TISSUE TRANSGLUTAMINASE--REGULATION AND DYSREGULATION

组织谷氨酰胺转氨酶——调节和失调

• 批准号: 6055391
• 负责人: Gail V. W. Johnson
• 金额: $ 18.19万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-25 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6055391
• 关键词: Alzheimer's disease; amyloid proteins; biological signal transduction; calcium flux; calpain; endoplasmic reticulum; enzyme activity; enzyme inhibitors; enzyme substrate; guanine nucleotide binding protein; guanosine triphosphate; human tissue; inositol phosphates; neurofibrillary tangles; presenilin; protein glutamine gamma glutamyltransferase; proteolysis; tau proteins; tissue /cell culture

Tissue transglutaminase (tTG) is a novel, dual function protein that is both a calcium-dependent transamidating enzyme and a signal transducing GTP-binding protein (Galpha h). As a transamidating enzyme, tTG catalyzes the formation of isopeptide bonds between specific substrate proteins to produce insoluble polymeric structures. A defining characteristic of Alzheimer's disease brain is the presence of intracellular (neurofibrillary tangles [NFTs]) and extracellular (senile plaques) filamentous proteinaceous aggregates that are highly insoluble. Studies from this, and other laboratories, have demonstrated that tau, the major protein of the NFTs, and Abeta (1-40), a primary peptide of the senile plaques, are both excellent in vitro substrates of tTG. Recent studies from the applicants laboratory have demonstrated that in cerebral cortex, where NFTs and senile plaques are prevalent, but in cerebellum which is virtually devoid of these lesions, tTG levels and TG activity are elevated significantly in Alzheimer's disease brain compared to age-matched controls. In addition, it has been hypothesized that tTG maybe involved in the neurodegeneration of codon reiteration diseases, such as Huntington's disease, by facilitating the formation of insoluble neuronal inclusions. These and other findings indicate that tTG could contribute to the formation of the insoluble, pathological lesions in certain neurodegenerative disorders. The focus of this proposal, which is a competing continuation, is on investigating the direct and indirect in situ regulation of tTG, predominantly by calcium and GTP, and how these processes may be disrupted, especially in conditions associated with Alzheimer's disease. This focus on the modulation of tTG, in situ represents a significant advance compared to the many previous in vitro studies. The applicants comprehensive working hypothesis is that in situ tTG is tightly regulated, and that perturbations of these regulatory processes results in inappropriate increases in the levels and transamidating activity of tTG and this contributes to the neurodegenerative processes of Alzheimer's disease. In this proposal the majority of experiments will be carried out in human neuroblastoma cells, although primary cell cultures of rat cerebral cortical neurons, as well as hippocampal neurons, will also be used in some studies. The goals of this proposal are to test the following hypotheses (1) that GTP and calcium work in concert to regulate tTG activity through direct and indirect mechanisms, (2) that receptor-mediated mobilization of calcium from the endoplasmic reticulum (ER) plays a significant role in modulating the transamidating activity of tTG, (3) that activation of the transamidating activity of tTG results in the modification of tau, and these modifications are associated with specific alterations in the metabolism, function and subcellular distribution of tau, (4) that GTP modulates tTG interactions with specific proteins which direct the localization and determine the function of tTG (i.e., as a transamidating enzyme or signal transducing G protein), and (5) that Abeta and/or Alzheimer's presenilin mutants increase the transamidating activity of tTG by direct and/or indirect mechanisms. These studies will increase our understanding of the regulation of tTG significantly and are likely to provide insight into its putative role in neurodegenerative processes.

组织转谷氨酰胺酶 (tTG) 是一种新型双功能蛋白， 既是钙依赖性转酰胺酶又是信号转导酶 GTP 结合蛋白 (Galpha h)。 作为转酰胺基酶，tTG 催化特定底物之间异肽键的形成 蛋白质产生不溶性聚合结构。 一个定义 阿尔茨海默病的大脑特征是存在 细胞内（神经原纤维缠结[NFT]）和细胞外（老年性纤维缠结） 斑块）高度不溶的丝状蛋白质聚集体。 该实验室和其他实验室的研究表明，tau、 NFT 的主要蛋白质，以及 Abeta (1-40)（NFT 的主要肽） 老年斑都是 tTG 的优良体外底物。 申请人实验室的最新研究表明， 大脑皮层，NFT 和老年斑在那里普遍存在，但 小脑实际上没有这些病变，tTG 水平和 阿尔茨海默病大脑中 TG 活性显着升高 与年龄匹配的对照组相比。 此外，还假设 tTG可能参与密码子重复的神经变性 疾病，例如亨廷顿病，通过促进形成 不溶性神经元包涵体。 这些和其他发现表明 tTG 可能有助于形成不溶性物质， 某些神经退行性疾病的病理病变。 该提案是一个竞争性的延续，其重点是 研究 tTG 的直接和间接原位调节， 主要由钙和 GTP 组成，以及这些过程如何进行 被破坏，特别是在与阿尔茨海默病相关的情况下。 这种对 tTG 调节的关注，原位代表了一个重要的意义。 与之前的许多体外研究相比，取得了进步。 申请人 全面的工作假设是原位 tTG 紧密结合 受到监管，并且这些监管过程的扰动会导致 的水平和转酰胺基活性的不适当增加 tTG 会导致神经退行性过程 阿尔茨海默病。 在这个提案中，大多数实验将 可以在人神经母细胞瘤细胞中进行，尽管原代细胞 大鼠大脑皮质神经元以及海马的培养物 神经元，也将用于一些研究。 该提案的目标是检验以下假设 (1) GTP 和钙协同作用，通过直接调节 tTG 活性 和间接机制，（2）受体介导的动员 内质网 (ER) 中的钙在 调节 tTG 的转酰胺基活性，(3) tTG 的转酰胺基活性导致 tau 的修饰， 这些修改与特定的更改相关 tau 的代谢、功能和亚细胞分布，(4) GTP 调节 tTG 与特定蛋白质的相互作用，从而指导 定位并确定 tTG 的功能（即，作为 转酰胺基酶或信号转导G蛋白），以及（5） Abeta 和/或阿尔茨海默病早老素突变体增加转酰胺基作用 通过直接和/或间接机制检测 tTG 的活性。 这些研究 将显着增加我们对 tTG 调控的理解 并且可能会深入了解其假定的作用 神经退行性过程。