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）代谢对于正常的人类发育和生存至关重要。内吞摄取进入细胞并转化为活性辅酶甲基ca胺和腺苷胆汁素之前，必须通过溶酶体运输钴胺素（CBL）。在称为CBLF和CBLJ的CBL代谢的两个极为罕见的先天误差中，自由的未结合CBL积聚在溶酶体中，CBL辅酶合成受到阻碍。在先前的研究中，我们证明了LMBRD1中的突变是编码溶酶体膜蛋白LMBD1的基因，负责CBL代谢的CBLF缺陷。在当前的DFG资金期间，我们表明患者中存在的LMBRD1突变导致LMBD1蛋白表达丧失。此外，我们能够证明ABCD4中的突变导致CBLJ缺陷。因为ABCD4和LMBD1蛋白都没有显示出放射性标记的CBL体外标记的转运活性，并且由于两种蛋白似乎都不相互作用，因此我们提出了另一种衔接蛋白的存在，这促进了溶酶体CBL转运。在当前项目的延续阶段，我们现在旨在识别该衔接子蛋白，并在溶酶体CBL转运方面表征其功能。我们将使用LMBD1和ABCD4抗体进行免疫沉淀研究，并在患者和控制成纤维细胞中与蛋白A和G和下拉分析结合，以识别推定的相互作用伴侣。沉淀蛋白将通过质谱分辨。如果我们无法识别直接相互作用的蛋白质，我们将对源自患者和控制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转运的可能刺激剂。所鉴定蛋白的功能作用将以过表达和敲低辅酶合成分析中各个蛋白质的过表达和敲低，而在不同细胞系中的CBL转运测定中。我们预计，鉴定溶酶体CBL转运机制会进一步了解包括阿尔茨海默氏病和帕金森病在内的更常见疾病的发病机理，因为在这些疾病中，溶酶体转运过程也受到损害。