Lysosomal Enzymes and Associated Human Genetic Diseases

描述(由申请人提供)： 这项提案的总体目标是开发和实施一种系统的方法，以识别和表征溶酶体蛋白，并了解它们在人类疾病中的作用。这种方法依赖于这样的发现，即大多数溶酶体蛋白包含一个共同的翻译后修饰，即甘露糖6-磷酸(M6P)识别标记，它将它们与其他类型的蛋白质区分开来。有三个具体目标。 具体目标1是利用双向凝胶电泳法和基于液-质联用的方法鉴定人类基因组编码的M6P糖蛋白的谱。这将确定一些以前未发现的候选溶酶体蛋白，并将创建一个蛋白质组学资源来研究它们在人类疾病中的作用。 具体目标2是开始表征以前未知的M6P糖蛋白的功能，这将需要确定它们的亚细胞分布，以验证它们的溶酶体位置，以及产生用于结构和功能研究的试剂。 具体目的3是研究病因不明的溶酶体贮积性疾病的分子基础。将使用两种方法：1)“疾病到蛋白质”方法将需要使用在特定目的1中创建的资源对正常和疾病标本中的M6P糖蛋白进行全球比较。该方法的扩展可能适用于开发溶酶体储存性疾病的临床筛查方法。2)蛋白质到疾病的方法将创建一个新表征的M6P糖蛋白属性的数据库，并使用这些信息来确定相关疾病的候选对象。使用任何一种方法识别的潜在疾病基因都将使用分子遗传分析进行仔细检查。鉴于这种方法已经确定了几种以前没有特征的溶酶原蛋白和三种遗传性疾病的分子基础，预计这一基于发现的研究的扩展将导致更多的重要发现。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop and implement a systematic approach that will result in the identification and characterization of lysosomal proteins and to understand their role in human disease. This approach relies on the finding that most lysosomal proteins contain a common post-translational modification, the mannose 6-phosphate (M6P) recognition marker, which distinguishes them from other types of proteins. There are three specific aims. Specific Aim 1 is to identify the spectrum of M6P glycoproteins encoded by the human genome using both two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry based approaches. This will identify a number of previously undiscovered candidate lysosomal proteins and will create a proteomics resource to investigate their role in human disease. Specific Aim 2 is to begin to characterize the function of previously unidentified M6P glycoproteins, This will entail determining their subcellular distribution to verify their lysosomal location as well as generating reagents for structural and functional studies, Specific Aim 3 is to investigate the molecular bases for lysosomal storage diseases of unknown etiology. Two approaches will be used: 1) The "disease-to-protein" approach will entail global comparisons of M6P glycoproteins in normal and disease specimens using the resources created in Specific Aim 1. Extensions of this approach may be applicable towards development of clinical screening methods for lysosomal storage diseases. 2) The protein-to-disease approach will create a database of the properties of newly characterized M6P glycoproteins and use this information to identify candidates for associated diseases. Potential disease genes identified using either approach will be scrutinized using molecular genetic analysis. Given that this approach has resulted in the identification of several previously uncharacterized lysosornal proteins and the molecular basis for three hereditary diseases, it is anticipated that extensions of this discovery-based research will result in additional important findings.