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

 描述(由申请人提供)：这个项目解决了这样一个基本问题：“什么样的‘人类’蛋白酶机制负责产生活性多肽神经递质，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，人类特有的组织蛋白酶V与非人类动物模型中确定的神经肽产生的蛋白水解酶相结合，参与产生活性神经肽。人类产生神经肽的蛋白酶机制的基础知识与神经肽在整个衰老过程中对神经和心理健康的调节有关，并受到包括滥用药物在内的环境条件的影响。在小鼠身上的研究已经证明了小鼠组织蛋白酶L在分泌囊泡中的重要作用，以产生阿片类药物和其他神经肽。有趣的是，人类特有的组织蛋白酶V是与小鼠组织蛋白酶L最接近的人类同源物，这表明组织蛋白酶V在神经肽的产生中发挥了作用。人类特有的组织蛋白酶V基因在老鼠或其他物种中不存在。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水解酶参与脑啡肽的产生。这些发现导致了这个以人类为中心的项目的目标，即确定组织蛋白酶V与组织蛋白酶L以及在非人类物种中起作用的神经肽产生的PC1/3和PC2转换酶相结合的人类蛋白酶产生神经肽的机制。人诱导多能干细胞(HiPSC)神经元能产生大量神经肽，有望成为研究人类神经肽生物合成中的蛋白水解酶机制的创新模型。对人类大脑富含神经肽的区域中神经肽和加工酶的平行研究将支持从HiPSC模型中获得的发现。创新的神经多肽质谱仪将以无偏见和高通量的方式识别神经肽图谱。第一个目的是通过基因沉默和表达，结合质谱肽鉴定，确定组织蛋白酶V在HIPSC神经元产生阿片神经肽中的细胞作用，并与组织蛋白L、PC1/3和PC2蛋白酶进行比较。第二个目标是进行组织蛋白酶V加工阿片前神经肽的体外生化研究，与组织蛋白酶L、PC1/3和PC2进行比较，以确定切割位点、肽产物和动力学效率，并在人脑区域进行平行分析。第三个目标将通过HiPSC神经元和人脑组织的神经肽代谢来研究不同的神经肽，以评估这些人的蛋白水解酶在产生不同的神经肽中的作用。这些结果将确定人类产生多肽神经递质的基本蛋白酶机制，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.