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 这项建议的目的是从机制和病理上了解人类GlcNAc-1-1- 磷酸转移酶(PTase)，一种修饰溶酶体水解酶的酶。溶酶体包含几个 十几种酸性水解酶，可以分解不需要的蛋白质和脂肪。溶酶体酶获得一种甘露糖- 以UDP-GlcNAc为供体底物的PTase作用于高尔基体中的6-P部分。这个 甘露糖-6-P的功能就像一个邮政编码，引导这些酶传递到溶酶体。PTase是 由GNPTAB和GNPTG基因编码，组装了α2β2γ2 PTase的340 kDa杂六角体 选择性地修饰通过内质网-高尔基体系统的溶酶体(但不是非溶酶体)糖蛋白。它有 一直不清楚PTase是如何催化GlcNAc-P转移反应的，以及它如何区分溶酶体和 非溶酶体糖蛋白。PTase编码基因的突变会导致粘脂病II和III，并导致 身体结构异常，智力低下，以及几种神经疾病。在初步研究中，我们 确定了载脂蛋白形式的二聚体α-2-β-2亚络合物催化核心的原子结构。我们 已经确定了一种抑制性曲棍球杆基元，它可以在没有 底物。我们还得到了全酶的纳米分辨率低温电子显微镜三维图谱。建立在这些基础上 经过严格的初步研究，我们建议确定与给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，并进行结构导向 诱变和体外活性测定。这些催化核心的结构-功能研究将使我们能够 阐述了独特的磷酸-糖基转移反应机理。我们还建议开展系统性的 突变和功能分析，以检查许多已报道的致病突变。最后，我们 建议研究大的溶酶体水解酶结合外围区和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。这个 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PTase突变如何导致相关人类疾病的分子洞察力。