Modulation of Monocarboxylic Acid Transporter Kinetic Function for Stroke Therapy

描述（由申请人提供）：拟议工作的总体目标是表征蛋白激酶A （PKA）调节大鼠脑内皮细胞中单羧酸转运蛋白1 （MCT1）功能的精确分子机制。我们假设PKA通过一种称为转导体的多蛋白调节复合体来调节MCT1。这项工作意义重大，因为脑血管内皮细胞MCT1是乳酸通过血脑屏障的脑血管内皮运输的唯一促进者。因此，在脑代谢中，乳酸是一种重要的能量底物，在缺血性中风、高血糖性中风和脑损伤等病理条件下，脑乳酸酸中毒已被证明是细胞损伤病因学的关键指标，它在脑代谢中很重要。通过更好地了解控制乳酸酸中毒的因素，可以促进中风和脑损伤的新药物治疗的发展。多蛋白信号复合物或转导体的组织在许多细胞系统中无处不在地协调PKA的功能，但在脑血管内皮中尚未得到很好的表征；因此，一个重要的附加项目是阐明转导体在脑血管内皮细胞中组织PKA依赖性MCT1调控的机制。我们的广泛假设是MCT1是一个转导体复合体的一部分，该复合体通过促进MCT1磷酸化和囊泡运输来协调其PKA依赖性调节。这种调节作用依赖于肌动蛋白细胞骨架的动态变化，肌动蛋白促进运输并作为转导体的锚点。在假设的复合体中，MCT1的c端与与AKAP蛋白相关的PDZ适配器蛋白结合。AKAP蛋白结合β -肾上腺素能受体PKA，并将转导体连接到形态动态的肌动蛋白细胞骨架上。我们已经证明，肌动蛋白在PKA激活后的细胞骨架变形是MCT1调控的一个必要因素。该复合物的功能是促进PKA磷酸化MCT1所需的蛋白质相互作用的空间组织，并在依赖PDZ适配器蛋白和/或AKAP和细胞骨架形态的途径中介导PKA依赖的MCT1的囊泡运输。我们发现，cAMP类似物刺激PKA可对MCT1功能产生双峰效应；胰蛋白酶化后3小时抑制，胰蛋白酶化后24小时增加。我们假设PKA的这种双峰调节作用是由胰蛋白酶破坏细胞质后转导体复合体的时间依赖性形成引起的。通过检查我们的自变量在3和24小时时间点的影响，我们将利用这种二分法进一步阐明有关转导体的细节。我们提出的第一个特定假设的实验将确定MCT1 C或n端是否与PDZ适配器或AKAP结合伙伴相互作用，作为PKA调节的功能要求。绑定伙伴将被识别。我们的第二个假设将通过显示PKA刺激是否导致MCT1和转导体蛋白磷酸化、MCT1的囊泡运输、MCT1调控中细胞骨架形态依赖的变化，以及细胞骨架形态依赖的转导体内关键蛋白-蛋白相互作用的变化，更密切地研究转导体及其功能机制。最后，这项工作将在普韦布洛的科罗拉多州立大学以学生为中心的环境中完成。本科生和研究生将对这项工作做出重大贡献，无论是在研究实验室，还是在课堂上，这将是拟议研究的重要溢出。科罗拉多州立大学普韦布洛分校是科罗拉多州唯一一所联邦承认的西班牙裔服务大学，拟议的工作将对建设该地区少数民族学生获得医学研究的能力产生重大影响。这项拨款将大大加强我们机构的研究能力，并将使一大批不同的学生接触到与生物计量学相关的重要研究。

DESCRIPTION (provided by applicant): The over-arching goal of the proposed work is to characterize the precise molecular mechanism by which Protein Kinase A (PKA) regulates the function of Monocarboxylic Acid Transporter 1 (MCT1) in rat brain endothelial cells. We hypothesize that PKA regulates MCT1 through a multi-protein regulatory complex called a transducisome. This work is significant because cerebrovascular endothelial cell MCT1 is the sole facilitator of lactic acid transport across the blood brain barrier's cerebrovascular endothelium. Therefore it is important in brain metabolism where lactate is an important energy substrate, and during pathological conditions such as ischemic stroke, hyperglycemic stroke, and brain injury, where brain lactic acidosis has been shown to be a key indicator in the etiology of cell damage. The development of new pharmacological treatments for stroke and brain injury could be advanced by better understanding factors that control lactic acidosis. The organization of multi-protein signaling complexes, or transducisomes, is ubiquitous in coordinating the functions of PKA in numerous cell systems, but is not well characterized in the cerebrovascular endothelium; therefore, an important additional item of significance is to elucidate the mechanism by which a transducisome organizes PKA dependent MCT1 regulation in cerebrovascular endothelial cells. Our broad hypothesis is that MCT1 is part of a transducisome complex that coordinates its PKA dependent regulation by facilitating MCT1 phosphorylation and vesicular trafficking. This regulatory action is dependent upon dynamic changes in the Actin cytoskeleton which facilitates trafficking and serves as an anchor for the transducisome. In the hypothesized complex, the C-terminus of MCT1 binds to a PDZ adapter protein which is associated with an AKAP protein. The AKAP protein binds to a beta-adrenergic receptor, PKA, and links the transducisome to the morphologically dynamic Actin cytoskeleton. We have shown that Actin cytoskeletal morphing upon PKA activation is a required factor in the regulation of MCT1. The complex is proposed to function by facilitating the spatial organization of protein interactions required for MCT1 phosphorylation by PKA, and also by mediating PKA dependent vesicular trafficking of MCT1 in a pathway dependent on the PDZ adapter protein, and/or the AKAP, and cytoskeletal morphology. We have discovered that PKA stimulation by cAMP analogues elicits a bimodal effect on MCT1 function; inhibiting it at 3 hours post-trypsinization, and increasing it when assessed at 24 hours post-trypsinization. We hypothesize that this bimodal regulatory effect of PKA is caused by the time-dependent formation of the transducisome complex following cytoplasmic disruption by trypsin. By examining the effects of our independent variables at both 3 and 24 hour time points we will capitalize on this dichotomy to further elucidate details about the transducisome. The proposed experimentation for our first specific hypothesis will determine whether the MCT1 C or N-terminus interacts with a PDZ adapter or AKAP binding partner as a functional requirement for its regulation by PKA. The binding partner will be identified. Our second hypothesis will more closely examine the transducisome and its mechanism of function by showing whether PKA stimulation leads to MCT1 and transducisome protein phosphorylation, vesicular trafficking of MCT1, cytoskeleton-morphology dependent changes in MCT1 regulation, and cytoskeleton morphology dependent changes key protein-protein interactions within the transducisome. Finally, this work will be done in a student-centered environment at Colorado State University in Pueblo. Undergraduate and graduate students will make significant contributions to this work, both in the research laboratory, and in the classroom where there will be significant spill-over of the proposed research. Colorado State University-Pueblo is the only federally recognized Hispanic serving university in Colorado, and the proposed work will have a major impact in building capacity for minority student access to medical research in the region. This grant would significantly strengthen the research abilities of our institution and would expose a large group of diverse students to biometrically relevant and important research. PUBLIC HEALTH RELEVANCE: The results of these studies will produce basic scientific information needed to better understand the mechanism by which Protein Kinase A (PKA) regulates Monocarboxylic Acid Transporter 1 (MCT1) in cerebrovascular endothelial cells. Understanding the regulation of MCT1 in these cells is fundamental for understanding the control of monocarboxylate levels in brain. This, in turn, is important for brain energy metabolism and for diseases such as stroke and brain injury where lactic acidosis is a key indicator of the severity of brain damage. Such knowledge will help us to begin developing concepts for inventing new pharmacological-based therapies for stroke and brain injury patients. The rationale for the research is that during stroke, brain cell damage resulting directly from lactic acidosis could be reduced by agents that prevent or reduce it. Our aims focus on understanding the regulation of cerebrovascular MCT1 because it is the major molecular determinant of cerebral lactic acid efflux from brain in stroke (Smith and Drewes, 2006). The research will build a foundation for extending and developing these new therapeutic approaches, and will make a significant contribution in the training of new biomedical research scientists from under-represented minority groups.