TISSUE TRANSGLUTAMINASE--REGULATION AND DYSREGULATION

组织转氨酶（tTG）是一种新型的双功能蛋白， 钙依赖性转酰胺酶和信号转导 GTP结合蛋白（Galfa h）。 作为一种转酰胺酶，tTG 催化特定底物之间形成异肽键 蛋白质以产生不溶性聚合物结构。 一个决定性 阿尔茨海默病的特征是大脑中存在 细胞内（神经元缠结[NFT]）和细胞外（老年性 斑块）高度不溶的丝状蛋白质聚集体。 这个实验室和其他实验室的研究表明，tau蛋白， NFT的主要蛋白，和Abeta（1-40），NFT的主要肽， 老年斑都是tTG的优良体外底物。 来自申请人实验室的最近研究已经证明， 大脑皮层，NFT和老年斑普遍存在，但在 小脑几乎没有这些病变，tTG水平和 阿尔茨海默病脑组织TG活性显著升高 与年龄匹配的对照组相比。 此外，有人假设 tTG可能参与密码子重复的神经变性 疾病，如亨廷顿病，通过促进形成 不溶的神经内含物 这些和其他调查结果表明， tTG可能有助于形成不溶物， 某些神经退行性疾病的病理损伤。 这一建议是一个相互竞争的延续，其重点是 研究tTG的直接和间接原位调节， 主要由钙和GTP，以及这些过程如何可能是 破坏，特别是在与阿尔茨海默病有关的情况下。 这种对tTG的原位调节的关注代表了一种重要的 与许多以前的体外研究相比，这是一个进步。 申请人 综合工作假设是，在原位tTG是紧密的 受监管，这些监管过程的扰动导致 在不适当的水平和transamading活动的增加， tTG，这有助于神经退行性过程， 老年痴呆症 在这项提案中，大多数实验将 在人神经母细胞瘤细胞中进行，尽管原代细胞 培养大鼠大脑皮层神经元，以及海马 神经元，也将用于一些研究。 本提案的目标是检验以下假设（1）， GTP和钙协同作用，通过直接作用调节tTG活性。 和间接机制，（2）受体介导的动员， 来自内质网（ER）的钙在 调节tTG的转酰胺活性，（3）激活 tTG的转酰胺活性导致tau的修饰， 这些修饰与特定的改变有关， 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， 定位并确定tTG的功能（即，作为 转酰胺酶或信号转导G蛋白），和（5） Abeta和/或阿尔茨海默氏早老素突变体增加转酰胺作用， 通过直接和/或间接机制的tTG活性。 这些研究 将大大增加我们对tTG调节的理解 并有可能提供洞察其假定的作用， 神经退化过程

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.