POMC Processing and Beta-Endorphine-Related Opioid Peptides

描述（由申请人提供）：内源性β-内啡肽阿片肽的生物合成需要其POMC（阿黑皮素原）前体的蛋白水解加工。β-内啡肽是镇痛、行为和压力的关键调节因子。因此，确定将POMC转化为活性β-内啡肽所需的蛋白水解途径是至关重要的。我们的研究已经确定了分泌囊泡组织蛋白酶L作为POMC的关键加工酶，基于组织蛋白酶L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囊泡中纯化、活性位点亲和标记和肽微测序来鉴定组织蛋白酶L的加工活性而获得的。组织蛋白酶L定位于含有POMC的分泌囊泡以及含有神经肽的分泌囊泡。这些新结果暗示了分泌囊泡组织蛋白酶L在β-内啡肽和神经肽产生中的重要作用。组织蛋白酶L对二元加工位点的切割特异性产生具有NH 2-末端碱性残基的肽中间体，这表明Arg/Lys氨肽酶是去除这些碱性残基所必需的。Arg/Lys氨肽酶活性与β-内啡肽和神经肽共定位于神经分泌囊泡中。这些新的结果表明，组织蛋白酶L和精氨酸/赖氨酸氨肽酶作为一个新的蛋白酶途径激素原加工，除了众所周知的枯草杆菌蛋白酶样PC 1和PC 2和羧肽酶E/H途径。因此，该建议的目标将是确定分泌囊泡组织蛋白酶L和Arg/Lys氨肽酶与PC 1和PC 2相比在将POMC加工成β-内啡肽中的作用。这一目标将通过四个具体目标来实现：（1）确定（a）与PC 1和PC 2敲除小鼠相比，在垂体和脑中，在组织蛋白酶L敲除小鼠中酶活性降低对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siRNA实验，以及直接化学抑制组织蛋白酶L的实验，（2）评估组织蛋白酶L与β-内啡肽和PC酶在垂体和脑的分泌囊泡中的定位，（3）确定每种加工酶在（a）PC 12神经内分泌细胞中的细胞POMC加工中的作用（和GH 3细胞），和（B）POMC加工的体外动力学和切割位点研究，和（4）获得用于β-内啡肽产生的Arg/Lys氨肽酶的生物化学和分子分析。结果将建立分泌囊泡组织蛋白酶L和精氨酸/赖氨酸氨肽酶加工途径在β-内啡肽的生物合成中的作用。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.