TRPML1通道在溶酶体生理功能和疾病机理的分子基础

The goal of this project is to study the structure, regulation, physiological functions and disease-causing mechanisms of the TRPML1 channel, a novel and unique member of the transient receptor potential mucolipin (TRPML) subfamily. TRPML1 channels are present in many cell types and are localized primarily in intracellular organelles, especially late endosomes and lysosomes. These channels play crucial roles in the endocytic pathway, as demonstrated by the fact that over twenty mutations in TRPML1 gene cause mucolipidosis type IV (ML IV), a severe lysosomal storage disorder characterized by mental and psychomotor retardation, retinal degeneration and corneal opacity, iron deficiency, and achlorhydria. The physiological functions of TRPML1 in lysosomes and how its dysfunction leads to ML IV are not understood. TRPML1 channels exhibit a number of novel and unique biophysical properties, including a complex regulation by luminal Ca2+, Mg2+ and pH (lysosomes have 0.5 mM Ca2+, 1 mM Mg2+, and a low pH of ~4.5), permeation to Ca2+ and Fe2+ (these divalent ions are concentrated in lysosomes), and a possible lipase activity (lipids are enriched in lysosomes). These properties maybe essential for the physiological functions of TRPML1 channels, but the molecular and biophysical mechanisms underlying these properties are virtually unclear. Obtaining high-resolution structures of functionally important domains of TRPML1 would greatly enhance our understanding of TRPML1 channel physiology. In preliminary studies, we have solved the crystal structure of a ~210-amino acid linker (named the I-II linker) between the first two transmembrane segments of TRPML1. This linker, which harbors 3 single amino acid mutations that cause ML IV, faces the lysosome lumen and is critical for TRPML1 channel function. The structure shows that the I-II linker forms a tetramer with a pore (called the luminal pore) in the center. The luminal pore is wide (with a diameter of ~14 ?) and is lined by a luminal pore-loop containing 3 aspartate residues and a putative serine lipase catalytic site. In this proposal, we will use this crystal structure as a blueprint and carry out systematic structure-guided mutagenesis to study the molecular and biophysical mechanisms of the novel biophysical properties of TRPML1 channels mentioned above (i.e., regulation by luminal Ca2+, Mg2+ and pH, Ca2+ and Fe2+ permeation, and lipase activity) and investigate what roles these channel properties play in lysosome physiology. Furthermore, we will investigate the effects of the 3 disease-causing mutations in the I-II linker on TRPML1 channel functions and on lysosome physiology and use the crystal structure we have obtained as a context to understand these effects. These studies will provide mechanistic insights into TRPML1 channel functions, shed light on the pathogenic mechanisms of ML IV, and lay a foundation for the development of better treatment strategies for this devastating disease.

溶酶体是细胞的回收和消化中心，含有数十种酸性水解酶，对细胞生理极为重要。溶酶体功能缺陷破坏细胞稳态，导致各种溶酶体贮积症，包括粘脂质贮积症（ML IV)。MLIV表现为智力低下，运动障碍，铁和胃酸缺乏，伴随退行性病变最终导致死亡。多达21种TRPML1基因突变体与ML IV有关。TRPML1是瞬时受体电位TRP通道成员，主要在溶酶体表达。该通道有三个独特的功能特征：对Ca2+ 和 Fe2+通透，受溶酶体内pH 和Ca2+调节，有脂酶活性。这些功能特征的分子机制、TRPML1在溶酶体的生理功能、及TRPML1突变的致病机理尚不清楚。前期工作中我们解析了TRPML1一个位于溶酶体内的重要功能片段的晶体结构。本项目拟以此结构及丰富的初步功能研究结果为基础，深入研究上述课题。这一研究将大力促进对TRPML1生物物理特性和生理功能的认识，为防治ML LV提供更加扎实的理论基础。

溶酶体是细胞的回收和消化中心，对细胞生理极为重要，溶酶体功能缺陷破坏细胞稳态，导致各种溶酶体贮积症，包括粘脂质贮积症（ML IV)。TRPML通道亚家族属于瞬时受体电位TRP通道家族，主要分布在溶酶体，对溶酶体功能至关重要，有三个成员，TRPML1,TRPML2和TRPML3。TRPML1有21种基因突变体与ML IV有关，虽然目前尚未发现TRPML3与人类疾病相关，但小鼠有两个自发性功能增强型TRPML3突变体能够导致听觉丧失及毛色弱化的生理表型，表明TRPML3通道在细胞生理过程中发挥重要作用。TRPML通道有许多独特的功能特征，包括对Ca2+通透，受溶酶体内pH 和Ca2+调节，可被膜脂分子PIP2调节等。这些功能特征的分子机制、TRPML通道在溶酶体的生理功能、及TRPML1突变的致病机理尚不清楚。本课题解析了TRPML1一个被称为I-II linker的重要功能域在三种不同pH状况下的晶体结构。结构显示其为旋转对称的四聚体，四聚体中央形成一个直径为14 Å的腔孔（luminal pore），通过由16个氨基酸残基组成的孔腔环连接，孔腔环含3个天门冬氨酸残基。我们用生化方法和冷冻电子显微镜验证了该功能域的结构存在于全长通道。我们发现孔腔环的天门冬氨酸残基对pH 和Ca2+调节TRPML1起关键性作用。这一研究将大力促进对TRPML1生物物理特性和生理功能的认识，为防治ML LV提供更加扎实的理论基础。我们通过使用单颗粒冷冻电子显微镜解析了全长人源性TRPML3离子通道在关闭状态、激动剂结合导致的开放状态及低pH抑制状态下的高分辨率（分别为4.06、3.62及4.65 Å）结构，发现激动剂ML-SA1结合于跨膜结构域S5与S6之间并诱导打开S6的门控组件。我们的研究提示了一种新的通道调控机制，即腔内低pH及其它生理调控因子（如PIP2），通过导致S1及S2的构象变化，对TRPML3通道功能进行调节。这一工作揭示了TRPML3通道全新的结构特点及通道激活与调控中发生的构象变化，为进一步研究TRPML3通道调控机制和生理功能提供了结构基础。除上述研究外，我们还解析了首个真核细胞环核苷酸门控通道的全长高分辨率三维结构，并开展了传统西药和中医的作用和副作用的分子机理方面的研究。