Molecular mechanisms for sorting lysosomal proteins

溶酶体蛋白分选的分子机制

基本信息

批准号：
10521596
负责人：
Huilin Li
金额：
$ 47.5万
依托单位：
VAN ANDEL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-10 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10521596
关键词：
3-Dimensional Acids Active Sites Address Affect Binding Biochemical Biological Assay Catalysis Catalytic Domain Code Cryoelectron Microscopy Data Dementia Disease EF Hand Motifs Enzymes Genes Glycobiology Glycoproteins Goals Golgi Apparatus Hockey Holoenzymes Human Hydrolase Hyperactivity I-Cell Disease In Vitro Intellectual functioning disability Lead Link Lipids Location Lysosomes Mannose Mannosides Maps Membrane Mental Retardation Metabolic Diseases Modeling Molecular Mutagenesis Mutate Mutation N-acetylglucopyranosylamine Neurodegenerative Disorders Pathologic Peripheral Phosphotransferases Polysaccharides Proteins Pseudo-Hurler Polydystrophy Reaction Reporting Research Resolution Series Signal Transduction Sorting - Cell Movement Structure System Transferase Universities Vertebrates Washington Work autism spectrum disorder chemical reaction conformational conversion design dimer disease-causing mutation enzyme activity flexibility human disease in vitro Assay in vitro activity in vivo insight lysosomal proteins mannose 6 phosphate methyl mannoside mutant nervous system disorder novel poly-N-acetyl glucosamine recruit

项目摘要

Project Summary The objective of this proposal is to gain mechanistic and pathological understanding of human GlcNAc-1- phosphotransferase (PTase), an enzyme which modifies lysosomal hydrolases. Lysosomes contain several dozen acid hydrolases that break down unwanted proteins and lipids. Lysosomal enzymes acquire a mannose- 6-P moiety in the Golgi apparatus by the action of PTase, which uses UDP-GlcNAc as a donor substrate. The mannose-6-P functions like a zip code that guides the delivery of these enzymes to the lysosomes. PTase is encoded by the GNPTAB and GNPTG genes and assembles a 340-kDa heterohexamer of α2β2γ2. PTase selectively modifies lysosomal (but not non-lysosomal) glycoproteins transiting the ER–Golgi system. It has been unclear how PTase catalyzes the GlcNAc-P transfer reaction and how it distinguishes lysosomal from non-lysosomal glycoproteins. Mutations in the genes encoding PTase cause mucolipidosis II and III and lead to abnormal body structure, mental retardation, and several neurological diseases. In preliminary studies, we have determined the atomic structure of the catalytic core of the dimeric α2β2 subcomplex in the apo form. We have identified an inhibitory hockey stick motif that moves in and out of the catalytic pocket in the absence of substrates. We have also derived a nm-resolution cryo-EM 3D map of the holoenzyme. Building on these strong preliminary studies, we propose to determine the structures of the catalytic core bound to the donor substrate UDP-GlcNAc and the acceptor substrate mimic α-methylmannoside, and to perform structure-guided mutagenesis and in vitro activity assays. These structure-function studies of the catalytic core will enable us to formulate the unique phospho-glycosyl transfer reaction mechanism. We also propose to carry out systematic mutational and functional assays to examine the many reported disease-causing mutations. Finally, we propose to study the structures of a large lysosomal hydrolase-binding peripheral region and the γ-subunit of the PTase holoenzyme and to explore how they interacts with selected substrate lysosomal hydrolases. The proposed research will address the molecular mechanism of a key signaling pathay in glycobiology and will provide molecular insights into how mutations in the PTase cause the associated human diseases.

项目概要该提案的目的是获得对人类 GlcNAc-1- 的机制和病理学理解磷酸转移酶（PTase），一种修饰溶酶体水解酶的酶，溶酶体含有多种酶。分解不需要的蛋白质和脂质的十几种酸性水解酶获得甘露糖。高尔基体中的 6-P 部分通过 PTase 的作用，使用 UDP-GlcNAc 作为供体底物。甘露糖-6-P 的功能类似于邮政编码，引导这些酶递送至溶酶体。由 GNPTAB 和 GNPTG 基因编码，并组装 PTase 的 340 kDa 异六聚体。选择性修饰通过内质网-高尔基体系统的溶酶体（但不是非溶酶体）糖蛋白。目前尚不清楚 PTase 如何催化 GlcNAc-P 转移反应以及它如何区分溶酶体和非溶酶体糖蛋白编码 PTase 的基因突变会导致粘脂沉积症 II 和 III 并导致在初步研究中，我们发现身体结构异常、智力低下和一些神经系统疾病。确定了 apo 形式二聚 α2β2 亚复合物催化核心的原子结构。已经确定了一种抑制性曲棍球棒基序，该基序在没有催化袋的情况下进出催化袋我们还基于这些底物得出了全酶的纳米分辨率冷冻电镜 3D 图。经过强有力的初步研究，我们建议确定与供体结合的催化核心的结构底物UDP-GlcNAc和受体底物模拟α-甲基甘露糖苷，并进行结构引导诱变和体外测定活性的这些催化核心的结构功能研究将使我们能够我们还提出了系统化的独特的磷酸糖基转移反应机制。突变和功能测定来检查许多报道的致病突变。提议研究大溶酶体水解酶结合外围区域和 γ 亚基的结构 PTase 全酶并探索它们如何与选定的底物溶酶体水解酶相互作用。拟议的研究将解决糖生物学中关键信号通路的分子机制，并将提供关于 PTase 突变如何导致相关人类疾病的分子见解。