Opioid Peptide Synthesizing Enzymes

前激素转换酶PC1/3和PC2分别由基因PCSK1和PCSK2编码，是 负责从较大前体中释放阿片活性多肽的内蛋白水解酶 蛋白质。前激素转换酶不仅在阿片肽介导的疼痛信号中发挥重要作用 但也在许多其他神经元回路中发挥作用，包括奖赏通路和下丘脑 参与进食和能量动态平衡的回路。例如，无论是罕见的还是常见的 PCSK1是人类肥胖的主要危险因素，可能是由于下丘脑缺乏所致 多肽能加工。与临床医生合作，他们已经发现了带有新突变的儿童 在PCSK1中，我们最近确定突变的人和鼠的PC1/3蛋白受到 针对可能导致下丘脑蛋白应激的缺陷。根据我们之前的发现 小鼠PC1/3蛋白在合成过程中寡聚，我们认为显性-负性相互作用 在人类PC1/3杂合子肥胖表型中起主要作用，影响前体的加工 与饱腹感信号有关的生物活性多肽。我们认为，外部压力甚至会加剧 轻度的PC1/3构象障碍，使PC1/3的C-末端裂解变得更小、更多 活动表单。这些过程最终将汇聚到强烈损害前驱体加工，例如。 原阿片黑素皮质素裂解成β-内啡肽，促肾上腺皮质激素，最重要的是，裂解成厌食性肽α- MSH。在目前的提案中，我们将使用CRISPR工程细胞模型来阐明细胞生物学和 已知强相关的三个人类PC1/3变异体和突变体的前体加工效应 肥胖的风险也会增加。其次，我们将创建两个常见的人类PCSK1的小鼠模型 肥胖突变，以及罕见但高度受损的突变的第三个模型，以扩展在细胞中的发现 对实际的分泌组织进行培养，并确定导致 PCSK1介导的肥胖表型。最后，我们将测试我们的假设，即在 前阿片黑素皮质素合成神经元通过选择性消除Pcsk1而成为肥胖表型的基础 利用Pcsk1缺失的小鼠模型在表达前阿片黑素皮质素的细胞中表达。结果是 这些研究将阐明控制下丘脑多肽产生的生物合成机制。 这有助于人类对各种疾病的易感性，从肥胖到药物奖励途径 上瘾。

The prohormone convertases PC1/3 and PC2, encoded by the genes PCSK1 and PCSK2 respectively, are the endoproteolytic enzymes responsible for the liberation of opioid-active peptides from larger precursor proteins. Prohormone convertases play important roles not only in opioid peptide-mediated pain signaling but also function in many other neuronal circuits, including in reward pathways and in hypothalamic circuits involved in feeding and energy homeostasis. For example, both rare and common variations in PCSK1 function as major risk factors for human obesity, potentially due to deficiencies in hypothalamic peptidergic processing. In collaboration with clinicians who have identified children with novel mutations in PCSK1, we have recently determined that mutant human and mouse PC1/3 proteins are subject to targeting defects which are likely to result in hypothalamic proteostatic stress. Based on our prior finding that mouse PC1/3 proteins oligomerize during synthesis, we propose that dominant-negative interactions play a major role in human PC1/3 heterozygote obesity phenotypes, affecting precursor processing to bioactive peptides involved in satiety signaling. We propose that external stressors will exacerbate even mild forms of PC1/3 conformational distress, impairing C-terminal cleavage of PC1/3 to the smaller, more active forms. These processes will ultimately converge to strongly impair precursor processing, eg. proopiomelanocortin cleavage to beta-endorphin, ACTH, and most importantly, to the anorexic peptide α- MSH. In the present proposal we will use CRISPR-engineered cell models to elucidate the cell biology and precursor processing efficacy of three human PC1/3 variants and mutants known to be strongly associated with increased risk of obesity. Secondly, we will create mouse models of two common human PCSK1 obesity mutants, and a third model of a rare but highly impaired mutant, to extend findings made in cell culture to actual secretory tissues, and to identify the specific physiologic alterations which underlie the PCSK1-mediated obesity phenotype. Lastly, we will test our hypothesis that processing deficits in proopiomelanocortin-synthesizing neurons underlie the obesity phenotype by selectively eliminating Pcsk1 expression in proopiomelanocortin-expressing cells using a floxed Pcsk1 null mouse model. The results of these studies will illuminate the biosynthetic mechanisms controlling hypothalamic peptide production that contribute to human susceptibility to a variety of diseases, from obesity to reward pathways in drug addiction.