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Modulation of Monocarboxylic Acid Transporter Kinetic Function for Stroke Therapy

用于中风治疗的单羧酸转运蛋白动力学功能的调节

基本信息

批准号：
7980693
负责人：
JEFFREY Paul SMITH
金额：
$ 30.38万
依托单位：
COLORADO STATE UNIVERSITY-PUEBLO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-15 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7980693
关键词：
A kinase anchoring protein Actins Binding Binding Proteins Biomedical Research Blood - brain barrier anatomy Brain Brain Diseases Brain Injuries Cells Cerebrum Colorado Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoskeleton Development Disease Endothelial Cells Endothelium Energy Metabolism Environment Etiology Foundations Goals Grant Herpes zoster disease Hispanics Hour Hyperglycemia Institution Intervention Ischemic Stroke Kinetics Knowledge Laboratory Research Lactic Acidosis Lactic acid Link Mediating Medical Research Minority Minority Groups Molecular Monocarboxylic Acid Transporters Morphology N-terminal Pathology Pathway interactions Patients Pharmacological Treatment Phosphorylation Play Protein Binding Protein Binding Domain Proteins Proteomics Pueblo Race Rattus Reducing Agents Regulation Research Role Scientist Severities Signal Pathway Signal Transduction Signaling Protein Stroke Structure Students System Time Training Trypsin Underrepresented Minority Universities Vascular Endothelial Cell Work adapter protein analog base beta-adrenergic receptor brain cell brain metabolism cell injury cerebrovascular genetic regulatory protein graduate student novel therapeutic intervention prevent protein protein interaction public health relevance receptor stroke therapy trafficking

项目摘要

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.

描述（由申请人提供）：拟定工作的主要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（MCT 1）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物调节MCT 1。这项工作是有意义的，因为脑血管内皮细胞MCT 1是乳酸转运穿过血脑屏障的脑血管内皮的唯一促进剂。因此，它在脑代谢中是重要的，其中乳酸是重要的能量底物，并且在诸如缺血性中风、高血糖中风和脑损伤的病理状况期间是重要的，其中脑乳酸酸中毒已被证明是细胞损伤病因学的关键指标。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协调PKA的功能是普遍存在的，但在脑血管内皮中没有很好的表征;因此，一个重要的附加意义是阐明转导体组织脑血管内皮细胞中PKA依赖性MCT 1调节的机制。我们的广泛假设是，MCT 1是转导体复合物的一部分，该复合物通过促进MCT 1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，肌动蛋白细胞骨架促进运输并充当转导体的锚。在假设的复合物中，MCT 1的C-末端结合与AKAP蛋白相关的PDZ衔接蛋白。AKAP蛋白与β-肾上腺素能受体PKA结合，并将转导体与形态学动态肌动蛋白细胞骨架连接。我们已经表明，肌动蛋白细胞骨架变形后PKA激活是一个必要的因素，在调节MCT 1。提出该复合物通过促进PKA磷酸化MCT 1所需的蛋白质相互作用的空间组织，以及通过介导依赖于PDZ衔接蛋白和/或AKAP和细胞骨架形态的途径中的MCT 1的PKA依赖性囊泡运输来发挥作用。我们已经发现，cAMP类似物对PKA的刺激对MCT 1功能产生双峰效应;在胰蛋白酶消化后3小时抑制MCT 1功能，并在胰蛋白酶消化后24小时评估时增加MCT 1功能。我们推测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物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。我们第一个特定假设的拟议实验将确定MCT 1 C或N末端是否与PDZ衔接子或AKAP结合配偶体相互作用，作为PKA对其调节的功能要求。将鉴定结合伴侣。我们的第二个假设将通过显示PKA刺激是否导致MCT 1和转导体蛋白磷酸化、MCT 1的囊泡运输、MCT 1调节中的细胞骨架形态依赖性变化以及转导体内关键蛋白-蛋白相互作用的细胞骨架形态依赖性变化来更仔细地检查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。本科生和研究生将为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，都将有重大的溢出效应。科罗拉多州立大学普韦布洛分校是科罗拉多唯一一所联邦政府承认的西班牙裔大学，拟议的工作将对该地区少数民族学生获得医学研究的能力建设产生重大影响。这笔赠款将大大加强我们机构的研究能力，并将使一大群不同的学生接触到生物统计学相关和重要的研究。公共卫生关系：这些研究的结果将产生更好地理解蛋白激酶A（PKA）调节脑血管内皮细胞单羧酸转运蛋白1（MCT 1）的机制所需的基本科学信息。了解这些细胞中MCT 1的调节对于了解大脑中单羧酸水平的控制至关重要。反过来，这对脑能量代谢和中风和脑损伤等疾病很重要，其中乳酸酸中毒是脑损伤严重程度的关键指标。这些知识将帮助我们开始发展概念，为中风和脑损伤患者发明新的基于药理学的疗法。研究的基本原理是，在中风期间，乳酸酸中毒直接导致的脑细胞损伤可以通过预防或减少它的药物来减少。我们的目标集中在了解脑血管MCT 1的调节，因为它是中风时脑乳酸流出脑的主要分子决定因素（Smith和Drewes，2006）。该研究将为扩展和开发这些新的治疗方法奠定基础，并将为培训来自代表性不足的少数群体的新生物医学研究科学家做出重大贡献。