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Lipid sensing tips the balance for a key cholesterol synthesis enzyme

Open AccessPublished:May 05, 2020DOI:https://doi.org/10.1194/jlr.ILR120000801
      Squalene monooxygenase (SM) is a rate-limiting enzyme of cholesterol synthesis beyond HMGCR, anchored in the membranes of the endoplasmic reticulum (ER). The first 100 amino acids (SM N100) represent a lipid-responsive degron (degradation signal) capable of sensing two lipids of the cholesterol synthesis pathway in the ER, squalene and cholesterol (
      • Gill S.
      • Stevenson J.
      • Kristiana I.
      • Brown A.J.
      Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
      ,
      • Yoshioka H.
      • Coates H.W.
      • Chua N.K.
      • Hashimoto Y.
      • Brown A.J.
      • Ohgane K.
      A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
      ). The role of the SM N100 degron is simple; it enables SM to be degraded when the catalytic activity of SM is no longer required, stopping the synthesis pathway at this step. When does this phenomenon occur? Cholesterol, the end-product of the pathway, feeds back on SM and accelerates its degradation (right). Thus, excess cholesterol shuts down the synthesis pathway, accumulating squalene (right) (
      • Gill S.
      • Stevenson J.
      • Kristiana I.
      • Brown A.J.
      Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
      ). However, degradation is attenuated when cholesterol levels are low and squalene levels increase (left). It was originally observed that SM levels increased in cells treated with the SM inhibitor NB-598. Further investigation revealed this occurred due to squalene accumulation, which stabilized the SM N100 degron (left) (
      • Yoshioka H.
      • Coates H.W.
      • Chua N.K.
      • Hashimoto Y.
      • Brown A.J.
      • Ohgane K.
      A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
      ). Hence, squalene, the substrate of SM, allosterically stabilizes SM, thereby providing a feedforward mechanism to drive cholesterol synthesis and meet the cellular demand for cholesterol (left) (
      • Yoshioka H.
      • Coates H.W.
      • Chua N.K.
      • Hashimoto Y.
      • Brown A.J.
      • Ohgane K.
      A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
      ). Importantly, the SM N100 degron is absent in lower organisms such as yeast, which produce ergosterol rather than cholesterol (
      • Gill S.
      • Stevenson J.
      • Kristiana I.
      • Brown A.J.
      Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
      ). From an evolutionary perspective, the degron may have evolved to impart cholesterol-sensing in human SM (
      • Gill S.
      • Stevenson J.
      • Kristiana I.
      • Brown A.J.
      Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
      ). Indeed, transferring the human SM N100 degron to green fluorescent protein enabled cholesterol-mediated degradation (right), whereas the less conserved SM N100 degron from chicken, zebrafish, and lamprey could not impart the same cholesterol-responsiveness (
      • Gill S.
      • Stevenson J.
      • Kristiana I.
      • Brown A.J.
      Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
      ,
      • Yoshioka H.
      • Coates H.W.
      • Chua N.K.
      • Hashimoto Y.
      • Brown A.J.
      • Ohgane K.
      A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
      ,
      • Chua N.K.
      • Howe V.
      • Jatana N.
      • Thukral L.
      • Brown A.J.
      A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.
      ). Mechanistically, squalene stabilizes the SM N100 degron by preventing the binding of MARCH6, an E3 ubiquitin ligase that adds ubiquitin onto the degron (
      • Yoshioka H.
      • Coates H.W.
      • Chua N.K.
      • Hashimoto Y.
      • Brown A.J.
      • Ohgane K.
      A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
      ). This subsequently reduces ubiquitination of the degron, a modification typically marking proteins for degradation (
      • Yoshioka H.
      • Coates H.W.
      • Chua N.K.
      • Hashimoto Y.
      • Brown A.J.
      • Ohgane K.
      A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
      ). On the other hand, cholesterol acts on the degron by inducing structural changes that facilitate degradation, notably on the reentrant loop and the amphipathic helix (62-QFALFSDILSGL-73) (
      • Chua N.K.
      • Howe V.
      • Jatana N.
      • Thukral L.
      • Brown A.J.
      A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.
      ). Excess cholesterol ejects the spring-loaded amphipathic helix from the ER membrane and misfolds the helix (
      • Chua N.K.
      • Hart-Smith G.
      • Brown A.J.
      Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase.
      ), initiating the proteasomal degradation of SM by ubiquitination of atypical residues (serines rather than lysines) that flank the amphipathic cholesterol sensor (
      • Chua N.K.
      • Hart-Smith G.
      • Brown A.J.
      Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase.
      ). Thus, the fate of SM hangs in the balance as it senses two lipids of the pathway through its lipid-responsive degron, ultimately determining the rate at which cholesterol synthesis proceeds.
      EQUIPMENT: Odyssey CLx (LI-COR Biosciences)
      REAGENTS: V5 Tag Monoclonal Antibody (#R960-25, ThermoFisher Scientific), Anti-α-Tubulin Monoclonal Antibody (#T5168, Sigma-Aldrich), IRDye 800CW Donkey anti-Mouse IgG (H + L) (#926-32212, LI-COR Biosciences)

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