Lysosomal Enzymes and Associated Human Genetic Diseases

描述（由申请人提供）： 该提案的总体目标是开发和实施一种系统的方法，该方法将导致溶酶体蛋白的鉴定和表征，并了解它们在人类疾病中的作用。这种方法依赖于发现大多数溶酶体蛋白含有一个共同的翻译后修饰，甘露糖6-磷酸（M6 P）识别标记，这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是使用基于二维凝胶电泳和液相色谱-质谱的方法鉴定由人类基因组编码的M6 P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，以研究它们在人类疾病中的作用。 具体目标2是开始表征先前未鉴定的M6 P糖蛋白的功能，这将需要确定其亚细胞分布以验证其溶酶体位置以及产生用于结构和功能研究的试剂， 具体目标3是研究病因不明的溶酶体贮积病的分子基础。将采用两种方法：1）“疾病-蛋白质”方法将需要使用特定目标1中创建的资源对正常和疾病样本中的M6 P糖蛋白进行全局比较。这种方法的扩展可能适用于开发溶酶体贮积病的临床筛查方法。2)蛋白质到疾病的方法将创建一个新表征的M6 P糖蛋白特性的数据库，并使用这些信息来识别相关疾病的候选者。将使用分子遗传分析来仔细检查使用任一方法鉴定的潜在疾病基因。鉴于这种方法已经鉴定了几种以前未表征的lysosornal蛋白和三种遗传性疾病的分子基础，预计这种基于发现的研究的扩展将导致额外的重要发现。

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.