The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression

Abstract

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 G 0 /G 1 . Moreover, serum-starved cells display reduced hRpn13 and Uch37 protein levels with hallmarks of G 0 /G 1 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 G 2 arrest by using fluorescence-activated cell sorting. Uch37 deprivation further indicated a reduction of DNA replication and G 0 /G 1 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.

RA190, a Proteasome Subunit ADRM1 Inhibitor, Suppresses Intrahepatic Cholangiocarcinoma by Inducing NF-KB-Mediated Cell Apoptosis

“…In the present study, we found that RA190 significantly suppressed cell proliferation and colony formation in both established ICC cell lines and primary ICC cells, and targeting ADRM1 by RA190 in these ICC cells could induce G 2 -M phase cell cycle arrest and the accumulation of WEE1 and p21, which was similar to previous findings [28].…”

“…RA190 only blocks the degradation of specific substrates regulated by ADRM1. This selectively might provide a high degree of safety of RA190 for clinical application [14,19,28].…”

“…Previous studies demonstrated that ADRM1 was required for proper cell cycle progression [28]. We explored whether RA190 has an effect on the cell cycle of ICC cells.…”

Meddling with Fate: The Proteasomal Deubiquitinating Enzymes

“…Uch37 has been implicated in DNA replication and cell cycle [141], TGF-β signaling [142–145], WNT signaling via Tcf7 [146], longevity [147], cell migration and invasion [148], and the reversal of ubiquitination of proteasome subunits [138]. It is difficult to resolve common themes from the physiological studies reported to date.…”

Isolation and Characterization of RNA Aptamers against a Proteasome‐Associated Deubiquitylating Enzyme UCH37

“…The amount of His-UCH37 was gradually decreased to achieve more stringent conditions: 2 mg (rounds 2-5), 1 mg (rounds 6-10), and 0.5 mg (rounds [11][12][13][14][15]. The RNA-His-UCH37 complexes were then washed three times with phosphate-buffered saline (PBS) and eluted in binding buffer supplemented with imidazole (250 mm).…”