Enhanced O-GlcNAcylation Mediates Cytoprotection under Proteasome Impairment by Promoting Proteasome Turnover in Cancer Cells

The proteasome is a therapeutic target in cancer, but resistance to proteasome inhibitors often develops owing to the induction of compensatory pathways. Through a genome-wide siRNA screen combined with RNA sequencing analysis, we identified hexokinase and downstream O-GlcNAcylation as cell survival factors under proteasome impairment. The inhibition of O-GlcNAcylation synergistically induced massive cell death in combination with proteasome inhibition. We further demonstrated that O-GlcNAcylation was indispensable for maintaining proteasome activity by enhancing biogenesis as well as proteasome degradation in a manner independent of Nrf1, a well-known compensatory transcription factor that upregulates proteasome gene expression. Our results identify a pathway that maintains proteasome function under proteasome impairment, providing potential targets for cancer therapy

Redundant Roles of Rpn10 and Rpn13 in Recognition of Ubiquitinated Proteins and Cellular Homeostasis

<div><p>Intracellular proteins tagged with ubiquitin chains are targeted to the 26S proteasome for degradation. The two subunits, Rpn10 and Rpn13, function as ubiquitin receptors of the proteasome. However, differences in roles between Rpn10 and Rpn13 in mammals remains to be understood. We analyzed mice deficient for Rpn13 and Rpn10. Liver-specific deletion of either Rpn10 or Rpn13 showed only modest impairment, but simultaneous loss of both caused severe liver injury accompanied by massive accumulation of ubiquitin conjugates, which was recovered by re-expression of either Rpn10 or Rpn13. We also found that mHR23B and ubiquilin/Plic-1 and -4 failed to bind to the proteasome in the absence of both Rpn10 and Rpn13, suggesting that these two subunits are the main receptors for these UBL-UBA proteins that deliver ubiquitinated proteins to the proteasome. Our results indicate that Rpn13 mostly plays a redundant role with Rpn10 in recognition of ubiquitinated proteins and maintaining homeostasis in <i>Mus musculus</i>.</p></div

Redundant roles of Rpn10 and Rpn13 in degradation of ubiquitinated proteins.

<p>(A) Immunoblot analysis of liver lysates from 3-week-old control and DKO mice with antibodies against the indicated proteins. (B) Real-time RT-PCR was performed to measure the mRNA expressions of the proteasome subunits α6 and Rpt4 in the liver of 2–4-week-old control and DKO mice. Data represent transcript levels in DKO livers relative to those in control livers and are expressed as means; error bars denote SEM. **p < 0.01 (n = 7 for each genotype). (C) Lysates from control and DKO livers were fractionated by glycerol gradient centrifugation (8 to 32% glycerol from fraction 1 to 30) and an equal amount of each fraction was used for immunoblot analysis using antibodies against the indicated proteins. Asterisks indicate nonspecific bands. (D) Each fraction of (C) was assayed for chymotrypsin-like activity using Suc-LLVY-AMC as a substrate. (E) The 26S proteasome fractions of (D) (fractions 20–23) were subjected to the assay of chymotrypsin-like activity (left panel). Degradation rates of ³⁵S-labeled cIAP1 with or without ubiquitination were measured and normalized by chymotrypsin-like activity (right panel). Data are mean ± standard deviations from triplicate experiments. **p < 0.01. (F) Lysates from HeLa cells transfected with indicated siRNAs were assayed for Suc-LLVY-AMC hydrolyzing activity (left panel) and degradation of ³⁵S-labeled cIAP1 with or without ubiquitination. cIAP degradation rates are normalized by Suc-LLVY-AMC hydrolyzing activity (right panel). Data are mean ± standard deviations from three experiments. *p < 0.05; **p < 0.01.</p

Loss of Rpn13 causes neonatal lethality in mice.

<p>(A) Genotype frequencies of embryos produced from <i>Adrm1</i>^<i>+/-</i> (<i>Rpn13</i>^<i>+/-</i>) mouse intercrosses. Numbers in parenthesis indicate resorbed fetuses or dead newborns. E: Embryonic day, P: Postnatal day. (B) Gross appearance of control and Rpn13KO littermates shortly after birth. (C) Skeletal analysis of E18.5 littermates by Alzarin red (bone) and Alcian blue (cartilage) staining. (D) Immunohistochemical analysis of sagittally sectioned E18.5 littermates by Rpn13 antibody. (E) Survival curves of newborn mice. Control and Rpn13KO mice were delivered by cesarean section.</p

Rpn13 deficiency in the liver impairs degradation of ubiquitinated proteins.

<p>(A) Immunoblot analysis of liver lysates from 8-week-old control and Rpn13^LKO mice with antibodies against the indicated proteins. Asterisk indicates a nonspecific band. (B) Lysates from control and Rpn13^LKO livers were fractionated by glycerol gradient centrifugation (8 to 32% glycerol from fraction 1 to 30) and an equal amount of each fraction was used for immunoblot analysis using antibodies against the indicated proteins. Asterisks indicate nonspecific bands. (C) Each fraction of (B) was assayed for chymotrypsin-like activity using Suc-LLVY-AMC as a substrate. (D) The 26S proteasome fractions of (C) (fractions 20–23) were subjected to the assay of chymotrypsin-like activity (left panel), and degradation of ³⁵S-labeled cIAP1 with or without ubiquitination was measured and normalized by chymotrypsin-like activity (right panel). Data are mean ±standard deviations from triplicate experiments. **p < 0.01. (E) The deubiquitinating activities of 26S proteasome fractions of (C) were measured using ubiquitin-AMC as a substrate. Data are mean ± standard deviations from triplicate experiments. **p < 0.01</p

Defective binding of ubiquitinated and UBL-UBA proteins to Rpn10ΔUIM/ΔRpn13 proteasomes.

<p>(A) Homogenates from mouse livers were immunoprecipitated with an anti-Rpt6 antibody and subjected to immunoblotting with the indicated antibodies. Values for the relative band intensities of ubiquitin normalized by tubulin (input) or Rpt6 (IP) are shown as A and B, with the control being set to one. Values for B/A indicate the relative amount of bound ubiquitinated proteins to the amount of input ubiquitinated proteins. (B) HEK293T cells were transfected with siRNA against Rpn10, Rpn13, or Uch37. Where indicated, cells were transfected with a mixture of siRNAs. After 96h, cell extracts were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies.</p

Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state.

Polyglutamine (polyQ)-expansion proteins cause neurodegenerative disorders including Huntington's disease, Kennedy's disease and various ataxias. The cytotoxicity of these proteins is associated with the formation of aggregates or other conformationally toxic species. Here, we show that the cytosolic chaperonin CCT (also known as TRiC) can alter the course of aggregation and cytotoxicity of huntingtin (Htt)-polyQ proteins in mammalian cells. Disruption of the CCT complex by RNAi-mediated knockdown enhanced Htt-polyQ aggregate formation and cellular toxicity. Analysis of the aggregation states of the Htt-polyQ proteins by fluorescence correlation spectroscopy revealed that CCT depletion results in the appearance of soluble Htt-polyQ aggregates. Similarly, overexpression of all eight subunits of CCT suppressed Htt aggregation and neuronal cell death. These results indicate that CCT has an essential role in protecting against the cytotoxicity of polyQ proteins by affecting the course of aggregation