Valosin-containing protein mediates the ERAD of squalene monooxygenase and its cholesterol-responsive degron

2019 ◽  
Vol 476 (18) ◽  
pp. 2545-2560 ◽  
Author(s):  
Ngee Kiat Chua ◽  
Nicola A. Scott ◽  
Andrew J. Brown
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholesterol Synthesis ◽  
Protein Quality ◽  
Extraction Process ◽  
Dependent Manner ◽  
Amphipathic Helix ◽  
Extraction Mechanism ◽  
Cholesterol Levels ◽  
Squalene Monooxygenase ◽  
Rate Limiting

Abstract Squalene monooxygenase (SM) is an essential rate-limiting enzyme in cholesterol synthesis. SM degradation is accelerated by excess cholesterol, and this requires the first 100 amino acids of SM (SM N100). This process is part of a protein quality control pathway called endoplasmic reticulum-associated degradation (ERAD). In ERAD, SM is ubiquitinated by MARCH6, an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). However, several details of the ERAD process for SM remain elusive, such as the extraction mechanism from the ER membrane. Here, we used SM N100 fused to GFP (SM N100-GFP) as a model degron to investigate the extraction process of SM in ERAD. We showed that valosin-containing protein (VCP) is important for the cholesterol-accelerated degradation of SM N100-GFP and SM. In addition, we revealed that VCP acts following ubiquitination of SM N100-GFP by MARCH6. We demonstrated that the amphipathic helix (Gln62–Leu73) of SM N100-GFP is critical for regulation by VCP and MARCH6. Replacing this amphipathic helix with hydrophobic re-entrant loops promoted degradation in a VCP-dependent manner. Finally, we showed that inhibiting VCP increases cellular squalene and cholesterol levels, indicating a functional consequence for VCP in regulating the cholesterol synthesis pathway. Collectively, we established VCP plays a key role in ERAD that contributes to the cholesterol-mediated regulation of SM.

scholarly journals MYC Enhances Cholesterol Biosynthesis and Supports Cell Proliferation Through SQLE

2021 ◽  
Vol 9 ◽  
Author(s):  
Fan Yang ◽  
Junjie Kou ◽  
Zizhao Liu ◽  
Wei Li ◽  
Wenjing Du
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cholesterol Synthesis ◽  
Cholesterol Metabolism ◽  
Cell Metabolism ◽  
Cholesterol Biosynthesis ◽  
Potential Therapeutic Target ◽  
Cholesterol Levels ◽  
Membrane Components ◽  
Rate Limiting

Oncogene c-Myc (referred in this report as MYC) promotes tumorigenesis in multiple human cancers. MYC regulates numerous cellular programs involved in cell growth and cell metabolism. Tumor cells exhibit obligatory dependence on cholesterol metabolism, which provides essential membrane components and metabolites to support cell growth. To date, how cholesterol biosynthesis is delicately regulated to promote tumorigenesis remains unclear. Here, we show that MYC enhances cholesterol biosynthesis and promotes cell proliferation. Through transcriptional upregulation of SQLE, a rate-limiting enzyme in cholesterol synthesis pathway, MYC increases cholesterol production and promotes tumor cell growth. SQLE overexpression restores the cellular cholesterol levels in MYC-knockdown cells. More importantly, in SQLE-depleted cells, enforced expression of MYC has no effect on cholesterol levels. Therefore, our findings reveal that SQLE is critical for MYC-mediated cholesterol synthesis, and further demonstrate that SQLE may be a potential therapeutic target in MYC-amplified cancers.

scholarly journals A subset of UPR-induced transmembrane proteins are prematurely degraded during lipid perturbation

2017 ◽  
Author(s):  
Benjamin S.H. Ng ◽  
Peter Shyu ◽  
Nurulain Ho ◽  
Ruijie Chaw ◽  
Seah Yi Ling ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Metabolic Disorders ◽  
Protein Quality ◽  
Transmembrane Domain ◽  
Biological Significance ◽  
Transmembrane Proteins ◽  
Dependent Manner ◽  
Alcoholic Fatty Liver ◽  
Unfolded Protein ◽  
The Unfolded Protein Response

ABSTRACTBackgroundPhospholipid homeostasis in biological membranes is essential to maintain functions of organelles such as the endoplasmic reticulum. Phospholipid perturbation has been associated to non-alcoholic fatty liver disease, obesity and other metabolic disorders. However, in most cases, the biological significance of lipid disequilibrium remains unclear. Previously, we reported that Saccharomyces cerevisiae adapts to lipid disequilibrium by upregulating several protein quality control pathways such as the endoplasmic reticulum-associated degradation (ERAD) pathway and the unfolded protein response (UPR).ResultsSurprisingly, we observed certain ER-resident transmembrane proteins (TPs), which form part of the UPR programme, to be destabilised under lipid perturbation (LP). Among these, Sbh1 was prematurely degraded by fatty acid remodelling and membrane stiffening of the ER. Moreover, the protein translocon subunit Sbh1 is targeted for degradation through its transmembrane domain in an unconventional Doa10-dependent manner.ConclusionPremature removal of key ER-resident TPs might be an underlying cause of chronic ER stress in metabolic disorders.

scholarly journals A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate

2020 ◽  
Vol 117 (13) ◽  
pp. 7150-7158 ◽  
Author(s):  
Hiromasa Yoshioka ◽  
Hudson W. Coates ◽  
Ngee Kiat Chua ◽  
Yuichi Hashimoto ◽  
Andrew J. Brown ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Cholesterol Synthesis ◽  
Cholesterol Biosynthesis ◽  
Squalene Epoxidase ◽  
Reporter Cell ◽  
Accelerated Degradation ◽  
Squalene Monooxygenase ◽  
Cholesterol Biosynthetic Pathway ◽  
The Stability ◽  
Rate Limiting

Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.

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 Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

2021 ◽  
Author(s):  
Vitalii Kryvenko ◽  
Olga Vagin ◽  
Laura A. Dada ◽  
Jacob I. Sznajder ◽  
István Vadász
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Signaling ◽  
Loss Of Function ◽  
Α Subunit ◽  
Rate Limiting Step ◽  
Atpase Subunits ◽  
A Cell ◽  
Health And Disease ◽  
And Function ◽  
Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

scholarly journals Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

2005 ◽  
Vol 169 (6) ◽  
pp. 897-908 ◽  
Author(s):  
Cosima Luedeke ◽  
Stéphanie Buvelot Frei ◽  
Ivo Sbalzarini ◽  
Heinz Schwarz ◽  
Anne Spang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Diffusion Barriers ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Protein Diffusion ◽  
Ultrastructural Studies ◽  
Bud Neck ◽  
Er Membrane

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

scholarly journals The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Network Formation ◽  
Binding Protein ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

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