Investigating Regulation of Integral Membrane Protein Cofactor Biosynthesis using

描述(申请人提供)：由蛋白质和作为辅因子的小分子组成的完整的膜蛋白复合体对生物体来说是普遍存在的，并且是至关重要的。例如，细胞色素氧化酶是一种线粒体复合体，它催化电子传递给氧气，从而允许细胞呼吸。视紫红质存在于包括人类在内的许多动物的眼睛中，由视黄素膜蛋白和维生素A视网膜辅因子组成。在这些系统中，蛋白质和辅因子保持着微妙的平衡。这种平衡的破坏被认为是阿尔茨海默氏症和视网膜色素变性等疾病的致病因素。这项拟议的项目描述了一种多学科的方法来表征一种新的协调合成蛋白质和辅因子的整体膜复合体的机制。我们将利用微生物盐生盐杆菌中简单的细菌视紫红质(BR)蛋白质辅因子复合体，潜在地为在更复杂的系统中如何实现协调提供洞察。在嗜盐古细菌H.salinarum中，BR作为光驱动的质子泵发挥作用，并允许有机体在低氧阻止有氧呼吸的条件下产生可用的细胞能量。BR是最简单的膜蛋白复合体，由单一的视网膜辅助因子与单一的细菌素(BO)结合而成。在低氧条件下，盐藻能迅速诱导BR的生物合成，这种诱导必然需要增加蛋白质BO和辅因子视网膜的产量。在这项拟议的工作中，我们探索了盐生H.salinarum蛋白质如何相互作用来协调BO和视网膜的合成。我们的初步结果表明，这种协调是通过一种新的机制发生的，其中BO不被视网膜结合，而是抑制另一条生化途径，只允许前体分子用于视网膜合成。为了研究这一机制，我们将首先使用遗传和生化方法来表征催化合成视网膜辅因子和相关分子的酶。然后，我们将确定哪些视网膜前体或酶与BO相互作用。最后，我们将从结构上表征BO与这些分子之间的相互作用。 与公共健康相关：由蛋白质和辅因子组成的分子复合体对我们的日常生活至关重要。例如，视紫红质是一种由蛋白质、视蛋白和维生素A分子组成的复合体，它们共同发挥作用，让我们能够看到东西。细胞色素氧化酶是几种蛋白质和一种血红素分子的复合体，它们聚集在一起，让我们从所吃的食物中获得能量。不幸的是，这些辅因子和蛋白质偶尔会失去平衡，导致视网膜色素变性和阿尔茨海默氏症等疾病。这项拟议的研究将研究微生物中的类似分子复合体，以阐明保持蛋白质及其辅因子正确平衡的机制。

DESCRIPTION (provided by applicant): Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors are ubiquitous and vitally important for organisms. For example, cytochrome oxidase is a mitochondrial complex that catalyzes the delivery of electrons to oxygen to allow cellular respiration. Rhodopsin is found in the eyes of many animals, including humans, and consists of an opsin membrane protein and a Vitamin A retinal cofactor. In these systems, an exquisite balance of protein and cofactor is maintained. Disruptions in this balance are thought to be causative factors for diseases such as Alzheimer's and retinitis pigmentosa. The proposed project describes a multidisciplinary approach to characterize a novel mechanism for coordinated synthesis of proteins and cofactors for integral membrane complexes. We will take advantage of the simple bacteriorhodopsin (BR) protein-cofactor complex in the microbe Halobacterium salinarum to potentially provide insight into how coordination is achieved in more complex systems. BR functions as a light-driven proton pump in the halophilic archaeon H. salinarum, and allows the organism to generate usable cellular energy under conditions where low oxygen prevents aerobic respiration. BR is the simplest possible membrane protein complex consisting of a single retinal cofactor bound to a single protein, bacterioopsin (BO). Under low oxygen conditions, H. salinarum rapidly induces BR biosynthesis, and this induction necessarily requires increased production of both the protein, BO, and the cofactor, retinal. In the proposed work, we explore how H. salinarum proteins interact to coordinate the synthesis of BO and retinal. Our preliminary results suggest that this coordination occurs by a novel mechanism where BO, not bound by retinal, inhibits an alternate biochemical pathway to only allow precursor molecules to be used for retinal synthesis. To investigate this mechanism, we will first use genetic and biochemical approaches to characterize enzymes that catalyze the synthesis of the retinal cofactor and related molecules. We will then identify which retinal precursors or enzymes interact with BO. Lastly, we will structurally characterize the interaction between BO and these molecules. PUBLIC HEALTH RELEVANCE: Molecular complexes consisting of proteins and cofactors our critical to our everyday life. For example, rhodopsin is a complex consisting of a protein, opsin, and a Vitamin A molecule that function together to allow us to see. Cytochrome oxidase is a complex of several proteins and a heme molecule that come together to allow us to get energy from the food we eat. Unfortunately, these cofactors and proteins can occasionally get out of balance leading to diseases such as retinitis pigmentosa and Alzheimer's. The proposed research will study a similar molecular complex in a microbe in order to elucidate the mechanisms that keep proteins and their cofactors in the correct balance.