Molecular mechanisms for sorting lysosomal proteins

溶酶体蛋白分选的分子机制

• 批准号: 10662534
• 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
• 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/10662534
• 关键词: 3-Dimensional; Acids; Active Sites; Address; Affect; Binding; Biochemical; Biological Assay; Catalysis; Catalytic Domain; 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; Link; Lipids; Location; Lysosomes; Mannose; Mannosides; Maps; Membrane; Mental Retardation; Metabolic Diseases; Modeling; Molecular; Mutagenesis; Mutate; Mutation; Neurodegenerative Disorders; Pathologic; Peripheral; Phosphorylation; Phosphotransferases; Polysaccharides; Proteins; Pseudo-Hurler Polydystrophy; Reaction; Reporting; Research; Resolution; Series; Signal Transduction; Sorting; Structure; System; Transferase; Universities; Vertebrates; Washington; Work; autism spectrum disorder; chemical reaction; conformational conversion; design; dimer; disease-causing mutation; enzyme activity; flexibility; glycosylation; human disease; in vitro Assay; in vitro activity; in vivo; insight; lysosomal proteins; mannose 6 phosphate; methyl mannoside; mutant; nervous system disorder; novel; recruit

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.

项目摘要 该提议的目的是获得对人GlcNAC-1-的机械和病理理解 磷酸转移酶（PTase），一种修饰溶酶体水解酶的酶。溶酶体含有几种 打破不需要的蛋白质和脂质的十二种酸水解酶。溶酶体酶获得甘露糖 - 通过PTase的作用，使用UDP-GLCNAC作为供体底物，在高尔基体中的6-P部分。这 Mannose-6-P的功能就像指导这些酶向溶酶体传递的邮政编码。 PTase是 由gnptab和gnptg基因编码，并组装了α2β2γ2的340 kDa杂聚糖。 PTase 有选择地修饰溶酶体（但没有非溶酶体）糖蛋白转移ER-GOLGI系统。它有 尚不清楚PTase如何催化GlcNAC-P转移反应及其如何区分溶酶体 非溶酶体糖蛋白。编码PTase的基因中的突变引起粘脂脂异常II和III，并导致 身体结构异常，智力低下和几种神经系统疾病。在初步研究中，我们 已经确定了Apo形式中二聚体α2β2子复合物的催化核心的原子结构。我们 已经确定了一个抑制性曲棍球棒图案，该图案在没有 基材。我们还得出了全酶的NM分辨率冷冻EM 3D图。建立在这些基础上 强大的初步研究，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-甲基诺糖苷，并执行结构引导 诱变和体外活性评估。这些催化核心的结构功能研究将使我们能够 制定独特的磷酸糖基转移反应机制。我们还建议进行系统 突变和功能测定，以检查许多报道的引起疾病的突变。最后，我们 研究大型溶酶体水解酶结合周围区域的结构和γ亚基的结构 PTase Holoenzyme并探索它们如何与选定的底物溶酶体水解酶相互作用。这 拟议的研究将解决糖生物学中关键信号传导曲折的分子机制，并将 提供分子见解，以了解PTase中的突变如何引起相关的人类疾病。