Membrane Structure and Enzyme Activity

描述(申请人提供)：蛋白质转位到磷脂 膜涉及特定的蛋白质结构，并可由 在界面上存在非磷脂类“第二信使”，如 二酰甘油这项研究的长期目标是了解 磷脂与非磷脂第二信使的相互作用 调节外周蛋白与界面的结合和随后的表达 催化活性。其焦点是胰腺三酰甘油脂肪酶(PTL)和 它的辅因子蛋白，Colipase(Col)，对它来说，二酰甘油等脂类是 活化剂以及脂肪酶底物。PTL及其基因的另外两个成员 脂蛋白脂肪酶和肝脂酶家族是脂蛋白脂酶和肝脂酶的主要调节因子。 脂类向周围组织和从周围组织的分布。因此，它们是 脂类动态平衡疾病治疗中的高度相关靶点 肥胖和动脉粥样硬化。为了正常发挥作用，脂质结合基序 PTL的N-末端结构域必须与其 催化高效或“开放”构象。C-末端的作用 PTL在其中的作用区域尚不清楚。在PTL所处的液脂界面 功能、磷脂和脂肪酶底物形成混合的动态复合体 用不复杂的脂类。我们假设，络合物抑制了 蛋白质的吸附，因此，脂肪分解。我们进一步假设一旦PTL 和Col结合，它们重新排列界面上的脂质物种，以帮助克服 这种抑制。载脂蛋白C-II，脂蛋白脂肪酶的辅助因子， 似乎与COL相似，尽管它缺乏结构上的同源性。至 检验这些假设，我们建议1)定义脂质复合体在 调节PTL的结构域和Col与接口的关联。至 为此，我们将使用辐射测量和荧光测量方法来测量 Col和PTL结构域结合的初始速率作为界面的函数 组成和包装；2)测定三种脂类各自的能力 结合PTL和COL基序扰乱脂质侧向组织。至 要做到这一点，我们将用荧光测定法来确定每一个 结合蛋白基序能够在侧向重新分布脂质物种 界面；3)确定绑定的界面要求 PTL在催化高效构象中的催化结构域。至 要做到这一点，我们将从化学和光谱上确定PTL如何 催化结构域的结合和构象受到调控；4)定义 PTL-Col脂解系统与血清的功能相似性 脂蛋白脂酶-载脂蛋白C-II系统。要实现这一点， AIMS 1-3中使用的技术将应用于这些蛋白质。以其独特的 关注脂类界面结构在调节蛋白质结合中的作用， 这项研究将提供一种具体的机制理解如何辅因子 蛋白质使脂肪分解发生在生理上相关的界面上。更多 广义地讲，它将有助于解释脂质第二信使如何调节蛋白质。 与细胞内的信号事件相关的易位。

DESCRIPTION (provided by applicant): Translocation of proteins to phospholipid membranes involves specific protein structures and can be triggered by the presence in the interface of a non-phospholipid lipid "second messenger," like diacylglycerol The long-range goal of this research is to understand how interactions between phospholipids and non-phospholipid second messengers regulate peripheral protein binding to interfaces and the subsequent expression of catalytic activity. Its focus is pancreatic triacylglycerol lipase (PTL) and its cofactor protein, colipase (COL), for which lipids like diacylglycerols are activators as well as lipase substrates. PTL and two other members of its gene family, lipoprotein lipase and hepatic lipase, are the primary regulators of the distribution of lipids to and from peripheral tissues. Hence, they are highly relevant targets in the treatment of diseases of lipid homeostasis like obesity and atherosclerosis. To function properly, the lipid-binding motif of the N-terminal domain of PTL must bind to the lipid-water interface in its catalytically-efficient or 'open' conformation. The role of the C-terminal domain of PTL in this is unclear. In the fluid lipid interfaces at which PTL functions, phospholipids and lipase substrates form dynamic complexes that mix with uncomplexed lipids. We hypothesize that complexes inhibit the rate of protein adsorption and, hence, lipolysis. We further hypothesize that once PTL and COL bind, they rearrange lipid species in the interface to help overcome the inhibition. Apolipoprotein C-II, the cofactor for lipoprotein lipase, appears to act similarly to COL despite its lack of structural homology. To test these hypotheses, we propose 1) to define the role of lipid complexes in regulating the association of PTL's domains and COL to interfaces. To accomplish this we will use radiometric and fluorometric methods to measure initial rates of COL and PTL domain binding as a function of interfacial composition and packing; 2) to determine the ability of each of the three lipid associating motifs of PTL and COL to perturb lipid lateral organization. To accomplish this we will fluorimetrically determine the extent to which each bound protein motif is able to laterally redistribute lipid species in interfaces; 3) to determine the interfacial requirements for the binding of PTL's catalytic domain in the catalytically-efficient conformation. To accomplish this we will chemically and spectroscopically determine how PTL catalytic domain binding and conformation are regulated; 4) to define the functional similarities of the PTL-COL lipolytic system with the serum lipoprotein lipase-apolipoprotein C-II system. To accomplish this the techniques used in aims 1-3 will be applied to these proteins. With its unique focus on the role of lipid interfacial structure in regulating protein binding, this research will provide a specific mechanistic understanding of how cofactor proteins enable lipolysis to occur at physiologically relevant interfaces. More broadly, it will help to explain how lipid second messengers regulate protein translocation associated with signaling events in cells.