Kim Beom-Jun, Chan Doug W, Jung Sung Yun, Chen Yue, Qin Jun, Wang Yi
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Cell Rep. 2017 May 30;19(9):1758-1766. doi: 10.1016/j.celrep.2017.05.027.
The breast- and ovarian-cancer-specific tumor suppressor BRCA1 and its heterodimeric partner BARD1 contain RING domains that implicate them as E3 ubiquitin ligases. Despite extensive efforts, the bona fide substrates of BRCA1/BARD1 remain elusive. Here, we used recombinant GST fused to four UBA domains to enrich ubiquitinated proteins followed by a Lys-ε-Gly-Gly (diGly) antibody to enrich ubiquitinated tryptic peptides. This tandem affinity purification method coupled with mass spectrometry identified 101 putative BRCA1/BARD1 E3 substrates. We identified the histone variant macroH2A1 from the screen and showed that BRCA1/BARD1 ubiquitinates macroH2A1 at lysine 123 in vitro and in vivo. Primary human fibroblasts stably expressing a ubiquitination-deficient macroH2A1 mutant were defective in cellular senescence compared to their wild-type counterpart. Our study demonstrates that BRCA1/BARD1 is a macroH2A1 E3 ligase and implicates a role for macroH2A1 K123 ubiquitination in cellular senescence.
乳腺癌和卵巢癌特异性肿瘤抑制因子BRCA1及其异源二聚体伴侣BARD1含有RING结构域，这表明它们是E3泛素连接酶。尽管进行了大量研究，但BRCA1/BARD1真正的底物仍然难以确定。在这里，我们使用与四个UBA结构域融合的重组GST来富集泛素化蛋白，随后用赖氨酸-ε-甘氨酸-甘氨酸（二甘氨酸）抗体来富集泛素化的胰蛋白酶肽段。这种串联亲和纯化方法与质谱联用鉴定出了101种可能的BRCA1/BARD1 E3底物。我们从筛选中鉴定出组蛋白变体macroH2A1，并表明BRCA1/BARD1在体外和体内均使macroH2A1的赖氨酸123位点发生泛素化。与野生型相比，稳定表达泛素化缺陷型macroH2A1突变体的原代人成纤维细胞在细胞衰老方面存在缺陷。我们的研究表明BRCA1/BARD1是一种macroH2A1 E3连接酶，并提示macroH2A1 K123位点的泛素化在细胞衰老中发挥作用。
