Molecular biology of TPPI/CLN2 protein

描述（由申请人提供）：本拟议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潜在因素和机制。TPP I中自然发生的突变导致致命的神经退行性溶酶体贮积症-经典的晚期婴儿神经元蜡样质脂褐质沉积症（CLN 2）。TPP I是一种溶酶体氨肽酶，从蛋白质的游离N-末端释放三肽。TPP I是广泛糖基化的。我们的初步数据表明，连接到Asn 286的N-聚糖对TPPI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运输中的作用，以及确定N-糖基化是否以及如何影响酶的折叠、活性和稳定性。这些实验将通过在哺乳动物细胞中表达和研究N-糖基化缺陷TPP I突变体来进行，所述表达和研究通过激光扫描共聚焦显微镜、蛋白质印迹、体内标记、免疫沉淀和酶测定来进行。TPP I是一种丝氨酸型蛋白酶，尽管它是非典型的，因为在活性位点中不是经典的Ser/His/Asp催化三联体，而是似乎含有Ser/Asp/Asp三联体。虽然TPP I能够在体外自动激活，但根据我们的数据，TPP I在体外的自动激活是一个分子内过程，在非常窄的pH范围内是有效的。此外，我们的研究表明，在体内，另一种丝氨酸蛋白酶参与TPP I的蛋白水解加工。我们计划进行的实验将评估各种因素的影响（例如，离子强度，带电化合物）对酶原自活化的影响;将阐明前结构域在酶的活化、活性和稳定性中的作用，并将鉴定负责TPP I体内加工的蛋白酶。对于这些研究，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。TPP I前结构域将从酵母毕赤酵母（Pichia pastoris）表达和纯化。将通过使用SDS-PAGE、蛋白质印迹、凝胶过滤色谱和荧光分光光度法分析酶的体外活性和稳定性。将通过使用生物化学纯化方法和活性位点突变体酶原作为底物来鉴定参与TPP I在体内加工的蛋白酶。最后，为了阐明TPP I的催化机制，我们打算通过CD光谱和高分辨率晶体学的野生型TPP I酶原和成熟酶以及活性位点突变体的手段来分析的二级和三维结构的酶。对野生型TPP I前体三维结构上天然存在的TPP I突变的定位分析将对CLN 2疾病过程的结构基础提供重要的见解。我们还预计，这些研究的完成将使我们能够更好地了解TPP I的分子生物学，并为CLN 2的发病机制提供新的线索，因此可能有助于未来开发这种毁灭性人类疾病的治疗方法。

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.