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. 2020 Apr 25;12(5):1065. doi: 10.3390/cancers12051065

Figure 1.

Figure 1

DDI2 is required for efficient proteasome re-synthesis in MM cells. (A) Current model of DDI2 function in the regulation of TCF11/Nrf1 transcriptional activity. Under normal conditions, TCF11/Nrf1 resides in the ER membrane, undergoes ubiquitination by HRD1, and is retrotranslocated by VCP/p97 to the cytosol, where it is degraded by the proteasome. When proteasome function is impaired, TCF11/Nrf1 is deglycosylated by N-glycanase 1 (NGLY1), extracted from the ER membrane by VCP/p97, and subsequently cleaved by DDI2. Active TCF11/Nrf1 translocates to the nucleus, binds to AREs, and activates proteasome gene expression. (B) Downregulation of DDI2 inhibits proteasome re-synthesis. RPMI8226 myeloma cells were treated with DDI2 CRISPRi lentiviral particles and BTZ (DDI2 CRISPRi + BTZ) for 16 h and compared to mock lentiviral particles and the BTZ control (BTZ + mock). Quantitative RT-qPCR with primers for the indicated genes was used to analyze the levels of DDI2 and proteasome gene expression. mRNA levels of GAPDH were used for normalization. The boxes show interquartile ranges, while whiskers denote minimal and maximal values (n = 4). (C) X-ray structure of the HIV-1 protease in an open conformation (Protein Data Bank code: 2pC0) [28] and X-ray structure of the retroviral protease domain (RVP) of DDI2 (Protein Data Bank code: 4rgh) [12]. (D) Left: Screening of HIV PIs used in clinical practice using a luciferase assay reporting TCF11/Nrf1 transcriptional activity. HEK293 cells stably expressing 3xPSMA4-ARE-Luc reporter were transfected with the renilla luciferase gene for normalization and co-treated with 1 μM MG132 and 10 μM HIV PIs. At 16 h post-transfection, a dual luciferase assay was used to measure luciferase activity. Normalized luciferase activity is shown. Error bars denote the SEM (n = 3). Right: Screening of HIV PIs with an N-end rule GFP reporter assay to measure proteasome activity. U2OS cells stably expressing UbG76V-GFP reporter were treated with 200 nM CFZ for 2 h. The cells were washed with the PBS and treated with HIV PIs at 10 μM. The GFP fluorescence (dependent on proteasome activity) was measured 24 h after HIV PI treatment and normalized to the CFZ-treated cells.