McGill University, Department of Pharmacology and Therapeutics and Cell Information Systems Group, Bellini Life Sciences Complex, 3649 Sir-William-Osler Promenade, Montreal H3G 0B1, Quebec, Canada.
Integrated Program in Neuroscience, McGill University, Montreal H3A 2B4, Quebec, Canada.
Biol Chem. 2018 Nov 27;399(12):1399-1408. doi: 10.1515/hsz-2018-0259.
Since the first genetic description of a rhomboid in Drosophila melanogaster, tremendous efforts have been geared towards elucidating the proteolytic mechanism of this particular class of intramembrane proteases. In particular, mammalian rhomboid proteases sparked our interest and we aimed to investigate the human homologue RHBDL4. In light of our recent finding of the amyloid precursor protein (APP) family as efficient substrates of RHBDL4, we were enticed to further study the specific proteolytic mechanism of this enzyme by comparing cleavage patterns of wild type APP and APP TMS chimeras. Here, we demonstrate that the introduction of positively charged amino acid residues in the TMS redirects the RHBDL4-mediated cleavage of APP from its ectodomain closer towards the TMS, possibly inducing an ER-associated degradation (ERAD) of the substrate. In addition, we concluded that the cytoplasmic tail and proposed palmitoylation sites in the ectodomain of APP are not essential for the RHBDL4-mediated APP processing. In summary, our previously identified APP ectodomain cleavages by RHBDL4 are a subsidiary mechanism to the proposed RHBDL4-mediated ERAD of substrates likely through a single cleavage near or within the TMS.
自从在黑腹果蝇中首次对菱形蛋白进行了基因描述以来，人们已经做出了巨大的努力来阐明这种特殊的跨膜蛋白酶的蛋白水解机制。特别是，哺乳动物的菱形蛋白酶引起了我们的兴趣，我们旨在研究人类同源物 RHBDL4。鉴于我们最近发现淀粉样前体蛋白（APP）家族是 RHBDL4 的有效底物，我们通过比较野生型 APP 和 APP TMS 嵌合体的切割模式，受到了进一步研究该酶的特定蛋白水解机制的诱惑。在这里，我们证明了在 TMS 中引入带正电荷的氨基酸残基会将 RHBDL4 介导的 APP 切割从其细胞外结构域更靠近 TMS，可能会诱导底物的 ER 相关降解（ERAD）。此外，我们得出结论，APP 细胞外结构域中的细胞质尾巴和拟议的棕榈酰化位点对于 RHBDL4 介导的 APP 加工不是必需的。总之，我们之前通过 RHBDL4 鉴定的 APP 细胞外结构域切割是一种辅助机制，可能通过在 TMS 附近或内部的单个切割来介导 RHBDL4 介导的底物 ERAD。
