POMC Processing and Beta-Endorphin-Related Opioid Peptides

描述(申请人提供)：内源性β-内啡肽阿片肽的生物合成需要对其前阿片皮质素(POMC)前体进行蛋白质分解处理。β-内啡肽是止痛、行为和压力的关键调节因子。因此，确定将POMC转化为活性β-内啡肽所需的蛋白分解途径(S)是至关重要的。我们的研究确定分泌囊泡组织蛋白酶L是POMC的关键处理酶，这是基于组织蛋白酶L基因敲除小鼠显示β-内啡肽水平降低的令人兴奋的新结果。这些新的结果是通过从分泌小泡中纯化、活性部位亲和标记和多肽显微测序鉴定为组织蛋白酶L的加工活性获得的。组织蛋白酶L定位于含有POMC的分泌小泡和含有神经肽的分泌小泡。这些新的结果表明，分泌囊泡组织蛋白L在β-内啡肽和神经肽的产生中具有重要作用。组织蛋白酶L对二碱性加工位点的切割专一性产生具有NH2末端碱性残基的多肽中间体，这表明精氨酸/赖氨酸氨基肽酶是去除这些碱性残基所必需的。精氨酸/赖氨酸氨基肽酶活性与β-内啡肽和神经肽共存于神经分泌小泡中。这些新的结果表明，组织蛋白酶L和精氨酸/赖氨酸氨基肽酶是除已知的类枯草杆菌毒素PC1和PC2和羧肽酶E/H途径外的一种新的激素前体加工的蛋白酶途径。因此，这项建议的目标将是确定分泌囊泡组织蛋白L和精氨酸/赖氨酸氨基肽酶，与PC1和PC2相比，在将POMC加工成β-内啡肽过程中所起的作用。这一目标将通过四个具体目标来实现：(1)确定组织蛋白酶L基因敲除小鼠(与PC1和PC2基因敲除小鼠相比)在脑垂体和脑中的酶活性降低对POMC加工的影响，(B)降低垂体细胞和脑神经元细胞中酶水平的小干扰RNA实验，以及组织蛋白酶L的直接化学抑制实验，(2)用β-内啡肽和PC酶评估组织蛋白酶L在垂体和脑的分泌囊中的定位。(3)确定各加工酶在(A)PC12神经内分泌细胞(和GH3细胞)的POMC加工中的作用，以及(B)POMC加工的体外动力学和裂解位点的研究，以及(4)对精氨酸/赖氨酸氨基肽酶产生β-内啡肽的生化和分子分析。这些结果将证实分泌囊泡组织蛋白L和精氨酸/赖氨酸氨基肽酶处理途径在β-内啡肽生物合成中的作用。该项目的新发现将加强我们对内源性阿片和神经肽系统生物合成机制的复杂性的了解。

DESCRIPTION (provided by applicant): The biosynthesis of the endogenous beta-endorphin opioid peptide requires proteolytic processing of its POMC (proopiomelanocortin) precursor. Beta-endorphin is a key regulator of analgesia, behavior, and stress. It is, therefore, critical to define the proteolytic pathway(s) required to convert POMC into active beta- endorphin. Our studies have identified secretory vesicle cathepsin L as a key processing enzyme for POMC, based on exciting new results from cathepsin L knockout mice showing reduced levels of beta-endorphin. These novel results were obtained by purification from secretory vesicles, active-site affinity labeling, and peptide microsequencing to identify the processing activity as cathepsin L. Cathepsin L is localized to POMC-containing secretory vesicles, as well as neuropeptide-containing secretory vesicles. These new results implicate a significant role for secretory vesicle cathepsin L in beta-endorphin and neuropeptide production. The cleavage specificity of cathepsin L for dibasic processing sites generates peptide intermediates with NH2-terminal basic residues, indicating that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Arg/Lys aminopeptidase activity is colocalized in neurosecretory vesicles with beta-endorphin and neuropeptides. These new results indicate cathepsin L and Arg/Lys aminopeptidase as a new protease pathway for prohormone processing, in addition to the well known subtilisin-like PC1 and PC2 and carboxypeptidase E/H pathway. Thus, the goal of this proposal will be to determine the role of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, compared to PC1 and PC2, for processing POMC into beta- endorphin. This goal will be achieved in four specific aims to (1) determine the effects of reduced enzyme activities on POMC processing in (a) cathepsin L knockout mice, compared to PC1 and PC2 knockout mice, in pituitary and brain, (b) siRNA experiments to reduce enzyme levels in pituitary cells and brain neuronal cells, as well as in experiments for direct chemical inhibition of cathepsin L, (2) assess localization of cathepsin L with beta-endorphin and PC enzymes in secretory vesicles of pituitary and brain, (3) determine the role of each processing enzyme in (a) cellular POMC processing in PC12 neuroendocrine cells (and GH3 cells) by cotransfection of each enzyme with POMC, and in (b) in vitro kinetic and cleavage site studies of POMC processing, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for beta- endorphin production. Results will establish roles for secretory vesicle cathepsin L and Arg/Lys aminopeptidase processing pathway in the biosynthesis of beta-endorphin. New findings from this project will enhance our knowledge of the complexity of biosynthetic mechanisms for endogenous opioid and neuropeptide systems.