Decision letter: Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Cholesterol synthesis is both energy- and oxygen-intensive, yet relatively little is known of the regulatory effects of hypoxia on pathway enzymes. We previously showed that the rate-limiting and first oxygen-dependent enzyme of the committed cholesterol synthesis pathway, squalene monooxygenase (SM), can undergo partial proteasomal degradation that renders it constitutively active. Here, we show hypoxia is a physiological trigger for this truncation, which occurs through a two-part mechanism: (1) increased targeting of SM to the proteasome via stabilization of the E3 ubiquitin ligase MARCHF6 and (2) accumulation of the SM substrate, squalene, which impedes the complete degradation of SM and liberates its truncated form. This preserves SM activity and downstream pathway flux during hypoxia. These results uncover a feedforward mechanism that allows SM to accommodate fluctuating substrate levels and may contribute to its widely reported oncogenic properties. Editor's evaluation Cholesterol biosynthesis is a highly oxygen-intensive process as the synthesis of one molecule of cholesterol consumes 11 molecules of oxygen. This valuable paper provides a new link between oxygen sensing and cholesterol synthesis by showing that under conditions of hypoxia (oxygen deprivation), a key cholesterol synthesis enzyme called squalene monooxygenase (SM) is partially degraded to a truncated form that is constitutively active. The supporting evidence is solid and suggests that unregulated activation of SM under oxygen-deficient conditions could reduce the toxicity of squalene and other sterol intermediates. https://doi.org/10.7554/eLife.82843.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Cells need cholesterol to work properly but too much cholesterol is harmful and can contribute to atherosclerosis (narrowing of blood vessels), cancer and other diseases. Cells therefore carefully control the activity of the enzymes that are involved in making cholesterol, including an enzyme known as squalene monooxygenase. When the level of cholesterol in a cell rises, a protein called MARCHF6 adds molecules of ubiquitin to squalene monooxygenase. These molecules act as tags that direct the enzyme to be destroyed by a machine inside cells, known as the proteasome, thereby preventing further (unnecessary) production of cholesterol. Previous studies found that squalene monooxygenase is sometimes only partially broken down to make a shorter (truncated) form of the enzyme that is permanently active, even when the level of cholesterol in the cell is high. However, it was unclear what triggers this partial breakdown. The process of making cholesterol uses a lot of oxygen, yet many cancer cells thrive in tumours with low levels of oxygen. Here, Coates et al. used biochemical and cell biology approaches to study the effect of low oxygen levels on the activity of squalene monooxygenase in human cells. The experiments revealed that low oxygen levels trigger squalene monooxygenase to be partially degraded to make the truncated form of the enzyme. Firstly, MARCHF6 accumulates and adds ubiquitin to the enzyme to accelerate its delivery to the proteasome. Secondly, as the proteasome starts to degrade the enzyme, a build-up of squalene molecules impedes further breakdown of the enzyme. This mechanism preserves squalene monooxygenase activity when oxygen levels drop in cells, which may compensate for temporary oxygen shortfalls and allow cells to continue to make cholesterol. Squalene monooxygenase is overactive in individuals with a wide variety of diseases including fatty liver and prostate cancer. Drugs that block squalene monooxygenase activity have been shown to stop cancer cells from growing, but unfortunately these drugs are also toxic to mammals. These findings suggest that reducing the activity of squalene monooxygenase in more subtle ways, such as stopping it from being partially degraded, may be a more viable treatment strategy for cancer and other diseases associated with high levels of cholesterol. Introduction Cholesterol is an essential component of mammalian cell membranes, yet its aberrant accumulation is detrimental (Baigent et al., 2010). Most cellular cholesterol arises from an energetically expensive biosynthetic pathway requiring eleven oxygen molecules and over one hundred ATP equivalents per molecule of product (Brown et al., 2021). Furthermore, many intermediates of this pathway are toxic in excess (Porter and Herman, 2011). Coordinated regulation of cholesterol synthesis enzymes is therefore vital to ensure the pathway is active only when required, and sufficient substrates and cofactors are available to maintain flux through the full length of the pathway. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE, EC:1.14.14.17) catalyzes the rate-limiting conversion of squalene to monooxidosqualene in the committed cholesterol synthesis pathway (Gill et al., 2011; Chua et al., 2020). This reaction is the first in the pathway to require molecular oxygen, with the introduced epoxide group ultimately forming the signature C3-hydroxyl group of cholesterol. SM can also act a second time on monooxidosqualene to produce dioxidosqualene, the precursor of the potent regulatory oxysterol 24(S),25-epoxycholesterol (Bai et al., 1992). As a flux-controlling enzyme, SM is subject to metabolic regulation at both the transcriptional level via sterol regulatory element-binding proteins (Horton et al., 2003) and the post-translational level via ubiquitination and proteasomal degradation (Gill et al., 2011). The latter is mediated by the N-terminal regulatory domain of SM (SM-N100), which senses lipid levels in the endoplasmic reticulum (ER) membrane and accelerates or attenuates SM degradation in response to excess cholesterol or squalene, respectively (Chua et al., 2017; Yoshioka et al., 2020). These reciprocal feedback and feedforward loops fine-tune SM activity according to metabolic supply and demand. SM is typically fully degraded by the proteasome; however, incomplete proteolysis produces a truncated form of SM (trunSM) that lacks a large portion of the lipid-sensing SM-N100 domain but retains the full catalytic domain (Coates et al., 2021). This renders trunSM cholesterol-resistant and therefore constitutively active. Although truncation is induced by the SM inhibitor NB-598, human cell lines express similar levels of full-length and truncated SM (Coates et al., 2021). This points to the existence of an unknown physiological trigger for truncation. Clarifying the mechanisms of SM regulation is particularly pertinent given the importance of the enzyme, and cholesterol more generally (Kuzu et al., 2016), in oncogenesis. Overexpression of the SM gene SQLE is associated with greater invasiveness and lethality in breast (Brown et al., 2016), prostate (Stopsack et al., 2016; Pudova et al., 2020), and pancreatic cancers (Bai et al., 2021), amongst others. At the protein level, aberrant SM expression is implicated in colorectal cancer progression (He et al., 2021; Jun et al., 2021) and the development of both nonalcoholic steatohepatitis and hepatocellular carcinoma (Liu et al., 2021; Liu et al., 2018). Given its key role in oxygen-dependent cholesterol synthesis, SM may be particularly critical for cancer cell survival during hypoxia, which is common in the poorly vascularized cores of solid tumors and often associated with poor prognosis (Rankin and Giaccia, 2016). In support of this idea, SM inhibition sensitizes breast and colorectal cancer cells to hypoxia-induced cell death (Haider et al., 2016). Although hypoxic cells tend to accumulate cholesterol, there are conflicting reports on changes in biosynthetic flux (Mukodani et al., 1990; Parathath et al., 2011; Wu et al., 2020). Furthermore, with the notable exception of the early pathway enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (Nguyen et al., 2007), the effects of hypoxia on individual biosynthetic enzymes are unknown. It is also unclear if these might be perturbed in a tumor context to favor continued cholesterol synthesis and cell proliferation. Here, we show hypoxic conditions induce SM truncation in a variety of cell lines through a combination of accelerated proteasomal degradation and inhibition of its complete proteolysis. This occurs due to the accumulation of both MARCHF6, the major E3 ubiquitin ligase for SM, and squalene, which impedes SM degradation through a mechanism involving the SM-N100 regulatory domain. Taken together, our findings point towards a role for the constitutively active trunSM in adaptations to hypoxic conditions and suggest it may contribute to the oncogenic impacts of SM activity. Results Oxygen availability regulates SM truncation We previously showed that SM is post-translationally regulated by its substrate squalene and pathway end-product cholesterol (Gill et al., 2011; Yoshioka et al., 2020). The enzyme also undergoes partial proteasomal degradation of its N-terminus to liberate a truncated protein (trunSM) that is cholesterol-resistant and thus constitutively active (Figure 1A; Coates et al., 2021), although physiological triggers are unknown. As SM is a rate-limiting enzyme of cholesterol synthesis and catalyzes its first oxygen-dependent reaction (Figure 1—figure supplement 1), we tested if SM protein levels are affected by oxygen availability. Incubation of HEK293T cells under hypoxic conditions (1% O2) stabilized hypoxia-inducible factor-1α (HIF1α; Figure 1B) and upregulated its target genes VEGF and CA9 (Figure 1—figure supplement 2A), confirming the induction of a hypoxic response. We also noted a striking increase in SM truncation caused by the disappearance of full-length SM and a four-fold accumulation of trunSM (Figure 1B). This led to trunSM becoming the predominant SM variant, as indicated by the elevated trunSM:SM ratio (Figure 1—figure supplement 2B). Hypoxia-induced truncation of SM increased over time (Figure 1C, Figure 1—figure supplement 2C) and according to the magnitude of oxygen deprivation (Figure 1D, Figure 1—figure supplement 2D), with the net result of increased total enzyme levels (expressed as the sum of full-length SM and trunSM levels; Figure 1—figure supplement 1C). Importantly, trunSM accumulation was greater under the severely hypoxic conditions characteristic of solid tumors (0.5–2% O2) than the ‘physoxic’ conditions experienced by normal human tissues in situ (3–7.5% O2) (Figure 1D, Figure 1—figure supplement 2D; McKeown, 2014). This suggested that increased SM truncation is a feature of pathophysiological hypoxia. We also noted our experiments, which for technical reasons used a variety of cell seeding densities, showed variation in the normoxic trunSM:SM ratio. Indeed, we confirmed SM truncation is increased at higher cell densities and accompanied by slight stabilization of HIF1α (Figure 1—figure supplement 2E), consistent with other reports (Sheta et al., 2001; Dayan et al., 2009). This further supported the phenomenon of hypoxia-induced truncation. Figure 1 with 3 supplements see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/82843/elife-82843-fig1-data1-v1.zip Download elife-82843-fig1-data1-v1.zip We also surveyed SM levels in a panel of cell lines and found hypoxia-induced accumulation of trunSM was generalizable to all, although full-length SM levels did not decline in MDA-MB-231 breast cancer cells (Figure 1—figure supplement 2F). As HIF1α and hypoxia-inducible factor-2α (HIF2α) transcriptionally regulate the cellular response to hypoxia, we next tested if their activity is required for SM truncation. However, knockdown of HIF1A and HIF2A expression to a level sufficient to reduce target gene activation (Sena et al., 2014) had no effect on the magnitude of hypoxia-induced SM truncation in HEK293T cells (Figure 1—figure supplement 3A, B). This ruled out the involvement of HIF1α, HIF2α and their target genes in this phenomenon. Hypoxia transcriptionally and post-translationally reduces full-length SM levels As SM is truncated via partial proteasomal degradation (Coates et al., 2021), we reasoned that hypoxia promotes this through a two-step mechanism: (1) targeting of full-length SM to the proteasome, and (2) inhibition of its complete proteolysis. To confirm the first step of this mechanism, we investigated the reason for the decline in full-length SM levels during hypoxia. SQLE transcripts were downregulated in hypoxic HEK293T cells, as were transcripts encoding the upstream cholesterol synthesis enzyme HMGCR (Figure 2A). Downregulation of SQLE transcripts was not observed in MDA-MB-231 cells (Figure 2—figure supplement 1A), accounting for the unchanged full-length SM levels in this cell line. Although the reduction in SQLE and HMGCR transcripts in HEK293T cells likely reflected a broad transcriptional suppression of cholesterol synthesis during hypoxia, as reported previously (Dolt et al., 2007; Cao et al., 2014), the magnitude of SQLE downregulation was unlikely to fully explain the large reduction in SM protein levels (Figure 1B). Moreover, levels of a constitutively expressed SM construct ([HA]3-SM-V5) were markedly reduced during extended hypoxic incubations with no associated change in mRNA levels (Figure 2B, Figure 2—figure supplement 1B). We concluded that hypoxia reduces the levels of full-length SM through both transcriptional downregulation and accelerated post-translational degradation. Figure 2 with 1 supplement see all Download asset Open asset Hypoxia transcriptionally and post-translationally reduces full-length SM levels. (A) HEK293T cells were incubated under normoxic or hypoxic conditions for 24 hr. Levels of the indicated 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) HEK293T cells were transfected with (HA)3-SM-V5 for 24 hr and incubated under normoxic or hypoxic conditions for the indicated times. (C) HEK SM-N100-GFP-V5 cells were treated with or without 20 µM MG132 and 20 nM bafilomycin A1 under normoxic or hypoxic conditions for 16 hr. (D) HEK293T cells were transfected with the indicated constructs for 24 hr and incubated under normoxic or hypoxic conditions for 16 hr. (B–D) Graphs depict densitometric quantification of protein levels normalized to the respective normoxic conditions for each timepoint, treatment or construct, which were set to 1 (dotted line). (A–D) Data presented as mean ± SEM from n=3–6 independent experiments (*, p≤0.05; **, p≤0.01; [A, B] two-tailed one-sample t-test vs. hypothetical mean of 1; [C, D] two-tailed ratio paired t-test). Figure 2—source data 1 Uncropped immunoblots for Figure 2. https://cdn.elifesciences.org/articles/82843/elife-82843-fig2-data1-v1.zip Download elife-82843-fig2-data1-v1.zip The basal and metabolically-regulated degradation of SM occurs through the ubiquitin-proteasome system and is mediated by the SM-N100 regulatory domain (Chua et al., 2017; Yoshioka et al., 2020). Therefore, we tested the effect of hypoxia on HEK293 cells stably expressing an SM-N100 fusion protein (SM-N100-GFP-V5). Like full-length SM, levels of SM-N100-GFP-V5 were reduced by hypoxic conditions (Figure 2C). Proteasomal inhibition using MG132 increased the levels of SM and SM-N100-GFP-V5 (Figure 2—figure supplement 1C) and blocked their hypoxia-induced degradation (Figure 2C), confirming this degradation occurs via the proteasome. We also noted that protein levels and hypoxia-induced accumulation of trunSM were ablated by MG132 (Figure 2—figure supplement 1C, D), consistent with this protein arising from partial proteasomal proteolysis of SM (Coates et al., 2021). Although hypoxia can trigger autophagy (Bellot et al., 2009), this did not play a role in SM degradation as inhibition of lysosomal acidification using bafilomycin A1 had no additive effect with MG132 (Figure 2C). To identify residues required for hypoxia-induced degradation of SM, we utilized protein constructs with mutations of previously identified ubiquitination sites. The magnitude of hypoxic degradation was blunted by disruption of Lys-82/90/100, a cluster of redundant ubiquitination sites previously found to promote truncation (Coates et al., 2021), but not by disruption of Lys-290 (Figure 2D; Hornbeck et al., 2015). Non-canonical cysteine, serine and threonine ubiquitination sites required for the cholesterol-induced degradation of SM (SM-N100 C/S/T) (Chua et al., 2019) also contributed to hypoxia-induced degradation, suggesting multiple ubiquitin signals are involved. Contrary to the expectation that loss of ubiquitination would stabilize SM, the mutation of Lys-82/90/100 reduced SM levels under normoxic conditions (Figure 2—figure supplement 1E). Therefore, this cluster of residues may be specifically involved in hypoxia-induced, rather than basal, degradation of SM. Hypoxia-induced degradation of full-length SM requires the E3 ubiquitin ligase MARCHF6 To investigate how hypoxia promotes SM ubiquitination, we considered the possible role of proline hydroxylation. This oxygen-dependent modification, catalyzed by prolyl hydroxylases, is required for the ubiquitination and degradation of HIF1α under normoxic conditions (Ivan et al., 2001), although there is conflicting evidence for the existence of substrates beyond the HIF proteins (Cockman et al., 2019). Indeed, treatment with the prolyl hydroxylase inhibitors DMOG and FG-4592 had no effect on the basal levels nor hypoxia-induced degradation of SM and SM-N100-GFP-V5, despite stabilizing HIF1α (Figure 3—figure supplement 1A). SM and SM-N100 are targeted for proteasomal degradation by the E3 ubiquitin ligase MARCHF6 (Foresti et al., 2013; Zelcer et al., 2014); therefore, we tested if increased MARCHF6 activity could account for the hypoxia-induced degradation of SM. To do so, we depleted MARCHF6 expression using siRNA that achieves a 60–70% reduction in transcript levels in HEK293 cells (Zelcer et al., 2014). SM and SM-N100-GFP-V5 levels were dramatically increased (Figure 3—figure supplement 1B) and the hypoxic decline in SM levels was blocked (Figure 3A), supporting the involvement of MARCHF6 in hypoxia-induced degradation. The basal levels and hypoxic accumulation of trunSM were also reduced (Figure 3—figure supplement 1B, C), consistent with MARCHF6 contributing to the proteasomal targeting, and therefore partial degradation, of SM (Coates et al., 2021). Hypoxia-induced accumulation of trunSM was not completely abolished, however, indicating SM can be truncated even when targeted to the proteasome by hypoxia-independent mechanisms. Surprisingly, there was no effect of MARCHF6 knockdown on hypoxia-induced degradation of SM-N100-GFP-V5 (Figure 3A), suggesting SM and the isolated SM-N100 domain are degraded through different proteasome-dependent routes under these conditions. We elected to further investigate the MARCHF6-dependent degradation of full-length SM, as the endogenous protein has greater physiological relevance. Figure 3 with 1 supplement see all Download asset Open asset Hypoxia-induced degradation of full-length SM requires the E3 ubiquitin ligase MARCHF6. (A) HEK SM-N100-GFP-V5 cells were transfected with control or MARCHF6 siRNA for 24 hr and incubated under normoxic or hypoxic conditions for 16 hr. (B) HEK MARCHF6-V5 cells were incubated under normoxic or hypoxic conditions for the indicated times. MARCHF6-V5 appears as two bands that were quantified collectively, as we have done previously (Sharpe et al., 2019). (A, B) Graphs depict densitometric quantification of protein levels normalized to the respective normoxic condition for each siRNA or timepoint, which were set to 1 (dotted line). Data presented as mean ± SEM from n=3–4 independent experiments (*, p≤0.05; **, p≤0.01; [A] two-tailed ratio paired t-test; [B] two-tailed one-sample t-test vs. hypothetical mean of 1). Figure 3—source data 1 Uncropped immunoblots for Figure 3. https://cdn.elifesciences.org/articles/82843/elife-82843-fig3-data1-v1.zip Download elife-82843-fig3-data1-v1.zip To study MARCHF6 levels in hypoxic cells, we used a previously generated HEK293 cell line stably expressing a V5-tagged form of the protein. This construct was used due to the poor performance of endogenous MARCHF6 antibodies (Sharpe et al., 2019) and to eliminate transcriptional effects on protein levels. We examined the response of MARCHF6-V5 to hypoxia and found it accumulated during prolonged hypoxic incubations, which correlated with the maximal decline in full-length SM levels (Figure 3B). Therefore, increased MARCHF6 protein levels and activity likely account for the accelerated ubiquitination and degradation of SM during hypoxia. Hypoxia-induced squalene accumulation promotes partial degradation of SM Having established that SM undergoes accelerated proteasomal degradation during hypoxia, we next investigated how low oxygen levels favor its partial rather than complete proteolysis to yield trunSM. As there is extensive precedent for metabolic regulation of cholesterol synthesis enzymes and the pathway contains multiple oxygen-dependent reactions, we considered if accumulation of a pathway intermediate might underlie this phenomenon. Hypoxia-induced accumulation of trunSM occurred in cells incubated under both lipoprotein-replete and lipoprotein-deficient conditions, in which the cholesterol synthesis pathway is active (Figure 4A). However, the magnitude of this accumulation was diminished when lipoprotein-deficient cells were co-treated with a statin to inhibit HMGCR and the early cholesterol synthesis pathway. By contrast, there was no effect of sterol depletion on the hypoxia-induced reduction in full-length SM. This indicated that an intermediate or end-product of cholesterol synthesis promotes the partial rather than complete degradation of SM at the proteasome. We therefore turned our attention to the SM substrate squalene, as it allosterically regulates SM degradation (Yoshioka et al., 2020) and is the substrate for the first oxygen-dependent step of cholesterol synthesis. Squalene accumulated over the course of a hypoxic incubation (Figure 4B, Figure 4—figure supplement 1), consistent with reduced SM activity under low-oxygen conditions. This accumulation was strikingly well-correlated with the previously observed increase in trunSM levels (Figure 4C), suggesting the two effects may be linked. Figure 4 with 4 supplements see all Download asset Open asset Hypoxia-induced squalene accumulation promotes partial degradation of SM. (A) HEK293T cells were incubated in medium containing fetal calf serum (FCS), lipoprotein-deficient FCS (LPDS) or LPDS containing 5 µM mevastatin and 50 µM mevalonolactone (LPDS +statin) for 8 hr, refreshed in their respective medium and incubated under normoxic or hypoxic conditions for 16 hr. (B) HEK293T cells were incubated under normoxic or hypoxic conditions for the indicated times. Non-saponifiable lipids were extracted, and squalene levels were determined using gas chromatography-mass spectrometry and adjusted relative to the normoxic condition, which was set to 1 (dotted line). The maximal squalene level detected was 0.66±0.12 ng per µg of total protein. (C) Pearson correlation between squalene levels in (B) and trunSM levels in Figure 1C. Blue line indicates linear regression. (D) HEK293T cells were treated with or without 300 µM squalene (squ.), monooxidosqualene (MOS) or dioxidosqualene (DOS) for 16 hr. (E) HEK293T SQLE-knockout (SQLE-KO) clone 10 (c10) cells were transfected with the indicated constructs for 24 hr, then treated with or without 1 µM NB-598 or 300 µM squalene for 16 hr. (A, D, E) Graphs depict densitometric quantification of trunSM or truncated protein levels normalized to the (A) respective normoxic conditions for each serum type or (D, E) vehicle conditions, which were set to 1 (dotted line). (A–E) Data presented as mean ± SEM from n=3–5 independent experiments (*, p≤0.05; **, p≤0.01; [A] two-tailed ratio paired t-test; [D, E] two-tailed one-sample t-test vs. hypothetical mean of 1). Figure 4—source data 1 Uncropped immunoblots for Figure 4. https://cdn.elifesciences.org/articles/82843/elife-82843-fig4-data1-v1.zip Download elife-82843-fig4-data1-v1.zip Delivery of exogenous squalene induced trunSM accumulation in normoxic HEK293T and Huh7 cells (Figure 4D, Figure 4—figure supplement 2A, B), confirming its ability to promote partial degradation of SM. Accumulation of trunSM was also induced by the oxygenated squalene derivatives monooxidosqualene and dioxidosqualene (Figure 4D) but not by its saturated analogue squalane (Figure 4—figure supplement 2C), which has similar biophysical properties (Hauß et al., 2002). This indicated truncation is promoted by squalene and its structurally related molecules in a specific manner, rather than through bulk membrane effects caused by lipid accumulation. To address the possibility that exogenous squalene is converted to a downstream product responsible for truncation, we generated SQLE-knockout HEK293T cells (Figure 4—figure supplement 3) and transfected them with a catalytically inactive SM Y195F mutant (Padyana et al., 2019) to prevent the metabolism of added squalene. The truncated form of the Y195F mutant accumulated upon squalene treatment in SQLE-knockout cells, confirming squalene alone can directly induce truncation (Figure 4—figure supplement 2D). There was no significant accumulation of the truncated fragment in cells transfected with wild-type SM, likely due to clearance of exogenous squalene by the overexpressed protein and downstream enzymes. To confirm if endogenously synthesized squalene is sufficient to trigger SM truncation, cells were treated with inhibitors of the relevant cholesterol synthesis enzymes (Figure 4—figure supplement 2A). The SM inhibitor NB-598 was excluded because of its ability to induce truncation through direct binding and stabilization of the SM catalytic domain that renders it resistant to proteasomal unfolding (Padyana et al., 2019; Coates et al., 2021). Inhibiting squalene synthesis from farnesyl diphosphate (TAK-475) increased trunSM levels but abolished its hypoxia-induced accumulation, whereas significant accumulation still occurred under conditions where squalene synthesis was preserved: inhibition of lanosterol synthesis from monooxidosqualene (BIBB 515), or inhibition of lanosterol demethylation (GR70585X) (Figure 4—figure supplement 2E). This confirmed that lanosterol, which also accumulates during hypoxia (Nguyen et al., 2007), has no effect on SM truncation. We further noted that the inhibition of squalene or lanosterol synthesis, but not lanosterol demethylation, increased the levels of full-length SM and SM-N100-GFP-V5 under normoxic conditions. This was consistent with our previous finding that farnesyl-containing molecules, including monooxidosqualene, dioxidosqualene and a squalene-derived photoaffinity probe, stabilize SM via its regulatory domain in a similar manner to squalene itself (Yoshioka et al., 2020). The increase in normoxic trunSM levels upon treatment with TAK-475 and BIBB 515 (Figure 4—figure supplement 2E) suggested farnesyl-containing cholesterol synthesis intermediates can also induce SM truncation. Nevertheless, as the primary substrate of oxygen-dependent SM catalysis, squalene is likely to be the major driver of this process under hypoxic conditions. SM contains two known squalene binding sites: the SM-N100 regulatory domain and the active site of the catalytic domain (Yoshioka et al., 2020). As the SM inhibitor NB-598 induces SM truncation by binding and stabilizing the catalytic domain (Coates et al., 2021), we considered if squalene exerts its effects on truncation through a similar mechanism. To eliminate the contribution of the SM-N100 domain, we transfected SQLE-knockout cells with an ectopic form of trunSM (SM[ΔN65]-V5). Consistent with past findings (Y