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. 2023 Jan 19;12:e82843. doi: 10.7554/eLife.82843

Figure 1. Oxygen availability regulates SM truncation.

(A) Simplified overview of SM truncation. Full-length SM contains an N-terminal domain mediating feedback regulation by cholesterol. Ubiquitinated SM is targeted to the proteasome, where proteolysis is prematurely halted within the regulatory domain. This liberates a truncated protein (trunSM) that no longer responds to cholesterol and is therefore constitutively active. (B) HEK293T cells were incubated under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 hr. (C) HEK293T cells were incubated under normoxic or hypoxic conditions for the indicated times. Changes in HIF1α levels over time are consistent with other reports (Jantsch et al., 2011; Bartoszewski et al., 2019). (D) HEK293T cells were incubated under the indicated oxygen concentrations for 24 hr. Each set of immunoblots was obtained in a separate experiment. (B–D) Immunoblotting was performed for SM and trunSM (red). Graphs depict densitometric quantification of SM and trunSM protein levels normalized to the normoxic condition, which was set to 1 (dotted line). In (D), oxygen concentrations considered hypoxic (hyp.), ‘physoxic’ (phys.) or normoxic (norm.) (McKeown, 2014) are indicated in blue. Data presented as mean ± SEM from n=3–4 independent experiments (**, p≤0.01; two-tailed one-sample t-test vs. hypothetical mean of 1).

Figure 1—source data 1. Uncropped immunoblots for Figure 1.

Figure 1.

Figure 1—figure supplement 1. Simplified schematic of the cholesterol synthesis pathway.

Figure 1—figure supplement 1.

Early cholesterol synthesis overlaps with the mevalonate pathway, which produces farnesyl diphosphate and other isoprenoid precursors. Its rate-limiting enzyme is HMG-CoA reductase (HMGCR), the target of statins. The late cholesterol synthesis pathway is committed to sterol production and begins with squalene synthase (SQS). Squalene is converted to monooxidosqualene by the rate-limiting enzyme squalene monooxygenase (SM, grey) and cyclized by lanosterol synthase (LSS). Lanosterol is then converted by lanosterol 14α-demethylase (LDM) to testis meiosis-activating sterol (T-MAS), which is ultimately converted to cholesterol. A second round of SM-catalyzed epoxidation converts monooxidosqualene to dioxidosqualene, the precursor for a parallel ‘shunt’ pathway producing the regulatory oxysterol 24(S),25-epoxycholesterol. Cholesterol synthesis is energy intensive and consumes a total of 11 O2 molecules: one by SM, three by LDM, and seven by downstream enzymes. Double-headed arrows indicate multiple enzymatic steps.
Figure 1—figure supplement 2. Hypoxia-induced truncation of SM is generalizable.

Figure 1—figure supplement 2.

(A) HEK293T cells were incubated under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 hr. Levels of the indicated HIF1α target gene transcripts were quantified, normalized to the levels of RPL11, GAPDH and ACTB housekeeping transcripts and adjusted relative to the normoxic condition, which was set to 1 (dotted line). (B) Quantification of trunSM:SM ratio in Figure 1B. (C) Quantification of trunSM:SM ratio and total SM levels (expressed as the sum of SM and trunSM levels) in Figure 1C. (D) Quantification of trunSM:SM ratio in Figure 1D. (E) HEK293T cells were seeded at the indicated densities and incubated for 48 hr. Graph depicts densitometric quantification of trunSM:SM ratio. (F) The indicated cell lines were incubated under normoxic or hypoxic conditions for 24 hr. (C, F) Graphs depict densitometric quantification of protein levels normalized to the respective normoxic conditions for each cell line, which were set to 1 (dotted line). (A–F) Data presented as mean ± SEM from n=3–4 independent experiments (*, p≤0.05; [A, F] two-tailed one-sample t-test vs. hypothetical mean of 1; [B, E, F] two-tailed ratio paired t-test).
Figure 1—figure supplement 2—source data 1. Uncropped immunoblots for Figure 1—figure supplement 2.
Figure 1—figure supplement 3. Hypoxia-induced truncation of SM is independent of hypoxia-inducible factors.

Figure 1—figure supplement 3.

HEK293T cells were transfected with the indicated siRNAs for 24 hr and incubated under normoxic or hypoxic conditions for 24 hr. (A) Levels of siRNA target transcripts in normoxic cells were quantified, normalized to the levels of PBGD housekeeping transcripts and adjusted relative to the control siRNA condition, which was set to 1 (dotted line). (B) Graphs depict densitometric quantification of protein levels normalized to the respective normoxic conditions for each siRNA, which were set to 1 (dotted line). (A, B) Data presented as mean ± SEM from n=3–4 independent experiments (*, p≤0.05; **, p≤0.01; [A] two-tailed one-sample t-test vs. hypothetical mean of 1; [B] two-tailed ratio paired t-test).
Figure 1—figure supplement 3—source data 1. Uncropped immunoblots for Figure 1—figure supplement 3.