Role of Human-Specific Cathepsin V Protease in the Production of Opioid and Related Peptide Neurotransmitters

人类特异性组织蛋白酶 V 蛋白酶在阿片类药物和相关肽神经递质生产中的作用

基本信息

批准号：
9007800
负责人：
Vivian Y. H Hook
金额：
$ 33.91万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9007800
关键词：
Absence of pain sensation Address Age-Years Aging Amino Acid Sequence Anabolism Animal Model Behavior Biochemical Brain Brain region Cathepsin L Cell model Chemicals Cognition Data Disease Dynorphins Enkephalins Environmental Impact Environmental Risk Factor Funding Opportunities Gene Expression Gene Silencing Generations Genes Goals Health Homologous Gene Human Immunofluorescence Microscopy In Vitro Kinetics Knowledge Lead Mass Spectrum Analysis Mental Health Modeling Mus Nervous System Physiology Neurologic Neurons Neuropeptides Neurosciences Neurotransmitters Opioid Pathway interactions Peptide Hydrolases Peptides Pharmaceutical Preparations Process Production Property Proprotein Convertase 1 Proprotein Convertase 2 Proprotein Convertases Protease Gene Protein Precursors Regulation Role Secretory Vesicles Site Stress beta-Endorphin brain tissue cathepsin V drug of abuse in vivo induced pluripotent stem cell innovation interest mind control neurotransmission prototype public health relevance response small molecule

项目摘要

DESCRIPTION (provided by applicant): This project addresses the fundamental question of "What `human' protease mechanisms are responsible for producing active peptide neurotransmitters, neuropeptides, that are a fundamental requirement for all brain and nervous system functions?" The unique hypothesis to address this question is that human-specific cathepsin V, combined with proteases identified in non-human animal models for neuropeptide production, participates in producing active neuropeptides. Fundamental knowledge of the human protease mechanisms for producing neuropeptides are relevant to neuropeptide regulation in neurological and mental health throughout aging, and which are impacted by environmental conditions including drugs of abuse. Studies in mice have demonstrated the important role of mouse cathepsin L in secretory vesicles for producing opioid and other neuropeptides. Interestingly, human-specific cathepsin V is the closest human homologue to mouse cathepsin L, suggesting a role for cathepsin V in neuropeptide production. The human-specific cathepsin V gene is not present in mouse or other species. We investigated human cathepsin V and found that it participates as a significant protease for enkephalin neuropeptide production. These findings lead to the goal of this `human focused' project to define the human protease mechanisms for neuropeptide production by cathepsin V, combined with cathepsin L and the PC1/3 & PC2 convertases that function in non-human species for neuropeptide production. Human induced pluripotent stem cell (hiPSC) neurons produce numerous neuropeptides and will be used as an innovative model for human protease mechanisms in neuropeptide biosynthesis. Parallel studies of neuropeptides and processing proteases in human brain regions rich in neuropeptides will support findings gained from the hiPSC model. Innovative neuropeptidomics mass spectrometry will identify neuropeptide profiles in an unbiased and high throughput manner. The first aim will define the cellular role in hiPSC neurons of cathepsin V in the production of opioid neuropeptides with comparison to cathepsin L, PC1/3, and PC2 proteases by gene silencing and expression, combined with mass spectrometry peptide identifications. The second aim will conduct in vitro biochemical studies of cathepsin V processing of opioid pro-neuropeptides compared to cathepsin L, PC1/3, and PC2, to define cleavage sites, peptide products, and kinetic efficiencies, with parallel analyses in human brain regions. The third aim will investigate diverse neuropeptides by neuropeptidomics of hiPSC neurons and human brain tissues to evaluate the role of these human proteases in producing diverse neuropeptides. Results will define the basic human protease mechanisms for peptide neurotransmitter production that is fundamental for brain and nervous system functions in health and disease.

描述（由申请人提供）：该项目解决了“什么‘人类’蛋白酶机制负责产生活性肽神经递质、神经肽，这是所有大脑和神经系统功能的基本要求？”的基本问题。这个问题是，人类特异性组织蛋白酶 V 与非人类动物模型中鉴定的用于神经肽生产的蛋白酶相结合，参与生产活性神经肽的人类蛋白酶机制的基础知识。神经肽与整个衰老过程中神经和心理健康的神经肽调节有关，并且受到包括滥用药物在内的环境条件的影响。小鼠研究表明，小鼠组织蛋白酶 L 在分泌囊泡中对产生阿片类药物和其他人类神经肽具有重要作用。特异性组织蛋白酶 V 是与小鼠组织蛋白酶 L 最接近的人类同源物，表明组织蛋白酶 V 在神经肽生成中发挥作用人类特异性组织蛋白酶 V 基因不存在于小鼠或其他物种中。我们研究了人类组织蛋白酶 V，发现它作为一种重要的蛋白酶参与脑啡肽神经肽的产生。这些发现导致了这个“以人类为中心”的项目的目标，即定义组织蛋白酶 V 与组织蛋白酶 L 和神经肽结合产生神经肽的人类蛋白酶机制。 PC1/3 和 PC2 转化酶在非人类物种中发挥神经肽生产作用，人类诱导多能干细胞 (hiPSC) 神经元可产生大量神经肽，并将用作神经肽生物合成中的人类蛋白酶机制的创新模型对富含神经肽的人脑区域中的神经肽和加工蛋白酶的并行研究将支持从 hiPSC 模型中获得的结果，首先以无偏见和高通量的方式识别神经肽谱。目的将与组织蛋白酶 L 相比，确定组织蛋白酶 V 在产生阿片类神经肽的 hiPSC 神经元中的细胞作用，通过基因沉默和表达，结合质谱肽鉴定，对 PC1/3 和 PC2 蛋白酶进行体外生化研究，与组织蛋白酶 L、PC1/3 和 PC2 相比，组织蛋白酶 V 处理阿片样前神经肽。确定切割位点、肽产物和动力学效率，并在人脑区域进行并行分析。第三个目标是通过神经肽组学研究不同的神经肽。 hiPSC 神经元和人类脑组织评估这些人类蛋白酶在产生不同神经肽中的作用，结果将定义肽神经递质产生的基本人类蛋白酶机制，这对于健康和疾病中的大脑和神经系统功能至关重要。