Biosynthesis of Enkephalin Opioid Peptides

描述（由申请人提供）：需要脑啡肽原（PE）的蛋白水解加工来产生调节镇痛、行为和免疫细胞功能的活性脑啡肽阿片肽。因此，确定将PE转化为活性脑啡肽所需的多步蛋白水解途径至关重要。我们对PE加工的研究已经确定了半胱氨酸蛋白酶，称为“激素原巯基蛋白酶”（PTP）作为含脑啡肽的嗜铬颗粒中主要的PE切割活性。值得注意的是，这个持续项目的最新进展利用活性位点亲和标记和质谱法肽微测序来鉴定组织蛋白酶L的PTP活性。组织蛋白酶L与脑啡肽共定位于分泌囊泡中，并且组织蛋白酶L在与天然PTP相同的切割位点切割含脑啡肽的底物。值得注意的是，组织蛋白酶L敲除小鼠显示脑啡肽水平降低。这些新的结果暗示了分泌囊泡组织蛋白酶L在脑啡肽产生中的关键作用。组织蛋白酶L和PTP的切割特异性产生具有NH 2末端碱性残基延伸的肽中间体。这些发现表明，精氨酸/赖氨酸氨肽酶，然后是必要的，以消除这些碱性残基。事实上，Arg/Lys氨肽酶活性与PE、脑啡肽和PTP/组织蛋白酶L共定位于嗜铬颗粒中。这些新发现的组织蛋白酶L和精氨酸/赖氨酸氨肽酶酶的PE加工补充了我们的早期研究结果，表明参与的枯草杆菌蛋白酶样PC 1和PC 2和羧肽酶E/H在处理PE。这些发现为本研究的目标提供了基础，该研究将研究分泌囊泡组织蛋白酶L和Arg/Lys氨肽酶与PC 1和PC 2在将PE加工成脑啡肽阿片肽所需的蛋白水解途径中的作用。该目标将在四个具体目标中实现，其将（1）通过（a）确定与PC 1和PC 2相比，分泌囊泡组织蛋白酶L体外加工PE的相对效率和切割位点，以及（B）评估在PC 12细胞中与PE共表达的酶期间的细胞PE加工，（2）评估组织蛋白酶L与脑啡肽和PC酶在嗜铬细胞、转染的PC 12细胞以及大鼠脑和神经内分泌组织中的共定位，（3）检测降低的酶活性对（a）经历反义酶表达的嗜铬细胞和皮质神经元中的PE加工的影响，以及用选择性化学抑制剂直接抑制组织蛋白酶L，和（B）组织蛋白酶L敲除和PC 2敲除小鼠，其显示脑内脑啡肽水平降低，和（4）获得用于脑啡肽产生的Arg/Lys氨基肽酶的生物化学和分子分析。结果可以建立两个新的蛋白酶组分，分泌囊泡组织蛋白酶L和精氨酸/赖氨酸氨肽酶，在脑啡肽阿片肽的生物合成的功能作用。这些发现将增强我们对内源性阿片系统复杂性的认识。

DESCRIPTION (provided by applicant): Proteolytic processing of proenkephalin (PE) is required to produce active enkephalin opioid peptides that regulate analgesia, behavior, and immune cell function. It is, therefore, critical to define the multi-step proteolytic pathway required to convert PE into active enkephalin peptides. Our studies of PE processing have identified the cysteine protease termed 'prohormone thiol protease' (PTP) as the major PE cleaving activity in enkephalin-containing chromaffin granules. Notably, recent progress on this continuing project utilized active site affinity labeling and peptide microsequencing by mass spectrometry to identify the responsible PTP activity as cathepsin L. Cathepsin L is colocalized in secretory vesicles with enkephalin, and cathepsin L cleaves enkephalin-containing substrates at identical cleavage sites as native PTP. Significantly, cathepsin L knockout mice show reduced levels of enkephalin in brain. These new results implicate a key role for secretory vesicle cathepsin L in enkephalin peptide production. The cleavage specificities of cathepsin L and PTP generate peptide intermediates with NH2-terminal basic residue extensions. These findings indicate that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Indeed, Arg/Lys aminopeptidase activity is colocalized in chromaffin granules with PE, enkephalin, and PTP/cathepsin L. These new discoveries of cathepsin L and Arg/Lys aminopeptidase enyzmes for PE processing complement our earlier findings showing participation of the subtilisin-like PC1 and PC2 and carboxypeptidase E/H in processing PE. These findings provide the basis for the goal of this proposal that will investigate the roles of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, with PC1 and PC2, in the proteolytie pathway required for processing PE into enkephalin opioid peptides. This goal will be achieved in four specific aims, which will (1) evaluate PE processing by (a) determining the relative efficiency and cleavage sites for in vitro processing of PE by secretory vesicle cathepsin L, compared to PC1 and PC2, and (b) assessing cellular PE processing during enzyme coexpression with PE in PC12 cells, (2) assess the colocalization of cathepsin L with enkephalin and PC enzymes in chromaffin cells, transfected PC12 cells, and rat brain and neuroendocrine tissues, (3) examine the effects of reduced enzyme activity on PE processing in (a) chromaffin cells and cortical neurons subjected to antisense enzyme expression, as well as direct inhibition of cathepsin L with a selective chemical inhibitor, and in (b) cathepsin L knockout and PC2 knockout mice that show reduced enkephalin levels in brain, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for enkephalin production. Results can establish functional roles for two new protease components, secretory vesicle cathepsin L and Arg/Lys aminopeptidase, in the biosynthesis of enkephalin opioid peptides. These findings will enhance our knowledge of the complexity of the endogenous opioid system.