The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

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The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

10.1101/391789 ◽

2018 ◽

Author(s):

Sam A. Menzies ◽

Norbert Volkmar ◽

Dick J. van den Boomen ◽

Richard T. Timms ◽

Anna S. Dickson ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Hmg Coa Reductase ◽

Accelerated Degradation ◽

Genome Wide ◽

Cholesterol Biosynthetic Pathway ◽

Coa Reductase ◽

Rate Limiting

ABSTRACTHMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78, independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR and Insig-1, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent ligase partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.

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Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase

The Journal of Lipid Research ◽

10.1194/jlr.ra120001006 ◽

2020 ◽

Vol 61 (12) ◽

pp. 1675-1686

Author(s):

Marco De Giorgi ◽

Kelsey E. Jarrett ◽

Jason C. Burton ◽

Alexandria M. Doerfler ◽

Ayrea Hurley ◽

...

Keyword(s):

Er Stress ◽

Mevalonate Pathway ◽

Critical Role ◽

Cellular Level ◽

Hmg Coa Reductase ◽

Biochemical Level ◽

Genetic Deletion ◽

Coa Reductase ◽

Rate Limiting ◽

Specific Deletion

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.

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3-Hydroxy-3-methylglutaryl-coenzyme A reductase in normal and dystrophic hamsters

Biochemical Journal ◽

10.1042/bj2010501 ◽

1982 ◽

Vol 201 (3) ◽

pp. 501-504 ◽

Cited By ~ 5

Author(s):

M C Greenough ◽

R J Boegman

Keyword(s):

Diurnal Variation ◽

Biosynthetic Pathway ◽

Dark Period ◽

Normal Male ◽

Hmg Coa Reductase ◽

Cholesterol Biosynthetic Pathway ◽

Normal Males ◽

Coa Reductase ◽

Rate Limiting ◽

The Brain

The activity and diurnal variation of 3-hydroxy-3-methyglutaryl-CoA reductase (EC 1.1.1.34; HMG-CoA reductase), the rate-limiting enzyme in the cholesterol-biosynthetic pathway, of normal and dystrophic hamsters was determined. Liver enzyme activity showed a diurnal pattern in the normal male, but not in the dystrophic male. Enzyme values in normal males at the midpoint of the 12 h dark period were 10 times those in dystrophic males. No evidence for diurnal variation in the HMG-CoA reductase of the brain was observed, and similar activities were found for normal and dystrophic animals. The apparent Km for HMG-CoA reductase from the liver of normal or dystrophic hamsters was approx. 9 microM, and the Vmax. was 5.9 and 21.7 pmol/min per mg of protein for dystrophic and normal hamsters respectively.

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Author response: The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

10.7554/elife.40009.038 ◽

2018 ◽

Author(s):

Sam A Menzies ◽

Norbert Volkmar ◽

Dick JH van den Boomen ◽

Richard T Timms ◽

Anna S Dickson ◽

...

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Author Response ◽

Hmg Coa Reductase ◽

Coa Reductase

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Decision letter: The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

10.7554/elife.40009.037 ◽

2018 ◽

Author(s):

Randy Y Hampton

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Hmg Coa Reductase ◽

Coa Reductase

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Faculty Opinions recommendation of The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.734621572.793554431 ◽

2018 ◽

Author(s):

Karin Romisch

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Hmg Coa Reductase ◽

Coa Reductase

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Faculty Opinions recommendation of Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1028177.335051 ◽

2005 ◽

Author(s):

Douglas Cyr

Keyword(s):

Ubiquitin Ligase ◽

Hmg Coa Reductase ◽

Coa Reductase

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Cul4-Ddb1 ubiquitin ligases facilitate DNA replication-coupled sister chromatid cohesion through regulation of cohesin acetyltransferase Esco2

10.1101/414888 ◽

2018 ◽

Cited By ~ 1

Author(s):

Haitao Sun ◽

Jiaxin Zhang ◽

Jingjing Zhang ◽

Zhen Li ◽

Qinhong Cao ◽

...

Keyword(s):

Dna Replication ◽

Ubiquitin Ligase ◽

Sister Chromatid ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Sister Chromatid Cohesion ◽

Vital Role ◽

Chromatid Cohesion ◽

Evolutionarily Conserved

AbstractCohesin acetyltransferases Esco1 and Esco2 play a vital role in establishing sister chromatid cohesion. How Esco1 and Esco2 are controlled to achieve this in a DNA replication-coupled manner remains unclear in higher eukaryotes. Here we show that Cul4-RING ligases (CRL4s) play a critical role in sister chromatid cohesion in human cells. Depletion of Cul4A, Cul4B or Ddb1 subunits substantially reduces normal cohesion efficiency. We also show that Mms22L, a vertebrate ortholog of yeast Mms22, is one of Ddb1 and Cul4-associated factors (DCAFs) involved in cohesion. Several lines of evidence suggest a selective interaction of CRL4s with Esco2, but not Esco1. Depletion of either CRL4s or Esco2 causes a defect in Smc3 acetylation which can be rescued by HDAC8 inhibition. More importantly, both CRL4s and PCNA act as mediators for efficiently stabilizing Esco2 on chromatin and catalyzing Smc3 acetylation. Taken together, we propose an evolutionarily conserved mechanism in which CRL4s and PCNA regulate Esco2-dependent establishment of sister chromatid cohesion.Author summaryWe identified human Mms22L as a substrate specific adaptor of Cul4-Ddb1 E3 ubiquitin ligase. Downregulation of Cul4A, Cul4B or Ddb1 subunit causes reduction of acetylated Smc3, via interaction with Esco2 acetyltransferase, and then impairs sister chromatid cohesion in 293T cells. We found functional complementation between Cul4-Ddb1-Mms22L E3 ligase and Esco2 in Smc3 acetylation and sister chromatid cohesion. Interestingly, both Cul4-Ddb1 E3 ubiquitin ligase and PCNA contribute to Esco2 mediated Smc3 acetylation. To summarise, we demonstrated an evolutionarily conserved mechanism in which Cul4-Ddb1 E3 ubiquitin ligases and PCNA regulate Esco2-dependent establishment of sister chromatid cohesion.

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Inhibition of cIAP1 as a strategy for targeting c-MYC–driven oncogenic activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807711115 ◽

2018 ◽

Vol 115 (40) ◽

pp. E9317-E9324 ◽

Cited By ~ 10

Author(s):

Haoyan Li ◽

Yanjia Fang ◽

Chunyi Niu ◽

Hengyi Cao ◽

Ting Mi ◽

...

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Pharmacological Agents ◽

Protein Levels ◽

Ubiquitin Ligase Activity ◽

Molecule Inhibitor ◽

Smac Mimetics ◽

High Throughput Assay ◽

In Cells

Protooncogenec-MYC, a master transcription factor, is a major driver of human tumorigenesis. Development of pharmacological agents for inhibiting c-MYC as an anticancer therapy has been a longstanding but elusive goal in the cancer field. E3 ubiquitin ligase cIAP1 has been shown to mediate the activation of c-MYC by destabilizing MAD1, a key antagonist of c-MYC. Here we developed a high-throughput assay for cIAP1 ubiquitination and identified D19, a small-molecule inhibitor of E3 ligase activity of cIAP1. We show that D19 binds to the RING domain of cIAP1 and inhibits the E3 ligase activity of cIAP1 by interfering with the dynamics of its interaction with E2. Blocking cIAP1 with D19 antagonizes c-MYC by stabilizing MAD1 protein in cells. Furthermore, we show that D19 and an improved analog (D19-14) promote c-MYC degradation and inhibit the oncogenic function of c-MYC in cells and xenograft animal models. In contrast, we show that activating E3 ubiquitin ligase activity of cIAP1 by Smac mimetics destabilizes MAD1, the antagonist of MYC, and increases the protein levels of c-MYC. Our study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting vs. activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity.

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