Combined treatment of human multiple myeloma cells with bortezomib and doxorubicin alters the interactome of 20S proteasomes

The proteasome is the key player in targeted degradation of cellular proteins and serves as a therapeutic target for treating several blood malignancies. Although in general, degradation of proteins via the proteasome requires their ubiquitination, a subset of proteins can be degraded independently of their ubiquitination by direct interaction with subunits of the 20S proteasome core. Thus, investigation of the proteasome-associated proteins may help identify novel targets of proteasome degradation and provide important insights into the mechanisms of malignant cell proteostasis. Here, using biochemical purification of proteasomes from multiple myeloma (MM) cells followed by mass-spectrometry we have uncovered 77 proteins in total that specifically interacted with the 20S proteasome via its PSMA3 subunit. Our GST pull-down assays followed by western blots validated the interactions identified by mass-spectrometry. Eleven proteins were confirmed to bind PSMA3 only upon apoptotic conditions induced by a combined treatment with the proteasome inhibitor, bortezomib, and genotoxic drug, doxorubicin. Nine of these eleven proteins contained bioinformatically predicted intrinsically disordered regions thus making them susceptible to ubiquitin-independent degradation. Importantly, among those proteins five interacted with the ubiquitin binding affinity matrix suggesting that these proteins may also be ubiquitinylated and hence degraded via the ubiquitin-dependent pathway. Collectively, these PSMA3-interacting proteins represent novel potential substrates for 20S proteasomes upon apoptosis. Furthermore, these data may shed light on the molecular mechanisms of cellular response to chemotherapy. Abbreviations: BD: bortezomib/doxorubicin treatment; CDK: cyclin-dependent kinases; CHCA: α-cyanohydroxycinnamic acid; IDP: intrinsically disordered proteins; IDR: intrinsically disordered regions; IPG: immobilized pI gradient; MALDI TOF/TOF: matrix-assisted laser desorption/ionization time-of-flight tandem mass-spectrometry; MM: multiple myeloma; ODC: ornithine decarboxylase; PI: proteasomal inhibitors; PSMA: alpha-type 20S proteasome subunits; PTMs: post-translational modifications; SDS-PAGE: sodium dodecylsulphate polyacrylamide gel electrophoresis; UIP: ubiquitin-independent proteasomal proteolysis.蛋白酶体（proteasome）是细胞蛋白质靶向降解的核心效应分子，同时也是多种血液恶性肿瘤的治疗靶点。通常而言，蛋白酶体介导的蛋白质降解依赖于底物蛋白的泛素化修饰，但存在一类蛋白质可通过直接结合20S蛋白酶体核心颗粒的亚基，实现不依赖泛素化的降解。因此，对蛋白酶体关联蛋白质的系统性研究，有助于发掘全新的蛋白酶体降解靶点，并为解析恶性细胞蛋白质稳态的调控机制提供重要见解。本研究通过对多发性骨髓瘤（multiple myeloma, MM）细胞中的蛋白酶体进行生化纯化，结合质谱分析，共鉴定出77种可通过PSMA3亚基特异性结合20S蛋白酶体的蛋白质。后续通过谷胱甘肽S-转移酶下拉实验结合蛋白质印迹验证了质谱鉴定得到的蛋白质相互作用。其中11种蛋白质仅在蛋白酶体抑制剂硼替佐米（bortezomib）与基因毒性药物多柔比星（doxorubicin）联合处理诱导的细胞凋亡条件下，才会与PSMA3发生结合。上述11种蛋白质中有9种含有生物信息学预测的内在无序区域（intrinsically disordered regions, IDR），这一结构特征使其具备不依赖泛素化被蛋白酶体降解的潜力。值得关注的是，其中5种蛋白质可结合泛素结合亲和基质，提示这类蛋白质可能同时发生泛素化修饰，进而通过泛素依赖途径被降解。综上，这些与PSMA3结合的蛋白质代表了凋亡状态下20S蛋白酶体的新型潜在底物。此外，本研究数据或可为阐明细胞对化疗药物的应答分子机制提供新的研究视角。缩写说明：BD：硼替佐米/多柔比星联合处理；CDK：细胞周期蛋白依赖性激酶（cyclin-dependent kinases）；CHCA：α-氰基肉桂酸（α-cyanohydroxycinnamic acid）；IDP：内在无序蛋白质（intrinsically disordered proteins）；IDR：内在无序区域；IPG：固相pH梯度（immobilized pI gradient）；MALDI TOF/TOF：基质辅助激光解吸电离飞行时间串联质谱（matrix-assisted laser desorption/ionization time-of-flight tandem mass-spectrometry）；MM：多发性骨髓瘤；ODC：鸟氨酸脱羧酶（ornithine decarboxylase）；PI：蛋白酶体抑制剂（proteasomal inhibitors）；PSMA：20S蛋白酶体α型亚基（alpha-type 20S proteasome subunits）；PTMs：翻译后修饰（post-translational modifications）；SDS-PAGE：十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecylsulphate polyacrylamide gel electrophoresis）；UIP：不依赖泛素化的蛋白酶体降解途径（ubiquitin-independent proteasomal proteolysis）