Molecular biology of TPPI/CLN2 protein

TPPI/CLN2蛋白的分子生物学

• 批准号: 6705225
• 负责人: ADAM A GOLABEK
• 金额: $ 18.81万
• 依托单位: INSTITUTE FOR BASIC RES IN DEV DISABIL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6705225
• 关键词: catalyst; confocal scanning microscopy; endopeptidases; enzyme mechanism; enzyme structure; fluorescence spectrometry; gel filtration chromatography; gene mutation; glycosylation; immunoprecipitation; inborn lysosomal enzyme disorder; protein structure function; protein transport; proteolysis; three dimensional imaging /topography; western blottings; yeasts

DESCRIPTION (provided by applicant): The long-term goal of this proposed project is to characterize the factors and mechanisms underlying maturation, stability, and catalytic activity of tripeptidyl-peptidase I (TPP I). Naturally occurring mutations in TPP I lead to a fatal neurodegenerative lysosomal storage disorder- classic late-infantile neuronal ceroid lipofuscinosis (CLN2). TPP I is a lysosomal aminopeptidase that releases tripeptides from a free N-terminus of proteins. TPP I is extensively glycosylated. Our preliminary data suggest that N-glycan attached to Asn286 is critical for lysosomal targeting and activity of TPP I. We propose to further elucidate the role of N-glycosylation in the trafficking of TPP I as well as to determine whether and how N-glycosylation affects folding, activity, and stability of the enzyme. These experiments will be performed by expressing and studying N-glycosylation defective TPP I mutants in mammalian cells by means of laser-scanning confocal microscopy, Western blotting, in vivo labeling, immunoprecipitation and enzymatic assay. TPP I is a serine-type protease, although it is atypical in that instead of classical Ser/His/Asp catalytic triad in the active site, it appears to contain Ser/Asp/Asp triad. Although TPP I is able to autoactivate in vitro, according to our data, autoactivation of TPP I in vitro is an intramolecular process that is efficient in a very narrow pH range. Furthermore, our studies suggest that in vivo, another serine protease is involved in proteolytic processing of TPP I. Experiments we are planning to perform will assess the effect of various factors (e.g., ionic strenght, charged compounds) on autoactivation of the proenzyme; will elucidate the role of the prodomain in the activation, activity, and stability of the enzyme and will identify the protease responsible for in vivo processing of TPP I. For these studies, we will use human TPP I purified from conditioned media of overexpressing CHO cells. TPP I prodomain will be expressed and purified from yeast, Pichia pastoris. The activity and stability of the enzyme in vitro will be analyzed by using SDS-PAGE, Western blotting, gel filtration chromatography, and spectrofluorometry. Protease involved in processing of TPP I in vivo will be identified by using the biochemical purification approach and active-site mutant proenzyme as a substrate. Finally, to elucidate the catalytic mechanism of TPP I, we intend to analyze the secondary and three-dimensional structure of the enzyme by means of CD spectroscopy and high-resolution crystallography of wild-type TPP I proenzyme and mature enzyme as well as active-site mutants. Analysis of the localization of naturally occurring TPP I mutations on three-dimensional structure of the wild-type TPP I precursor will give important insights into the structural basis of the CLN2 disease process. We also anticipate that completion of these studies will allow us to better understand the molecular biology of TPP I and shed new light on the pathogenesis of the CLN2 and thus may be useful for development in the future of therapy for this devastating human disorder.

描述（由申请人提供）：本项目的长期目标是表征三肽基肽酶I （TPP I）成熟、稳定性和催化活性的因素和机制。自然发生的TPP I突变可导致致死性神经退行性溶酶体贮积症——典型的婴儿晚期神经性ceroid脂褐病（CLN2）。TPP I是一种溶酶体氨基肽酶，从蛋白质的游离n端释放三肽。TPP I被广泛糖基化。我们的初步数据表明，附着在Asn286上的n -聚糖对TPP I的溶酶体靶向和活性至关重要。我们建议进一步阐明n -糖基化在TPP I转运中的作用，并确定n -糖基化是否以及如何影响该酶的折叠、活性和稳定性。这些实验将通过激光扫描共聚焦显微镜、Western blotting、体内标记、免疫沉淀和酶分析等方法，在哺乳动物细胞中表达和研究n -糖基化缺陷的TPP I突变体。TPP I是一种丝氨酸型蛋白酶，但它的非典型特征在于其活性位点不具有经典的Ser/His/Asp催化三联体，而是含有Ser/Asp/Asp三联体。虽然TPP I能够在体外自激活，但根据我们的数据，TPP I在体外的自激活是一个分子内过程，在非常窄的pH范围内有效。此外，我们的研究表明，在体内，另一种丝氨酸蛋白酶参与了TPP的蛋白水解过程。我们计划进行的实验将评估各种因素（例如，离子强度、带电化合物）对原酶自激活的影响；将阐明原结构域在酶的激活、活性和稳定性中的作用，并将确定负责TPP I体内加工的蛋白酶。在这些研究中，我们将使用从过表达CHO细胞的条件培养基中纯化的人TPP I。将从酵母、毕赤酵母中表达和纯化TPP I原域。酶的活性和稳定性将通过SDS-PAGE， Western blotting，凝胶过滤层析和荧光光谱法进行分析。利用生物化学纯化方法和活性位点突变前酶作为底物，将在体内鉴定参与TPP I加工的蛋白酶。最后，为了阐明TPP I的催化机制，我们打算利用CD光谱和高分辨率晶体学对野生型TPP I原酶和成熟酶以及活性位点突变体进行二级和三维结构分析。分析自然发生的TPP I突变在野生型TPP I前体三维结构上的定位，将为了解CLN2疾病过程的结构基础提供重要见解。我们还预计，这些研究的完成将使我们更好地了解TPP I的分子生物学，并为CLN2的发病机制提供新的线索，从而可能有助于开发这种毁灭性人类疾病的治疗方法。