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

描述（由申请人提供）：该申请将为Steven Bark博士提供使用分析和化学技术进行多肽神经传递的神经生物学和神经化学方面的培训。凭借巴克博士在分析和蛋白质化学方面的丰富经验，此次K01培训将专注于多肽神经传导的神经科学。该培训将在神经系统蛋白酶处理领域的著名研究员Vivian Hook博士的指导下进行。胡克博士的实验室位于加州大学圣地亚哥分校的斯卡格斯药学院和制药科学学院。该机构和附近的联合机构为神经科学各个领域的研讨会、课程和合作互动提供了非凡的机会。神经传递多肽机制方面的培训将增强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-内啡肽是阿片类神经肽，与精神状态、慢性疼痛、药物和酒精滥用、行为、焦虑和抑郁等不良神经系统疾病有关。这些阿片肽的生物合成需要前体蛋白的蛋白水解处理。分泌囊组织蛋白酶L是近年来在体内发现的一种新的、重要的产生脑啡肽和β -内啡肽的蛋白酶途径。这些新发现表明，除了众所周知的枯草杆菌样激素原转化酶1和2用于神经肽的产生外，还有一种独特的半胱氨酸蛋白酶途径。现在重要的是确定组织蛋白酶L在产生这些类阿片神经肽中的作用。因此，本研究的目的是明确由组织蛋白酶L介导的产生脑啡肽和β -内啡肽的蛋白酶机制。在Specific Aim 1中，我们将通过组织蛋白酶L和proenkephalin或proopiomelanocortin在PC12细胞中的共表达来确定脑啡肽、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Specific Aim 2中，我们将扩展这些实验，以评估Cathepsin L敲除小鼠体内神经肽和前体衍生中间体蛋白水解的改变，从而导致脑啡肽和-内啡肽的产生。在Specific Aim 3中，我们将定义这些神经递质及其加工中间体在PC12和原代染色质细胞中刺激分泌或激活后的变化。这些质谱实验将提供(1)在神经肽的明确鉴定和定量方面取得重大进展；(2)在健康和神经系统疾病条件下，使用这些关键信息来定义活性脑啡肽和-内啡肽生物合成的蛋白水解机制。

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.