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Mechanisms Of Er-To-Golgi Transport Of Lysosomal Enzymes

溶酶体酶从 Er 到高尔基体的转运机制

基本信息

批准号：
10376728
负责人：
Marco Sardiello
金额：
$ 31.5万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10376728
关键词：
Acids Aging Amino Acid Motifs Biogenesis CLN6 gene CLN8 gene COP-Coated Vesicles Carbohydrates Cell Death Cells Cellular biology Client Communities Complement Data Defect Degenerative Disorder Disease Endoplasmic Reticulum Engineering Enzymes Future Goals Golgi Apparatus Growth Health Homeostasis Homo Human Hydrolase Immune System Diseases Impairment Integral Membrane Protein Investigation Knock-in Knockout Mice Lysosomes Malignant Neoplasms Mammalian Cell Maps Mediating Metabolism Methods Missense Mutation Modeling Molecular Molecular Biology Mus Mutate Mutation Organ Organelles Pathogenesis Pathogenicity Pathway interactions Proteins Proteomics Research Role Signal Transduction Signaling Protein Sorting - Cell Movement Spielmeyer-Vogt Disease Structural Models Surface System Tertiary Protein Structure Testing Therapeutic Tissues Transportation Work base biochemical tools cancer type cohort disease-causing mutation human disease in vivo interdisciplinary approach macromolecule molecular modeling mutant overexpression receptor recruit therapy design trafficking vesicle transport

项目摘要

PROJECT SUMMARY/ABSTRACT Lysosomes control a substantial part of cellular metabolism by acting as the main catabolic hub of the cell and serving as a platform for the integration of numerous signals that modulate cell death, growth and proliferation. Most lysosomal functions rely on a set of more than 50 acid hydrolases that degrade a wide variety of macromolecules. Lysosomal enzymes are trafficked to the lysosome in two stages: transport of the newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi complex, and their subsequent receptor-assisted transfer from the Golgi to endolysosomal compartments. How lysosomal enzymes are transported from the ER to the Golgi complex is unknown and, to our knowledge, the simple model of a bulk, unregulated transportation has never been questioned. We have identified two candidate ER receptors, CLN6 and CLN8, whose deficiency results in altered maturation of lysosomal enzymes and lysosomal storage disorder-like diseases. We propose to study how CLN6 and CLN8 function in the pathway of maturation of lysosomal enzymes. First, we will test the hypothesis that CLN6 and CLN8 directly interact with lysosomal enzymes and that such interaction is disrupted by disease-associated mutations on either CLN6/CLN8 or on the surface of lysosomal enzymes (Aim 1). Second, we will examine the trafficking and maturation of newly synthesized lysosomal enzymes to identify the exact step that is disrupted by CLN6 and CLN8 deficiency. We will also define CLN6 and CLN8 functions in vivo by carrying out detailed tissue-specific analyses of lysosomal composition in CLN6- and CLN8-deficient mouse lines by LC-MS/MS-based proteomics. To this aim, we have generated a knock-in Lamp1FLAG mouse line to efficiently isolate lysosomes from the desired tissues (Aim 2). Third, we will identify the protein domains and motifs that are involved in CLN6/CLN8 interaction and that direct their sorting across the compartments of the early secretory pathway via COP-coated vesicles (Aim 3). We will accomplish our goals with a multi-disciplinary approach that uses the tools of biochemistry, molecular biology, cell biology and mouse engineering and we will also develop a new method of in vivo lysosome isolation from mouse tissues. Our results are likely to have important consequences for our understanding of the mechanisms governing lysosomal biogenesis and of the molecular pathogenesis of numerous human diseases. Some of the regulatory mechanisms we uncover may serve in the future as targets for modulating lysosomal biogenesis in diseases resulting from impaired lysosomal function or in conditions, such as certain types of cancer, that are characterized by aberrant or unrestricted lysosomal activation.

项目总结/摘要溶酶体通过充当细胞的主要分解代谢中心来控制细胞代谢的实质部分，作为整合调节细胞死亡、生长和增殖的多种信号的平台。大多数溶酶体功能依赖于一组超过50种的酸性水解酶，其降解各种各样的溶酶体酶通过两个阶段运输到溶酶体：运输新的大分子，从内质网（ER）到高尔基复合体合成蛋白质，以及它们随后的受体介导的从高尔基体到内溶酶体的转移。从内质网运输到高尔基复合体是未知的，据我们所知，简单的散装模型，我们已经确定了两个候选ER受体，CLN 6 和CLN 8，其缺陷导致溶酶体酶成熟和溶酶体储存的改变我们建议研究CLN 6和CLN 8如何在细胞成熟的途径中发挥作用，首先，我们将检验CLN 6和CLN 8直接与溶酶体酶相互作用的假设。这些相互作用被CLN 6/CLN 8或CLN 9上的疾病相关突变破坏。第二，我们将研究新的溶酶体酶的运输和成熟。合成溶酶体酶，以确定被CLN 6和CLN 8缺陷破坏的确切步骤。还将通过对溶酶体进行详细的组织特异性分析来确定CLN 6和CLN 8的体内功能。通过基于LC-MS/MS-ESI的蛋白质组学方法，我们研究了CLN 6-β 1和CLN 8-β 2缺陷小鼠系中的蛋白质组成。产生敲除Lamp 1FLAG小鼠系以有效地从所需组织分离溶酶体（目的2）。第三，我们将鉴定参与CLN 6/CLN 8相互作用的蛋白质结构域和基序，它们通过COP-1包被的囊泡在早期分泌途径的隔室中进行分选（目的3）。通过使用生物化学，分子生物学，细胞生物学和小鼠工程，我们还将开发一种新的方法，在体内溶酶体分离，我们的研究结果可能对我们理解溶酶体生物发生的机制和许多人类肿瘤的分子发病机制我们发现的一些调节机制可能在未来作为调节疾病的靶点。在由受损的溶酶体功能引起的疾病中或在诸如某些病症的病症中的溶酶体生物发生，以异常或不受限制的溶酶体激活为特征的癌症类型。