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

 说明（由申请人提供）：该项目解决的基本问题是“什么`人类'蛋白酶机制负责生产活性肽神经递质，神经肽，这是所有大脑和神经系统功能的基本要求？“解决这个问题的唯一假设是，人类特异性组织蛋白酶V与非人类动物模型中鉴定的蛋白酶结合，参与产生活性神经肽。用于产生神经肽的人类蛋白酶机制的基本知识与整个衰老过程中神经和精神健康中的神经肽调节相关，并且其受到环境条件（包括滥用药物）的影响。 在小鼠中的研究已经证明小鼠组织蛋白酶L在分泌囊泡中产生阿片样物质和其他神经肽的重要作用。有趣的是，人类特异性组织蛋白酶V是最接近人类同源小鼠组织蛋白酶L，这表明组织蛋白酶V在神经肽生产中的作用。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PC 1/3 & PC 2转化酶结合产生神经肽的人类蛋白酶机制，这些转化酶在非人类物种中发挥神经肽产生的作用。 人诱导多能干细胞（hiPSC）神经元产生许多神经肽，并将用作神经肽生物合成中的人蛋白酶机制的创新模型。在富含神经肽的人脑区域中对神经肽和加工蛋白酶的平行研究将支持从hiPSC模型获得的发现。创新的神经肽组学质谱法将以无偏和高通量的方式鉴定神经肽谱。第一个目标将通过基因沉默和表达，结合质谱肽鉴定，与组织蛋白酶L、PC 1/3和PC 2蛋白酶相比，确定组织蛋白酶V在hiPSC神经元中产生阿片类神经肽的细胞作用。第二个目标将进行阿片类前神经肽的组织蛋白酶V加工的体外生物化学研究相比，组织蛋白酶L，PC 1/3和PC 2，以确定切割位点，肽产物和动力学效率，在人脑区域的平行分析。第三个目标将通过hiPSC神经元和人脑组织的神经肽组学研究多种神经肽，以评估这些人类蛋白酶在产生多种神经肽中的作用。 结果将定义肽神经递质生产的基本人类蛋白酶机制，这是健康和疾病中大脑和神经系统功能的基础。

 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.