Proteolytic Mechanisms for Production of the Dynorphin Peptide Neurotransmitter

描述(申请人提供)：阿片肽神经递质强啡肽是通过较大的前强啡肽前体蛋白的蛋白分解而合成的。强啡肽在调节疼痛和物质滥用机制方面的作用得到了充分的证明。因此，更好地理解这些重要的过程可以通过阐明不活跃的前强啡肽被切割以产生活性强啡肽的蛋白分解途径来实现。在成熟的多肽神经递质或神经肽的合成中，有两条主要的蛋白酶途径，包括最近确定的分泌小泡中的组织蛋白酶L半胱氨酸蛋白酶，以及众所周知的前蛋白转换酶(PC)酶PC1/3和PC2。新的结果表明，新的组织蛋白酶L半胱氨酸蛋白酶途径参与了强啡肽生物合成的分泌囊泡中。值得注意的是，组织蛋白酶L基因敲除小鼠显示，大脑中强啡肽的水平降低了75%。此外，组织蛋白酶L的表达导致PC12细胞产生强啡肽。根据这些新发现，推测组织蛋白酶L是生产强啡肽的一种重要的加工酶。这项建议的目标是评估组织蛋白酶L从前强啡肽生产强啡肽的效果，这一目标将通过三个具体目标来实现。具体目的1将结合神经内分泌PC12细胞中组织蛋白酶L与前强啡肽原共表达过程中强啡肽的产生，结合组织蛋白酶L与强啡肽共定位于分泌小泡的细胞评价。组织蛋白酶L的体内补充研究将在特定目标2中实现，该研究将评估组织蛋白酶L基因敲除小鼠的强啡肽和前强啡肽衍生产物，并与PC1/3和PC2基因敲除小鼠进行比较。结果将表明这些加工酶中的每一个对强啡肽神经肽产生的影响。具体目标3将评估组织蛋白酶选择性抑制剂L在原代培养的嗜铬细胞和大脑皮层神经元产生强啡肽过程中的作用。结果将表明细胞系统如何利用组织蛋白酶L的加工途径来生产强啡肽。强啡肽在调节疼痛，包括慢性疼痛，以及与药物滥用相关的神经可塑性方面发挥着重要作用。从这项研究中获得的知识将增加我们对这些过程的理解，并为调节强啡肽产生的调节剂提供靶点，以促进其有益效果或减少其不良影响。

DESCRIPTION (provided by applicant): The opioid peptide neurotransmitter dynorphin is synthesized through the proteolysis of the larger prodynorphin precursor protein. Dynorphin has a well-documented role in modulating pain and mechanisms involved in substance abuse. A better understanding of these important processes could therefore emerge from elucidation of the proteolytic pathways through which inactive prodynorphin is cleaved to produce active dynorphin peptides. Two primary protease pathways are implicated in the synthesis of mature peptide neurotransmitters, or neuropeptides, consisting of the recently characterized cathepsin L cysteine protease in secretory vesicles, together with the well-known proprotein convertase (PC) proteases PC 1/3 and PC2. New results demonstrate participation of the novel cathepsin L cysteine protease pathway in secretory vesicles for dynorphin biosynthesis. Notably, cathepsin L gene knockout mice show that dynorphin levels in brain are reduced by 75%. Furthermore, expression of cathepsin L results in dynorphin production in PC12 cells. Based on these new findings, it is hypothesized that cathepsin L is an important processing protease for dynorphin production. The goal of this proposal is to evaluate the production of dynorphin from prodynorphin by cathepsin L This goal will be achieved in three specific aims. Specific aim 1 will assess dynorphin peptide production by cathepsin L during coexpression of prodynorphin and cathepsin L in neuroendocrine PC12 cells, combined with cellular evaluation of cathepsin L colocalization with dynorphin in secretory vesicles of the regulated secretory pathway. Complementary in vivo studies of cathepsin L will be achieved in specific aim 2 which will assess dynorphin and prodynorphin-derived products in cathepsin L gene knockout mice, with comparisons to PC1/3 and PC2 knockout mice. Results will indicate the impact of each of these processing proteases on dynorphin neuropeptide production. Specific aim 3 will assess the effects of a selective inhibitor of cathepsin L during dynorphin production in chromaffin cells and brain cortical neurons in primary culture. Results will indicate how cellular systems utilize the cathepsin L processing pathway for production of dynorphin. Dynorphin plays an important role in modulating pain, including chronic pain, and neuroplasticity associated with drug abuse. Knowledge gained from this study will increase our understanding of these processes and provide targets for regulatory agents that may modulate dynorphin production to facilitate its beneficial effects or reduce its adversive effects.