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Cathepsin L in the Biosynthesis of Enkephalins and beta-Endorphin Opioid Peptides

脑啡肽和 β-内啡肽阿片肽生物合成中的组织蛋白酶 L

基本信息

批准号：
8552301
负责人：
Steven Bark
金额：
$ 10.61万
依托单位：
UNIVERSITY OF HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8552301
关键词：
Affect Alcohol abuse Ally Anabolism Analytical Chemistry Anxiety Area Behavior Biological Biological Models CTSL gene California Cathepsin L Cells Chemicals Chromaffin Cells Complex Cyclic AMP-Dependent Protein Kinases Cysteine Protease Data Detection Disease Drug abuse Endorphins Enkephalins Epitopes Forskolin Genes Genetic Health In Vitro Institution Knockout Mice Knowledge Laboratories Lead Mass Spectrum Analysis Measures Mediating Mental Depression Mentorship Methods Molecular Nervous system structure Neurobiology Neurologic Neurons Neuropeptides Neurosciences Neurotransmitters Nicotine Opioid Opioid Peptide PC12 Cells Pathway interactions Peptide Hydrolases Peptides Pharmacologic Substance Pharmacy Schools Play Preparation Pro-Opiomelanocortin Process Production Proprotein Convertase 1 Protein Chemistry Protein Precursors Proteolysis Proteolytic Processing Radioimmunoassay Regulation Research Research Personnel Rest Role Sampling Science Scientist Secretory Vesicles Signal Transduction Site Specificity Subtilisins System Technology Tetradecanoylphorbol Acetate Tissue Extracts Training Universities base beta-Endorphin chronic pain comparative experience his6 tag human disease in vivo mental state nervous system disorder neurochemistry neurotransmission peptide A peptide structure proenkephalin protein aminoacid sequence research study vector

项目摘要

DESCRIPTION (provided by applicant): This application will provide training for Dr. Steven Bark in the neurobiology and neurochemistry of peptide neurotransmission using analytical and chemical technologies. With Dr. Bark's extensive experience in analytical and protein chemistry, this K01 training will focus on the neuroscience of peptides for neurotransmission. This training will occur under the mentorship of Dr. Vivian Hook, a noted researcher in protease processing in neurological systems. Dr. Hook's laboratory is in the Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California at San Diego. This institution and nearby allied institutions provide extraordinary opportunities for seminars, courses and collaborative interactions in all areas of Neuroscience. This training in peptide mechanisms of neurotransmission will enhance Dr. Bark's capabilities to identify and answer important questions about regulation of the nervous system in health and human disease as an independent research scientist. Enkephalin and beta-endorphin are opioid neuropeptides that have been implicated in adverse neurological disease conditions for mental state, chronic pain, drug and alcohol abuse, behavior, anxiety, and depression. The biosynthesis of these opioid peptides requires proteolytic processing of precursor proteins. Secretory vesicle Cathepsin L has recently been discovered as a new and significant protease pathway for production of enkephalins and beta-endorphin in vivo. These new findings indicate a distinct cysteine protease pathway, in addition to the well known subtilisin-like Prohormone Convertases 1 and 2 for production of neuropeptides. It is now important to define the role of Cathepsin L in producing these opioid neuropeptides. Therefore, the objective of this proposal is to define the protease mechanisms mediated by Cathepsin L for producing enkephalin and beta-endorphin. In Specific Aim 1, we will establish the identification and levels of enkephalin, beta-endorphin and their processing intermediates by coexpression of Cathepsin L and proenkephalin or proopiomelanocortin in PC12 cells. We will also optimize current mass spectrometry (MS) approaches for qualitative and quantitative analysis of neuropeptides. In Specific Aim 2, we will extend these experiments to evaluate Cathepsin L knockout mice for altered proteolysis of neuropeptides and precursor-derived intermediates that lead to production of enkephalin and beta-endorphin in vivo. In Specific Aim 3, we will define the changes in these neurotransmitters and their processing intermediates after stimulated secretion or activation in PC12 and primary chromaffin cells in culture. These mass spectrometry experiments will provide (1) significant advances for definitive identification and quantitation of neuropeptides and (2) use this critical information for defining proteolytic mechanisms for the biosynthesis of active enkephalin and beta-endorphin in health and in neurological disease conditions.

描述（由申请人提供）：本申请将为Steven Bark博士提供使用分析和化学技术进行肽神经传递的神经生物学和神经化学方面的培训。凭借Bark博士在分析和蛋白质化学方面的丰富经验，K 01培训将专注于神经传递肽的神经科学。这项培训将在Vivian Hook博士的指导下进行，Vivian Hook博士是神经系统蛋白酶处理的著名研究员。胡克博士的实验室位于圣地亚哥的加州大学斯卡格斯药学院。该机构和附近的联盟机构提供了在神经科学的所有领域的研讨会，课程和协作互动的非凡机会。这种神经传递肽机制的培训将提高Bark博士作为独立研究科学家识别和回答有关健康和人类疾病中神经系统调节的重要问题的能力。脑啡肽和β-内啡肽是阿片类神经肽，其与精神状态、慢性疼痛、药物和酒精滥用、行为、焦虑和抑郁的不良神经疾病状况有关。这些阿片肽的生物合成需要前体蛋白的蛋白水解加工。分泌囊泡组织蛋白酶L（Cathepsin L）是近年来发现的一种新的、重要的蛋白酶途径，可在体内产生脑啡肽和β-内啡肽。这些新发现表明，除了众所周知的枯草杆菌蛋白酶样激素原转化酶1和2外，还有一种独特的半胱氨酸蛋白酶途径用于产生神经肽。现在重要的是确定组织蛋白酶L在产生这些阿片类神经肽中的作用。因此，本提案的目的是确定由组织蛋白酶L介导的产生脑啡肽和β-内啡肽的蛋白酶机制。在具体目标1中，我们将通过在PC 12细胞中共表达组织蛋白酶L和脑啡肽原或阿黑皮素原来确定脑啡肽、β-内啡肽及其加工中间体的鉴定和水平。我们还将优化目前的质谱（MS）方法，用于神经肽的定性和定量分析。在具体目标2中，我们将扩展这些实验，以评估组织蛋白酶L基因敲除小鼠的神经肽和神经肽衍生中间体的蛋白水解改变，导致脑啡肽和β-内啡肽在体内的生产。在具体目标3中，我们将定义这些神经递质及其加工中间体在培养的PC 12和初级嗜铬细胞中刺激分泌或激活后的变化。这些质谱实验将提供（1）神经肽的确定性鉴定和定量的重大进展，以及（2）使用这些关键信息来定义健康和神经疾病条件下活性脑啡肽和β-内啡肽生物合成的蛋白水解机制。