TISSUE TRANSGLUTAMINASE--REGULATION AND DYSREGULATION

组织谷氨酰胺转氨酶(TTG)是一种新型的双功能蛋白 钙依赖的转胺酶和信号转导 GTP结合蛋白(Galpha H)。作为一种转胺酶，tTG 催化特定底物之间形成异肽键 蛋白质产生不溶解的聚合结构。一个有定义的 阿尔茨海默病的特征是大脑中存在 细胞内(神经原纤维缠结[NFTs])和细胞外(老年性 斑块)高度不溶的丝状蛋白质聚集体。 来自该实验室和其他实验室的研究已经证明， NFTs的主要蛋白质和Abeta(1-40)，Abeta(1-40)是NFTs的主肽 老年斑，都是良好的体外tTG底物。 申请者实验室最近的研究表明，在 大脑皮层，NFT和老年斑普遍存在，但在 几乎没有这些损害的小脑，tTG水平和 阿尔茨海默病患者脑组织中TG活性显著升高 与年龄匹配的对照组相比。此外，它还被假设为 TTG可能参与了密码子重复的神经退行性变。 疾病，如亨廷顿病，通过促进形成 不可溶的神经元内含物。这些和其他发现表明 TTG可以促进不溶物质的形成， 某些神经退行性疾病的病理损害。 这项提案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控， 主要是通过钙和GTP，以及这些过程可能是如何 精神错乱，尤其是在与阿尔茨海默病相关的情况下。 这种对tTG，原位调节的关注代表了一个重要的 与以往的许多体外研究相比取得了进展。申请者 综合工作假说是原位tTG紧密结合 受监管，而这些监管过程的扰动导致 在不适当的水平和变态活动的增加中 TTG，这有助于神经退行性变过程 阿尔茨海默氏症。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 大鼠大脑皮层神经元和海马神经元的培养 神经元，也将在一些研究中使用。 这项提议的目标是检验以下假设(1) GTP和钙通过直接作用调节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的转胺化活性，(3)激活 TTG的转胺化活性导致tau的修饰， 并且这些修改与 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，从而引导 定位并确定tTG的功能(即，作为 转胺酶或信号转导G蛋白)，以及(5) Aβ和/或阿尔茨海默病早老素突变体增加变态反应 TTG的活性通过直接和/或间接机制实现。这些研究 将大大增加我们对tTG调控的理解。 并可能提供对其在 神经退行性变过程。

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.