Randles Leah, Anchoori Ravi K, Roden Richard B S, Walters Kylie J
From the Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, NCI, National Institutes of Health, Frederick, Maryland 21702 and.
the Departments of Oncology.
J Biol Chem. 2016 Apr 15;291(16):8773-83. doi: 10.1074/jbc.M115.694588. Epub 2016 Feb 23.
Recently, we reported that bisbenzylidine piperidone RA190 adducts to Cys-88 of the proteasome ubiquitin receptor hRpn13, triggering accumulation of ubiquitinated proteins and endoplasmic reticulum stress-related apoptosis in various cancer cell lines. hRpn13 contains an N-terminal pleckstrin-like receptor for ubiquitin domain that binds ubiquitin and docks it into the proteasome as well as a C-terminal deubiquitinase adaptor (DEUBAD) domain that binds the deubiquitinating enzyme Uch37. Here we report that hRpn13 and Uch37 are required for proper cell cycle progression and that their protein knockdown leads to stalling at G0/G1 Moreover, serum-starved cells display reduced hRpn13 and Uch37 protein levels with hallmarks of G0/G1 stalling and recovery to their steady-state protein levels following release from nutrient deprivation. Interestingly, loss of hRpn13 correlates with a small but statistically significant reduction in Uch37 protein levels, suggesting that hRpn13 interaction may stabilize this deubiquitinating enzyme in human cells. We also find that RA190 treatment leads to a loss of S phase, suggesting a block of DNA replication, and G2 arrest by using fluorescence-activated cell sorting. Uch37 deprivation further indicated a reduction of DNA replication and G0/G1 stalling. Overall, this work implicates hRpn13 and Uch37 in cell cycle progression, providing a rationale for their function in cellular proliferation and for the apoptotic effect of the hRpn13-targeting molecule RA190.
最近，我们报道双苄叉哌啶酮RA190与蛋白酶体泛素受体hRpn13的Cys-88结合，在多种癌细胞系中引发泛素化蛋白的积累和内质网应激相关的凋亡。hRpn13包含一个用于泛素结构域的N端普列克底物蛋白样受体，该受体结合泛素并将其对接至蛋白酶体，以及一个C端去泛素化酶衔接子（DEUBAD）结构域，该结构域结合去泛素化酶Uch37。在此我们报道hRpn13和Uch37是细胞周期正常进展所必需的，它们的蛋白敲低会导致细胞停滞在G0/G1期。此外，血清饥饿细胞显示hRpn13和Uch37蛋白水平降低，具有G0/G1期停滞的特征，并且在从营养剥夺中释放后恢复到其稳态蛋白水平。有趣的是，hRpn13的缺失与Uch37蛋白水平的小幅但具有统计学意义的降低相关，这表明hRpn13的相互作用可能会在人类细胞中稳定这种去泛素化酶。我们还发现RA190处理会导致S期缺失，提示DNA复制受阻，并通过荧光激活细胞分选导致G2期停滞。Uch37缺失进一步表明DNA复制减少和G0/G1期停滞。总体而言，这项工作表明hRpn13和Uch37参与细胞周期进展，为它们在细胞增殖中的功能以及靶向hRpn13的分子RA190的凋亡作用提供了理论依据。
