A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis

Abstract

Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans.

Keywords: cholesterol; cholesterol regulation; degron; endoplasmic reticulum (ER); membrane protein; protein degradation; squalene monooxygenase.

Figure 1.

C-terminal half of SM N100 is required for cholesterol regulation. A, working model of the membrane topology of SM N100-GFP. SM N100 contains a re-entrant loop, which is embedded in the ER membrane. Residues between 11–18 and 41–43 (shown in red) are noted to be on the ER membrane–cytosol interface. The black dashed lines at the C terminus of SM N100 refer to the continuation of the protein with the linker region and GFP (not shown). The C-terminal half of SM N100 is cytosolically exposed along with GFP. B, schematic of truncation mutants of SM N100. All constructs contain GFP fused to the C-terminal end of SM N100 and contain a V5 epitope tag. C, CHO-7 cells were transfected for 24 h with the indicated SM N100 constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blot is representative of at least three independent experiments. D, densitometric analyses of the Western blot in C. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in cholesterol regulation or protein levels compared with WT SM N100 are indicated: *, p ≤ 0.05, or **, p ≤ 0.01.

Figure 2.

Truncations of SM N100 reveal a 20-residue region required for cholesterol regulation. A, schematic of truncation mutants of SM N100. All constructs contain GFP fused to the C-terminal end of SM N100 and contain a V5 epitope tag. B, CHO-7 cells were transfected for 24 h with the indicated SM N100 constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blot is representative of at least three independent experiments. C, densitometric analyses of Western blot in B. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in cholesterol regulation or protein levels compared with WT SM N100 are indicated: **, p ≤ 0.01.

Figure 3.

Leu-42 in SM N100 is required for normal cholesterol regulation. A, CHO-7 cells were transfected for 24 h with the indicated SM N100 constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blots are representative of at least three independent experiments, and the data are presented as mean ± S.E. B and C, densitometric analyses of Western blots in A. The gray-shaded regions represent an arbitrary range to choose a statistical significance threshold. The relative protein levels and cholesterol regulation were normalized to WT SM N100, which was set at 1 as shown by the black dashed lines. Significant changes in cholesterol regulation or protein levels compared with WT SM N100 are indicated: *, p ≤ 0.05, or **, p ≤ 0.01.

Figure 4.

Evolutionary variants of SM N100 display distinct protein expression levels. A, first 100 amino acids of human, chicken, zebrafish, and lamprey SM were aligned with Geneious 9.1.5 using default settings. Green bars indicate pairwise identity of 100%. Yellow bars indicate pairwise identity of 30–100%. Red bars indicate pairwise identity below 30%. B, pairwise comparisons of sequence identity and similarity in A were determined using Geneious 9.1.5. Boldface red numbers indicate percentage similarity and boldface black numbers refer to percentage identity. C, CHO-7 cells were transfected for 24 h with the indicated SM N100 species constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blot is representative of at least three independent experiments. D, densitometric analyses of Western blot in C. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in cholesterol regulation or protein levels compared with human SM N100 are indicated: *, p ≤ 0.05, or **, p ≤ 0.01. E, heat map showing the SM N100 protein sequence variability using the Wu-Kabat variability coefficient. F, heat map shows frequency of disordered regions in 10-residue windows for each SM N100 species variant.

Figure 5.

Four conserved degron residues of Deg1 are required for SM cholesterol-mediated turnover. A, conservation of the Deg1 degron with SM N100. Deg1 degron sequence belonging to the Matα2 protein was aligned with SM N100 in Geneious 9.1.5 with default settings. Red asterisks indicate the critical residues of Deg1, which matched SM N100. B and D, CHO-7 cells were transfected for 24 h with the indicated SM N100 constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector) or SM full-length constructs (1 μg of expression plasmid). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blot is representative of at least three independent experiments. C and E, densitometric analyses of Western blots in B and D, respectively. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in cholesterol regulation or protein levels compared with WT are indicated: *, p ≤ 0.05; **, p ≤ 0.01.

Figure 6.

Second half of SM N100 contains a helix. A, secondary structure prediction of full-length SM by PSIPRED version 3.3 with results from only the first 120 amino acids shown. The prediction (pred) row shows helices as pink rods and strands as yellow arrows. Amino acid residue (AA) positions are shown below each amino acid. The confidence level (conf) is shown on the top. Higher bars with darker shades of blue represent higher prediction confidence. B, residues 50–80 of SM N100 were analyzed in PEP-FOLD3. Secondary predictions with helical structures are highlighted in blue. C and D, two independent molecular dynamics simulations were performed with different ab initio modeled starting structures (see “Experimental procedures”). Secondary structure assignment of SM N100 for simulation 1 (C) and simulation 2 (D), respectively, is plotted as a function of time. The secondary structure is color-coded (white, coil; dark blue, α-helix; light blue, 3₁₀ helix; turquoise, π-helix; green, bend; orange, turn; red, β-bridge; dark red, β-sheet). The representative structures shown on the right were extracted from the last time frame at 1.3 μs for simulation 1 and 1.1 μs for simulation 2.

Figure 7.

Amphipathic helix of SM N100 is necessary for cholesterol regulation. A, D, and G, helical wheel representations of amphipathic helices generated from HeliQuest. The arrows indicate the magnitude and direction of the hydrophobic moment. The hydrophobicity (H) and hydrophobic moment (μ_H) from HeliQuest are also shown. B, E, and H, CHO-7 cells were transfected for 24 h with the indicated constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blots are representative of at least three independent experiments. C, F, and I, densitometric analyses of Western blots in B, E, and H. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in cholesterol regulation or protein levels compared with WT SM N100 are indicated: *, p ≤ 0.05; **, p ≤ 0.01.

Figure 8.

SM N100 cholesterol regulation is dependent on the hydrophobicity of residues 62–73. A, helical wheel representations of amphipathic helices generated from HeliQuest. The arrows indicate the magnitude and direction of the hydrophobic moment. The hydrophobicity (H) and hydrophobic moment (μ_H) from HeliQuest are also shown. B, CHO-7 cells were transfected for 24 h with the indicated constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blot is representative of at least three independent experiments. C, densitometric analyses of Western blot in B. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in cholesterol regulation or protein levels compared with SM N100 are indicated: **, p ≤ 0.01. D, data showing the hydrophobicity of residues 62–73 in human SM N100, non-human SM N100, and Deg1. For replacement of the amphipathic helix mutations, hydrophobicity is obtained from the new sequence that replaced the amphipathic helix. Cholesterol regulation is obtained from densitometric analyses of each construct from the Western blots. E, linear regression line obtained using hydrophobicity and cholesterol regulation in D. Non-human SM N100 constructs were not used to generate the line of best fit. Lamprey SM N100 was not included in the plot as it gave a negative value for cholesterol regulation. Linear regression R² and p value were obtained from GraphPad Prism 7.02.

Figure 9.

Evidence for amphipathic helix that forms with the presence of a membrane-like matrix. A and B, circular dichroism spectra of the amphipathic helix and disordered peptides at pH 7.0 in the presence (red line) or absence (black line) of n-dodecyl β-d-maltoside (DDM). Units are expressed as mean residue ellipticity.

Figure 10.

Disordered region from residues 50 to 60 is required for basal turnover of SM N100. A, CHO-7 cells were transfected for 24 h with the indicated constructs (0.25 μg of expression plasmid and 0.75 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, cells were treated for 8 h with or without Chol/CD (20 μg/ml). The Western blot is representative of at least three independent experiments. B, densitometric analyses of Western blot in A. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in cholesterol regulation or protein levels compared with WT SM N100 are indicated: *, p ≤ 0.05.

Figure 11.

Second half of SM N100 associates weakly with the ER membrane and can dissociate upon cholesterol addition. A, CHO-7 cells were transfected for 24 h with the indicated constructs (4.2 μg of expression plasmid and 12.7 μg of pTK-empty vector). Cells were then pre-treated overnight with compactin (5 μm) and mevalonate (50 μm). The next day, microsomal membranes were isolated and treated with or without Chol/CD (20 μg/ml) for 30 min at 37 °C. The Western blot is representative of at least three independent experiments. Pellet is represented by (P) and supernatant is represented by (S). B, experiment was carried out as in A except protein expression was matched by loading a quarter of SM N100. The Western blot is representative of at least three independent experiments. C, densitometric analyses of Western blots in A and B. Data are presented as mean + S.E. from at least three independent experiments. Significant changes in supernatant to pellet ratios are as indicated: *, p ≤ 0.05.

Figure 12.

Working model on how the disordered region and amphipathic helix combine in the presence of excess cholesterol to mediate turnover of SM N100. SM N100 has a characteristic re-entrant loop and an amphipathic helix that is proposed to associate transiently with the ER membrane. In theory, the ER membrane may exhibit membrane curvature because of protein insertions and phospholipid asymmetry (upper part of diagram). Cholesterol can thicken and condense the ER membrane, flattening the membrane, resulting in the amphipathic helix being displaced from the ER membrane (lower part of diagram). The unraveling of the amphipathic helix upon displacement exposes the hydrophobic patch that constitutes the amphipathic helix. This extends the disordered region length, resulting in a sufficient number of disordered residues to facilitate the cholesterol-mediated turnover of SM N100.