Molecular mechanisms for sorting lysosomal proteins

项目摘要 本提案的目的是获得人GlcNAc-1-GlcNAc- 磷酸转移酶（PTase），一种修饰溶酶体水解酶的酶。溶酶体含有几个 十几种酸性水解酶可以分解不需要的蛋白质和脂质。溶酶体酶获得甘露糖- 6-P部分在高尔基体中通过PTase的作用，其使用UDP-GlcNAc作为供体底物。的 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基因编码并组装α2β2γ2的340-kDa异六聚体。PTase 选择性地修饰通过ER-高尔基体系统的溶酶体（而不是非溶酶体）糖蛋白。它有 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的基因突变引起粘脂沉积症II和III， 身体结构异常、智力迟钝和几种神经系统疾病。在初步研究中，我们 已经确定了载脂蛋白形式的二聚体α2β2亚复合物的催化核心的原子结构。我们 已经确定了一种抑制性曲棍球棒基序，它在没有催化剂的情况下进出催化口袋。 印刷受体.我们还推导出全酶的nm分辨率冷冻EM 3D图。根据这些 强的初步研究，我们建议，以确定结构的催化核心结合的供体 底物UDP-GlcNAc和受体底物模拟α-甲基甘露糖苷，并进行结构引导的 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 提出了独特的磷酸-糖基转移反应机理。我们还建议进行系统的 突变和功能测定，以检查许多报告的致病突变。最后我们 建议研究一个大的溶酶体水解酶结合外周区和γ亚基的结构， PTase全酶，并探讨它们如何与选定的底物溶酶体水解酶相互作用。的 拟议的研究将解决糖生物学中关键信号通路的分子机制， 为PTase的突变如何导致相关的人类疾病提供了分子见解。

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.