Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada.
Mol Genet Metab. 2011 Dec;104(4):682-7. doi: 10.1016/j.ymgme.2011.09.027. Epub 2011 Sep 24.
Proprotein convertase 1/3 (PC1/3) is one of the endoproteases initiating the proteolytic activation of prohormones and proneuropeptides in the secretory pathway. It is produced as a zymogen that is subsequently modified by activity-determining cleavages at the amino and the carboxyl termini. In human, it is encoded by the PCSK1 locus on chromosome 5. Spontaneous inactivating mutations in its gene have been linked to obesity. Minor alleles of the common non-synonymous single-nucleotide polymorphisms (SNPs) rs6232 (T>C, N221D), rs6234 (G>C, Q665E) and rs6235 (C>G, S690T) have been associated with increased risk of obesity. We have shown that the variations associated with these SNPs are linked on minor PCSK1 alleles.
In this study, we examined the impact of amino acid substitutions specified by the minor PCSK1 alleles on PC1/3 biosynthesis and prohormone processing activity in cultured cells.
The common and variant isoforms of PC1/3 were expressed in transfected rat pituitary GH4C1 cells with or without proopiomelanocortin (POMC) as a substrate. Secreted PC1/3- or POMC-related proteins and peptides were analyzed by immunoblotting and immunoprecipitation.
When expressed in GH4C1 cells, the triple-variant PC1/3 underwent significantly more proteolytic processing at the amino and carboxyl termini than the common and double-variant isoforms. However, there was no detectable difference among these isoforms in their ability to process POMC in the transfected cells.
Since truncation of PC1/3 in its C-terminal region reportedly renders the enzyme unstable, we speculate that the accentuated processing of the triple variant in this region may, in vivo, create a subtle deficit of PC1/3 enzymatic activity in endocrine and neuroendocrine cells, causing impaired processing of prohormones and proneuropeptides to their bioactive forms.
蛋白水解酶 1/3（PC1/3）是一种内切蛋白酶，可在分泌途径中启动激素原和神经肽前体的蛋白水解激活。它作为酶原产生，随后在氨基和羧基末端通过活性决定的切割进行修饰。在人类中，它由染色体 5 上的 PCSK1 基因座编码。其基因中的自发失活突变与肥胖有关。常见的非同义单核苷酸多态性（SNP）rs6232（T>C，N221D）、rs6234（G>C，Q665E）和 rs6235（C>G，S690T）的次要等位基因与肥胖风险增加有关。我们已经表明，与这些 SNP 相关的变异与次要 PCSK1 等位基因上的变异有关。
在这项研究中，我们检查了由次要 PCSK1 等位基因指定的氨基酸取代对培养细胞中 PC1/3 生物合成和激素原加工活性的影响。
用或不用前阿黑皮素原（POMC）作为底物，在转染的大鼠垂体 GH4C1 细胞中表达常见和变体 PC1/3。通过免疫印迹和免疫沉淀分析分泌的 PC1/3 或 POMC 相关蛋白和肽。
当在 GH4C1 细胞中表达时，三重变体 PC1/3 在氨基和羧基末端经历了显著更多的蛋白水解加工，而常见和双变体同工型则没有。然而，在转染细胞中，这些同工型在加工 POMC 的能力方面没有可检测到的差异。
由于 PC1/3 在其 C 末端区域的截断据称使酶不稳定，我们推测该三重变体在该区域的加工增强可能在体内导致内分泌和神经内分泌细胞中 PC1/3 酶活性的轻微缺陷，导致激素原和神经肽前体加工为其生物活性形式。
