scholarly journals The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6

2020 ◽  
Vol 477 (2) ◽  
pp. 541-555 ◽  
Author(s):  
Nicola A. Scott ◽  
Laura J. Sharpe ◽  
Isabelle M. Capell-Hattam ◽  
Samuel J. Gullo ◽  
Winnie Luu ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Branch Point ◽  
Cholesterol Synthesis ◽  
E3 Ubiquitin Ligase ◽  
Biochemical Pathway ◽  
Lanosterol Synthase ◽  
Rate Limiting ◽  
Fine Tune

Cholesterol synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in cholesterol synthesis, also control levels of LDM and the terminal cholesterol synthesis enzyme, 24-dehydrocholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of cholesterol synthesis.

scholarly journals A MARCH6 and IDOL E3 ubiquitin ligase circuit uncouples cholesterol synthesis from lipoprotein uptake in hepatocytes

2015 ◽  
pp. MCB.00890-15 ◽  
Author(s):  
Anke Loregger ◽  
Emma Claire Laura Cook ◽  
Jessica Kristin Nelson ◽  
Martina Moeton ◽  
Laura Jane Sharpe ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Cholesterol Synthesis ◽  
Cholesterol Biosynthesis ◽  
E3 Ubiquitin Ligase ◽  
Cellular Level ◽  
Cholesterol Homeostasis ◽  
Pathological Conditions ◽  
Lipoprotein Uptake ◽  
Rate Limiting ◽  
Dependent Mechanism

Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate limiting enzymes HMGCR and SQLE. Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the SREBP transcriptional program. Accordingly, loss ofMARCH6increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the LDLR. Finally, we provide evidence that induction of the E3-ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake.

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

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sam A Menzies ◽  
Norbert Volkmar ◽  
Dick JH van den Boomen ◽  
Richard T Timms ◽  
Anna S Dickson ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Editorial Note ◽  
Hmg Coa Reductase ◽  
Genome Wide ◽  
Cholesterol Biosynthetic Pathway ◽  
Coa Reductase ◽  
Rate Limiting

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>).

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

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.

scholarly journals LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Yilin Fan ◽  
Marielle S. Köberlin ◽  
Nalin Ratnayeke ◽  
Chad Liu ◽  
Madhura Deshpande ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Division ◽  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Essential Gene ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Replication Forks ◽  
G2 Phase ◽  
Rate Limiting

After two converging DNA replication forks meet, active replisomes are disassembled and unloaded from chromatin. A key process in replisome disassembly is the unloading of CMG helicases (CDC45–MCM–GINS), which is initiated in Caenorhabditis elegans and Xenopus laevis by the E3 ubiquitin ligase CRL2LRR1. Here, we show that human cells lacking LRR1 fail to unload CMG helicases and accumulate increasing amounts of chromatin-bound replisome components as cells progress through S phase. Markedly, we demonstrate that the failure to disassemble replisomes reduces the rate of DNA replication increasingly throughout S phase by sequestering rate-limiting replisome components on chromatin and blocking their recycling. Continued binding of CMG helicases to chromatin during G2 phase blocks mitosis by activating an ATR-mediated G2/M checkpoint. Finally, we provide evidence that LRR1 is an essential gene for human cell division, suggesting that CRL2LRR1 enzyme activity is required for the proliferation of cancer cells and is thus a potential target for cancer therapy.

Parkin: a multifaceted ubiquitin ligase

2006 ◽  
Vol 34 (5) ◽  
pp. 749-753 ◽  
Author(s):  
D.J. Moore
Keyword(s):  
Ubiquitin Ligase ◽  
Dopaminergic Neurons ◽  
Autosomal Recessive ◽  
E3 Ubiquitin Ligase ◽  
Gene Products ◽  
Biochemical Pathway ◽  
Leucine Rich Repeat ◽  
Parkin Gene ◽  
Protein Substrates ◽  
Phosphatase And Tensin Homologue

Mutations in the parkin gene are a common cause of autosomal recessive early-onset parkinsonism. Parkin functions as an E3 ubiquitin ligase where it can polyubiquitinate a number of its protein substrates, thus targeting them for degradation by the 26 S proteasomal complex. Recent studies have demonstrated that alternative modes of parkin-mediated ubiquitination may serve other non-degradative regulatory roles. In addition, parkin appears to function as a multipurpose neuroprotectant in a number of toxic paradigms. Coupled with these observations, parkin may integrate other gene products associated with parkinsonism, including α-synuclein, LRRK2 (leucine-rich repeat kinase 2), DJ-1 and PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1], into a common biochemical pathway of potential relevance to disease pathogenesis. Parkin therefore represents a unique multifaceted ubiquitin ligase consistent with an important housekeeping role in maintaining the integrity or survival of dopaminergic neurons.

The Cholesterol Synthesis Enzyme Lanosterol 14α‐Demethylase is Post‐Translationally Regulated by the E3 Ubiquitin Ligase MARCH6

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Nicola A. Scott ◽  
Laura J. Sharpe ◽  
Isabelle M. Capell-Hattam ◽  
Samuel J. Gullo ◽  
Winnie Luu ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Cholesterol Synthesis ◽  
E3 Ubiquitin Ligase

scholarly journals Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase

2019 ◽  
Vol 294 (20) ◽  
pp. 8134-8147 ◽  
Author(s):  
Ngee Kiat Chua ◽  
Gene Hart-Smith ◽  
Andrew J. Brown
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Amphipathic Helix ◽  
Accelerated Degradation ◽  
Ubiquitination Site ◽  
Squalene Monooxygenase ◽  
Lysine Residues ◽  
Cholesterol Regulation ◽  
Rate Limiting

Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-mediated SM degradation and show that neither SM lysine residues nor the N terminus impart instability. Instead, we discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. Notably, these two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62–Leu-73) that together confer cholesterol responsiveness. In summary, our findings reveal the degron architecture of SM N100, introducing the role of non-canonical ubiquitination sites and deepening our molecular understanding of how SM is degraded in response to cholesterol.

scholarly journals BBX proteins promote HY5-mediated UVR8 signaling in Arabidopsis

2021 ◽  
Author(s):  
Roman Podolec ◽  
Timothee B. Wagnon ◽  
Manuela Leonardelli ◽  
Henrik Johansson ◽  
Roman Ulm
Keyword(s):  
Transcription Factors ◽  
Ubiquitin Ligase ◽  
Red Light ◽  
E3 Ubiquitin Ligase ◽  
Transcriptional Regulator ◽  
Cooperative Binding ◽  
Uv Resistance ◽  
Hypocotyl Growth ◽  
Rate Limiting ◽  
Pigment Accumulation

Plants undergo photomorphogenic development in the presence of light. Photomorphogenesis is repressed by the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which binds substrates through their valine-proline (VP) motifs. The UV RESISTANCE LOCUS8 (UVR8) photoreceptor senses UV-B and inhibits COP1 through cooperative binding of its own VP motif mimicry and its photosensing core to COP1, thereby preventing COP1 binding to substrates, including the bZIP transcriptional regulator ELONGATED HYPOCOTYL5 (HY5). As a key promoter of visible light and UV-B photomorphogenesis, HY5 functions together with the B-box family transcription factors BBX20-22 that were recently described as HY5 rate-limiting coactivators under red light. Here we describe a hypermorphic bbx21-3D mutant with enhanced photomorphogenesis, which carries a proline-314 to leucine mutation in the VP motif that impairs interaction with and regulation through COP1. We show that BBX21 and BBX22 are UVR8-dependently stabilized after UV-B exposure, which is counteracted by a repressor induced by HY5/BBX activity. bbx20 bbx21 bbx22 mutants under UV-B are impaired in hypocotyl growth inhibition, photoprotective pigment accumulation, and expression of several HY5-dependent genes. We conclude that BBX20-22 importantly contribute to HY5 activity in a subset of UV-B responses, but that additional, presently unknown coactivators for HY5 are functional in early UVR8 signaling.

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