POMC Processing and Beta-Endorphin-Related Opioid Peptides

描述（由申请人提供）：内源性-内啡肽阿片肽的生物合成需要对其POMC前体进行蛋白水解处理。-内啡肽是镇痛、行为和应激的关键调节因子。因此，确定将POMC转化为活性β -内啡肽所需的蛋白水解途径至关重要。我们的研究已经确定了分泌囊泡组织蛋白酶L是POMC的关键加工酶，基于组织蛋白酶L敲除小鼠的令人兴奋的新结果，显示β -内啡肽水平降低。这些新结果是通过从分泌囊泡中纯化、活性位点亲和标记和肽微测序来确定加工活性为组织蛋白酶L.获得的。组织蛋白酶L定位于含有pomc的分泌囊泡，以及含有神经多肽的分泌囊泡。这些新的结果暗示了分泌囊泡组织蛋白酶L在-内啡肽和神经肽的产生中的重要作用。组织蛋白酶L对双碱基加工位点的切割特异性产生了具有nh2末端碱性残基的肽中间体，这表明需要Arg/Lys氨基肽酶来去除这些碱性残基。精氨酸/赖氨酸氨基肽酶活性与-内啡肽和神经肽共定位于神经分泌囊泡。这些新结果表明，除了众所周知的枯草杆菌样PC1和PC2以及羧肽酶E/H途径外，组织蛋白酶L和精氨酸/赖氨酸氨基肽酶是激素原加工的新途径。因此，本提案的目标将是确定与PC1和PC2相比，分泌囊泡组织蛋白酶L和精氨酸/赖氨酸氨基肽酶在将POMC加工成β -内啡肽中的作用。这一目标将通过以下四个具体目标来实现：(1)确定酶活性降低对垂体和脑POMC加工的影响(a)与PC1和PC2敲除小鼠相比，cathepsin L敲除小鼠，(b) siRNA实验，降低垂体细胞和脑神经细胞中的酶水平，以及直接化学抑制cathepsin L的实验。(2)评估垂体和脑分泌囊泡中组织蛋白酶L与β -内啡肽和PC酶的定位；(3)通过将每种酶与POMC共转染，确定每种加工酶在(a) PC12神经内分泌细胞（和GH3细胞）中POMC加工中的作用；(b) POMC加工的体外动力学和裂解位点研究；(4)获得用于β -内啡肽生产的精氨酸/赖氨酸氨基肽酶的生化和分子分析。研究结果将确定分泌囊泡组织蛋白酶L和精氨酸/赖氨酸氨基肽酶加工途径在-内啡肽生物合成中的作用。该项目的新发现将增强我们对内源性阿片样物质和神经肽系统生物合成机制复杂性的认识。

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.