Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of amino acids and are associated with diabetic complications. One proposed pathomechanism is the impaired processing of AGE-modified proteins or peptides including prohormones. Two approaches were applied to investigate whether substrate modification with AGEs affects the processing of substrates like prohormones to the active hormones. First, we employed solid-phase peptide synthesis to generate unmodified as well as AGE-modified protease substrates. Activity of proteases towards these substrates was quantified. Second, we tested the effect of AGE-modified proinsulin on the processing to insulin. Proteases showed the expected activity towards the unmodified peptide substrates containing arginine or lysine at the C-terminal cleavage site. Indeed, modification with Nε-carboxymethyllysine (CML) or methylglyoxal-hydroimidazolone 1 (MG-H1) affected all proteases tested. Cysteine cathepsins displayed a reduction in activity by ∼50% towards CML and MG-H1 modified substrates. The specific proteases trypsin, proprotein convertases subtilisin–kexins (PCSKs) type proteases, and carboxypeptidase E (CPE) were completely inactive towards modified substrates. Proinsulin incubation with methylglyoxal at physiological concentrations for 24 h resulted in the formation of MG-modified proinsulin. The formation of insulin was reduced by up to 80% in a concentration-dependent manner. Here, we demonstrate the inhibitory effect of substrate-AGE modifications on proteases. The finding that PCSKs and CPE, which are essential for prohormone processing, are inactive towards modified substrates could point to a yet unrecognized pathomechanism resulting from AGE modification relevant for the etiopathogenesis of diabetes and the development of obesity.

中文翻译：

---

非交联高级糖基化终产物影响激素原加工

晚期糖基化终末产物 (AGE) 是氨基酸的非酶翻译后修饰，与糖尿病并发症相关。一种提出的病理机制是 AGE 修饰的蛋白质或肽（包括激素原）的加工受损。采用两种方法来研究 AGE 的底物修饰是否会影响激素原等底物对活性激素的加工。首先，我们采用固相肽合成来生成未修饰的以及 AGE 修饰的蛋白酶底物。量化了蛋白酶对这些底物的活性。其次，我们测试了 AGE 修饰的胰岛素原对胰岛素加工的影响。蛋白酶对 C 末端裂解位点含有精氨酸或赖氨酸的未修饰肽底物表现出预期的活性。事实上，用 Nε-羧甲基赖氨酸 (CML) 或甲基乙二醛-氢咪唑酮 1 (MG-H1) 进行的修饰会影响所有测试的蛋白酶。半胱氨酸组织蛋白酶对 CML 和 MG-H1 修饰底物的活性降低了约 50%。特定的蛋白酶胰蛋白酶、前蛋白转化酶枯草杆菌蛋白酶 (PCSK) 型蛋白酶和羧肽酶 E (CPE) 对修饰的底物完全没有活性。胰岛素原与生理浓度的甲基乙二醛一起孵育24小时导致MG修饰的胰岛素原的形成。胰岛素的形成以浓度依赖性方式减少高达 80%。在这里，我们证明了底物 AGE 修饰对蛋白酶的抑制作用。PCSK 和 CPE 对激素原加工至关重要，这一发现对修饰底物没有活性，这一发现可能指出与糖尿病发病机制和肥胖发展相关的 AGE 修饰所产生的尚未被认识的病理机制。