; funktionelle; Analysen; zum

Intact vitamin B12 (cobalamin) metabolism is essential for normal human development and survival. After endocytotic uptake into the cell and before conversion to its active coenzymes methylcobalamin and adenosylcobalamin, cobalamin (cbl) has to be transported through lysosomes. In two extremely rare inborn errors of cbl metabolism named cblF and cblJ, free unbound cbl accumulates in lysosomes and cbl coenzyme synthesis is hampered. In previous studies, we demonstrated that mutations in LMBRD1, a gene encoding for the lysosomal membrane protein LMBD1, are responsible for the cblF defect of cbl metabolism. During the current DFG funding period we showed that the LMBRD1 mutations present in the patients resulted in a loss of LMBD1 protein expression. Furthermore, we were able to prove that mutations in ABCD4 are causing the cblJ defect. Because neither the ABCD4 nor the LMBD1 protein shows transport activity for radioactively labeled cbl in vitro and because both proteins do not seem to interact, we propose the existence of another adaptor protein, which facilitates lysosomal cbl transport. In the continuation phase of the current project we now aim to identify this adaptor protein and to characterize its function with respect to lysosomal cbl transport. We will perform immunoprecipitation studies with LMBD1 and ABCD4 antibodies coupled to protein A and G and pulldown assays in patient and control fibroblasts to identify a putative interaction partner. The precipitating proteins will be indentified by mass spectrometry. If we fail to identify a directly interacting protein, we will perform a comparative proteome analysis of lysosomal extracts derived from patient and control fibroblasts to identify possible stimulators of lysosomal cbl transport. The functional role of the identified proteins will be characterized by overexpression and knockdown of the respective proteins in coenzyme synthesis assays and in cbl transport assays in different cell lines. We anticipate that the identification of the mechanism of lysosomal cbl transport sheds further light on the pathogenesis of more common disorders including Alzheimer and Parkinson disease, since also in these disorders lysosomal transport processes are impaired.

完整的维生素 B12（钴胺素）代谢对于人类正常发育和生存至关重要。被细胞内吞后，在转化为其活性辅酶甲钴胺和腺苷钴胺之前，钴胺素 (cbl) 必须通过溶酶体转运。在两种极为罕见的 cbl 代谢先天性错误（cblF 和 cblJ）中，游离的未结合 cbl 在溶酶体中积聚，并且 cbl 辅酶合成受到阻碍。在之前的研究中，我们证明了编码溶酶体膜蛋白LMBD1的基因LMBRD1的突变是导致cbl代谢的cblF缺陷的原因。在当前 DFG 资助期间，我们发现患者体内存在的 LMBRD1 突变导致 LMBD1 蛋白表达缺失。此外，我们能够证明 ABCD4 的突变导致 cblJ 缺陷。因为 ABCD4 和 LMBD1 蛋白在体外均不显示放射性标记的 cbl 的转运活性，并且因为这两种蛋白似乎不相互作用，所以我们提出存在另一种接头蛋白，其促进溶酶体 cbl 转运。在当前项目的继续阶段，我们现在的目标是鉴定这种接头蛋白并表征其在溶酶体 cbl 运输方面的功能。我们将使用与蛋白 A 和 G 偶联的 LMBD1 和 ABCD4 抗体进行免疫沉淀研究，并在患者和对照成纤维细胞中进行 Pulldown 测定，以确定假定的相互作用伙伴。沉淀的蛋白质将通过质谱法来鉴定。如果我们未能鉴定出直接相互作用的蛋白质，我们将对患者和对照成纤维细胞的溶酶体提取物进行比较蛋白质组分析，以鉴定溶酶体 cbl 运输的可能刺激物。已鉴定蛋白质的功能作用将通过在不同细胞系的辅酶合成测定和 cbl 转运测定中各蛋白质的过表达和敲低来表征。我们预计溶酶体 cbl 转运机制的鉴定将进一步阐明包括阿尔茨海默病和帕金森病在内的更常见疾病的发病机制，因为在这些疾病中溶酶体转运过程也受到损害。