Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125, USA.
Division of Biology and Biological Engineering, California Institute of Technology, USA.
J Proteomics. 2021 Jun 15;241:104197. doi: 10.1016/j.jprot.2021.104197. Epub 2021 Apr 10.
Proteasome inhibitors are an important class of chemotherapeutic drugs. In this study, we performed a large-scale ubiquitylome analysis of the three proteasome inhibitors MG132, bortezomib and carfilzomib. Although carfilzomib is currently being used for the treatment of multiple myeloma, it has not yet been subjected to a global ubiquitylome analysis. In this study, we identified more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. We introduced stringent criteria to determine the correct ubiquitylation site ratios and used five biological replicates to achieve increased statistical power. With the vast amount of data acquired, we made proteome-wide comparisons between the proteasome inhibitors and indicate candidate proteins that will benefit from further study. We find that in addition to the expected increase in ubiquitylation in the majority of proteins, unexpectedly a select few are specifically and significantly decreased in ubiquitylation at specific sites after treatment with proteasome inhibitors. We chose to follow-up on Mortality factor 4-like 1 (MORF4L1), which was significantly decreased in ubiquitylation at lysine 187 and lysine 104 upon proteasome inhibition, but increased in protein abundance by approximately two-fold. We demonstrate that the endogenous protein level of MORF4L1 is highly regulated by the ubiquitin proteasome system. SIGNIFICANCE: This study provides a highly curated dataset of more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. For the proper quantification of ubiquitylation sites, we introduced a higher standard by quantifying only those ubiquitylation sites that are not flanked by neighboring ubiquitylation, thereby avoiding the report of incorrect ratios. The sites identified will serve to identify important targets of the ubiquitin proteasome system and aid to better understand the repertoire of proteins that are affected by inhibiting the proteasome with MG132, bortezomib, and carfilzomib. In addition, we investigated the unusual observation that ubiquitylation of the tumor suppressor Mortality factor 4-like (MORF4L1) protein decreases rather than increases upon proteasome inhibition, which may contribute to an additional anti-tumor effect of bortezomib and carfilzomib.
蛋白酶体抑制剂是一类重要的化疗药物。在这项研究中，我们对三种蛋白酶体抑制剂（MG132、硼替佐米和卡非佐米）进行了大规模泛素组学分析。虽然卡非佐米目前用于治疗多发性骨髓瘤，但尚未对其进行全局泛素组学分析。在这项研究中，我们在 4400 多个蛋白质组中鉴定了超过 14000 个独特的泛素化位点。我们引入了严格的标准来确定正确的泛素化位点比例，并使用了五个生物学重复来提高统计能力。在获得了大量数据后，我们对蛋白酶体抑制剂进行了全蛋白质组比较，并指出了一些候选蛋白质，这些蛋白质可能需要进一步研究。我们发现，除了大多数蛋白质的泛素化增加之外，出乎意料的是，在使用蛋白酶体抑制剂处理后，少数几个蛋白质的特定位点的泛素化特异性和显著降低。我们选择继续研究凋亡相关因子 4 样蛋白 1（MORF4L1），在蛋白酶体抑制后，赖氨酸 187 和赖氨酸 104 处的泛素化明显减少，但蛋白质丰度增加了约两倍。我们证明，MORF4L1 的内源性蛋白质水平受到泛素蛋白酶体系统的高度调节。意义：本研究提供了一个超过 14000 个独特泛素化位点的高度精确的数据集，涉及 4400 多个蛋白质组。为了正确定量泛素化位点，我们引入了更高的标准，只定量那些不被相邻泛素化包围的泛素化位点，从而避免报告不正确的比例。鉴定出的这些位点将有助于确定泛素蛋白酶体系统的重要靶点，并有助于更好地理解受 MG132、硼替佐米和卡非佐米抑制蛋白酶体影响的蛋白质谱。此外，我们研究了一个不寻常的观察结果，即在蛋白酶体抑制后，肿瘤抑制因子凋亡相关因子 4 样蛋白 1（MORF4L1）的泛素化减少而不是增加，这可能有助于硼替佐米和卡非佐米的额外抗肿瘤作用。
