FUNCTIONAL/STRUCTURAL STUDIES OF MAMMALIAN NEURAMINIDASE

拟议的研究范围是研究哺乳动物溶酶体神经氨酸酶(Neur)，这是一种重要的水解酶，属于唾液酸酶超家族，在自然界中广泛分布。哺乳动物神经氨酸酶，包括胞浆、溶酶体和质膜形式，由于其丰度低、不稳定和膜结合的特点而受到限制。直到最近几年，关于这些酶的生理功能的重要线索才出现(S)。溶酶体神经通过去除唾液酸端残基来启动唾液酸糖结合物的水解。该酶在唾液酸酶中是独一无二的，因为它需要与保护性蛋白/组织蛋白A(PPCA)结合以实现细胞内路由和溶酶体激活，并且它与神经退行性代谢性疾病有关。涎腺病是由神经部位的结构性病变引起的，而半乳唾液酸病(GS)是由于缺乏PPCA而引起的神经和β-半乳糖苷酶(β-GAL)的联合缺陷。这两种疾病具有共同的临床和生化特征，可归因于神经功能的丧失。我们的总体目标是对溶酶体神经功能有更广泛的了解(S)，并将这些知识应用于涎腺病和GS的病理生理学研究，进而有助于设计适当的治疗方法。我们计划利用PPCA前体的3D结构，对这两种蛋白之间的潜在接触位点进行定向突变，并利用在涎腺病患者中发现的自然神经突变(S)，来研究NUR和PPCA之间的结构-功能关系。通过这种方法，我们将确定对Nur/PPCA相互作用、细胞内转运和激活至关重要的残基/结构域。我们还将对一种新的神经氨酸酶亚型进行生化表征，其想法是将特定的生化特性与功能联系起来。NURE基因敲除小鼠的特征将有助于确定由NURE缺陷引起的疾病的一般外显性，并将其与GS小鼠进行比较。这些体外和体内研究将与使用杆状病毒表达系统确定NURR 3D结构以大量生产NURR蛋白相结合。最后，如果我们将成功地产生稳定的Neur和PPCA的复合体，我们将启动这两种酶的复合体的结晶。PI的实验室处于开发这一研究路线的独特地位，因为它已经为拟议的研究建立了遗传和生化系统，并且可以依靠杰出的结构生物学家的专业知识来完成项目的结晶学部分。

The scope of the proposed research is to study mammalian lysosomal neuraminidase (neur), an essential hydrolase that belongs to the superfamily of sialidases found widely spread in nature. Information on the mammalian neuraminidases, including a cytosolic, a lysosomal and a plasma membrane form, has been limited by their apparent low abundance, instability and membrane-bound character. Only in recent years important clues have emerged as to the physiological function(s) of these enzymes. Lysosomal neur initiates the hydrolysis of sialo- glyconjugates by removing their terminal sialic acid residues. The enzyme is unique among sialidases in that it requires association with the protective protein/cathepsin A (PPCA) for intracellular routing and lysosomal activation, and it is associated with neurodegenerative diseases of metabolism. Sialidosis is caused by structural lesions at the neur locus, and galactosialidosis (GS), a combined deficiency of neur and beta- galactosidase (beta-gal) is caused by the absence of PPCA. The two diseases have common clinical and biochemical features that can be assigned to the loss of neur function. Our overall goal is to gain a broader understanding of lysosomal neur function(s), and to apply this knowledge to the study of the pathophysiology of sialidosis and GS which, in turn, could help in designing appropriate therapy. We plan to investigate the structure- function relationships between neur and PPCA, making use of the 3D structure of the PPCA precursor for targeted mutagenesis of potential contact sites between the two proteins, and of natural neur mutation(s) identified in patients with sialidosis. With this approach we will identify residues/domains crucial for neur/PPCA interaction, intracellular transport and activation. We will also biochemically characterize a novel neuraminidase isoform with the idea of linking specific biochemical properties with function. The characterization of neur knockout mice will be instrumental to determine the general penetrance of the disease that results from neur deficiency, and to compare it with the GS mouse. These in vitro and in vivo studies will be coupled to the determination of neur 3D structure using the baculovirus expression system to produce neur protein in large quantities. Finally, provided that we will be successful in generating stable complex of neur and PPCA we will initiate crystallization of the complex of the two enzymes. The PI's laboratory is in the unique position to develop this line of investigation as it has established genetic and biochemical systems for the proposed studies, and can rely on the expertise of an outstanding structural biologist for the crystallography part of the project.