Dietary cholesterol activates a Ral-dependent pathway driving LDLR turnover

Numerous studies have shown that dietary cholesterol produces a marked reduction in hepatic LDLR levels.

“Numerous studies have shown that dietary cholesterol produces a marked reduction in hepatic LDLR levels 3-6 .”

• cites [3] Applebaum-Bowden, D. et al. Down-regulation of the low-density lipoprotein receptor by dietary cholesterol. Am. J. Clin. Nutr. 39, 360–367 (1984). sourceNot checked — no full text of the source
• cites [4] Feingold, K. R. et al. in Endotext (eds K. R. Feingold et al.) https://www.ncbi.nlm.nih.gov/books/NBK570127/ (National Library of Medicine, 2000).Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [5] Horton, J. D., Cuthbert, J. A. & Spady, D. K. Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels. J. Clin. Invest. 92, 743–749 (1993). sourcePartly supported

  The Results report that dietary cholesterol reduced hepatic LDL receptor protein in hamsters, markedly so with coconut oil: levels fell to 52% and 36% of control at the two cholesterol doses. With safflower oil, levels remained at 93% and 80%, so the claim overstates the reduction by omitting its dependence on dietary fat. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [6] Lecerf, J. M. & de Lorgeril, M. Dietary cholesterol: from physiology to cardiovascular risk. Br. J. Nutr. 106, 6–14 (2011). sourceNot checked — no full text of the source

Deleterious missense variation in REPS1 is nominally associated with lower LDL-C (β = -0.00175, P = 0.00173).

“and lower LDL-C ( β = -0.00175, P = 0.00173) (Table 1).”

• cites [24] Karczewski, K. J. et al. Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes. Cell Genom. 2, 100168 (2022). sourceCould not verify

  The Results section discusses missense-variant associations generally and reports an LDL-cholesterol association for PCSK9, not REPS1 (page unavailable). Searches for REPS1 and the stated β and P values found no passage in our copy that verifies the claimed REPS1 association. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Deleterious missense variation in REPS1 is nominally associated with lower APOB (β = -0.00163, P = 0.00344).

“we found that deleterious missense variation in REPS1 was nominally associated with lower APOB ( β = -0.00163, P = 0.00344)”

• cites [24] Karczewski, K. J. et al. Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes. Cell Genom. 2, 100168 (2022). sourceCould not verify

  The Results section on rare-variant associations discusses deleterious missense variants in general, but does not report a REPS1–APOB association. Searches of our copy for REPS1, APOB and P = 0.00344 found no passage verifying the stated effect size or P value; page numbers were not available. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Deleterious missense variation in REPS1 is nominally associated with lower total cholesterol (β = -0.00164, P = 0.00277).

“lower total cholesterol ( β = -0.00164, P = 0.00277)”

• cites [24] Karczewski, K. J. et al. Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes. Cell Genom. 2, 100168 (2022). sourceCould not verify

  The Results section (‘Biological insights from rare-variant association results’; page unavailable) says the authors released their full association dataset, but the passage lists other genes and does not report a REPS1–total-cholesterol result. Searches for REPS1 and the stated β and P values found no matching passage in our copy, so the specific association cannot be verified from it. (LLM, conf=0.70)

  Experimental — accuracy not yet measured

Clearance of circulating PCSK9 is largely mediated by LDLR-dependent uptake.

“As the clearance of circulating PCSK9 is largely mediated by LDLR-dependent uptake 25 , higher plasma PCSK9 in RalGAPB HKO mice is probably due to reduced LDLR-mediated clearance, not increased PCSK9 production.”

• cites [25] Tavori, H., Rashid, S. & Fazio, S. On the function and homeostasis of PCSK9: reciprocal interaction with LDLR and additional lipid effects. Atherosclerosis 238, 264–270 (2015). sourceSupported

Chronic Ral activation causes mitochondrial fragmentation in adipocytes.

“and causing mitochondrial fragmentation in adipocytes 48 .”

• cites [48] Xia, W. et al. Obesity causes mitochondrial fragmentation and dysfunction in white adipocytes due to RalA activation. Nat. Metab 6, 273–289 (2024). sourceSupported

pLoF variants in CTSA were associated with lower total cholesterol (β = -0.00270, P = 0.0176).

“and lower total cholesterol ( β = -0.00270, P = 0.0176)”

• cites [24] Karczewski, K. J. et al. Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes. Cell Genom. 2, 100168 (2022). sourceCould not verify

  The Results section, “Biological insights from rare-variant association results” (page unavailable), reports a pLoF association between PCSK9 and LDL cholesterol, not CTSA and total cholesterol. Searches for CTSA, total cholesterol, β = -0.00270, and P = 0.0176 found no passage in our copy reporting the claimed association or statistics. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

pLoF variants in CTSA from UK Biobank exomes were associated with lower APOB (β = -0.00255, P = 0.0276).

“Analysis of protein-coding variation in CTSA from UK Biobank exomes revealed that pLoF variants were associated with lower APOB ( β = -0.00255, P = 0.0276)”

• cites [24] Karczewski, K. J. et al. Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes. Cell Genom. 2, 100168 (2022). sourceCould not verify

  The Results section describes UK Biobank exome association testing and gives examples involving other genes, but does not report a CTSA–APOB association. Searches for CTSA, APOB, β = -0.00255, and P = 0.0276 found no passage establishing the claimed direction or statistics in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

pLoF variants in CTSA showed a directionally concordant trend for lower LDL-C (β = -0.00220, P = 0.0573).

“with a directionally concordant trend for lower LDL-C ( β = -0.00220, P = 0.0573) (Table 1).”

• cites [24] Karczewski, K. J. et al. Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes. Cell Genom. 2, 100168 (2022). sourceCould not verify

  The Results section, “Biological insights from rare-variant association results” (page unavailable), reports a pLoF association between PCSK9 and LDL cholesterol, but does not report one for CTSA. Searches of our copy for CTSA, LDL-C, β = -0.00220, and P = 0.0573 found no passage establishing the claimed trend. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

SAR164653 (CTSAi) is a selective small-molecule inhibitor of CTSA.

“We next evaluated the effect of CTSA inhibition using the selective small-molecule inhibitor SAR164653 (CTSAi) 35,36 .”

• cites [35] Cowling, R. T. Cathepsin A inhibitors to treat heart disease: much potential, many questions. JACC Basic Transl. Sci. 4, 345–347 (2019). sourcePartly supported

  The main text (page not provided) identifies SAR164653 as a cathepsin A inhibitor and gives its chemical name, supporting its description as a small-molecule inhibitor. It does not state or establish that SAR164653 is selective, so that qualifier is unsupported by our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [36] Tillner, J. et al. Tolerability, safety, and pharmacokinetics of the novel cathepsin A inhibitor SAR164653 in healthy subjects. Clin. Pharmacol. Drug Dev. 5, 57–68 (2016). sourceNot checked — no full text of the source

Genes encoding components of this pathway are associated with changes in circulating cholesterol levels in human GWAS.

“Genes encoding components of this pathway are associated with changes in circulating cholesterol levels in human genome-wide association studies (GWAS).”

Overexpression of constitutively active Ral mutants in hepatocytes reduces LDLR levels.

“Constitutive activation of Ral via RalGAPB deletion or overexpression of constitutively active Ral mutants in hepatocytes reduces LDLR levels and impairs cholesterol clearance.”

mRNA levels of the RalGAP subunits Ralgapa1, Ralgapa2 and Ralgapb were unchanged in livers from NCD- and HCD-fed mice.

“mRNA levels of the RalGAP subunits Ralgapa1 , Ralgapa2 and Ralgapb were unchanged in livers from NCD- and HCD-fed mice (Extended Data Fig. 1k).”

RalA activity progressively increased at 12 and 20 weeks of HCD feeding.

“Although RalA and RalB activities did not change at 4 weeks of HCD feeding, RalA activity progressively increased at 12 and 20 weeks, whereas RalB activity was significantly increased at 20 weeks (Fig. 1b,c).”

Primary hepatocytes isolated from RalGAPB HKO mice showed a significant reduction in LDLR protein levels compared with RalGAPB f/f controls.

“Primary hepatocytes isolated from RalGAPB HKO mice showed a significant reduction in LDLR protein levels compared with RalGAPB f/f controls, and this reduction was further enhanced by LDL supplementation under lipoprotein-deficient serum (LPDS) conditions (Fig. 1d,e).”

Cholesterol loading in HepG2 cells increased RalA and RalB activities.

“Supporting this, cholesterol loading in HepG2 cells also increased RalA and RalB activities (Extended Data Fig. 1f).”

Constitutive activation of Ral via RalGAPB deletion or overexpression of constitutively active Ral mutants in hepatocytes impairs cholesterol clearance.

“Constitutive activation of Ral via RalGAPB deletion or overexpression of constitutively active Ral mutants in hepatocytes reduces LDLR levels and impairs cholesterol clearance.”

RalGAPA1 protein levels were increased in livers from HCD-fed mice.

“The protein levels of RalGAPA2 and RalGAPB were also comparable, whereas RalGAPA1 was increased (Extended Data Fig. 1m).”

RalA and RalB activities did not change at 4 weeks of HCD feeding.

“Although RalA and RalB activities did not change at 4 weeks of HCD feeding, RalA activity progressively increased at 12 and 20 weeks, whereas RalB activity was significantly increased at 20 weeks (Fig. 1b,c).”

LDLR protein levels remained unchanged at 4 weeks of HCD feeding.

“LDLR protein levels remained unchanged at 4 weeks (Fig. 1a, Extended Data Fig. 1d), coinciding with reduced PCSK9 expression at this time point (Extended Data Fig. 1e).”

The dominant-negative GDP-bound RalA mutant (S28N) had no effect on LDLR protein levels in HepG2 cells.

“Moreover, overexpression of either wild-type or the constitutively active GTP-bound RalA mutant (G23V) reduced LDLR protein levels in HepG2 cells, whereas the dominant-negative GDP-bound mutant (S28N) had no effect (Fig. 1f), indicating that RalA suppresses LDLR protein levels in a GTP-dependent manner.”

Ral activation directs CTSA towards lysosomes for maturation.

“Ral activation directs CTSA towards lysosomes for maturation while limiting its secretion, further promoting LDLR degradation in lysosomes.”

None of these manipulations altered Ldlr mRNA levels.

“Furthermore, none of these manipulations altered Ldlr mRNA levels (Extended Data Fig. 1j).”

RalB activity was significantly increased at 20 weeks of HCD feeding.

“Although RalA and RalB activities did not change at 4 weeks of HCD feeding, RalA activity progressively increased at 12 and 20 weeks, whereas RalB activity was significantly increased at 20 weeks (Fig. 1b,c).”

PCSK9 expression was reduced at 4 weeks of HCD feeding.

“LDLR protein levels remained unchanged at 4 weeks (Fig. 1a, Extended Data Fig. 1d), coinciding with reduced PCSK9 expression at this time point (Extended Data Fig. 1e).”

A previously unrecognized signalling pathway triggered by Ral activation controls LDLR trafficking and turnover.

“We show here that a previously unrecognized signalling pathway triggered by Ral activation controls LDLR trafficking and turnover.”

mRNA levels of RalGEFs, including Rgl1, Rgl2, Rgl3 and Ralgds, were similar between NCD- and HCD-fed mice.

“mRNA levels of RalGEFs, including Rgl1 , Rgl2 , Rgl3 and Ralgds , were similar between groups (Extended Data Fig. 1l).”

Protein levels of RalGAPA2 and RalGAPB were comparable between NCD- and HCD-fed mice.

“The protein levels of RalGAPA2 and RalGAPB were also comparable, whereas RalGAPA1 was increased (Extended Data Fig. 1m).”

A new pathway regulated by Ral GTPases links extracellular cholesterol signals to the intracellular trafficking machinery controlling LDLR turnover.

“Here we identify a new pathway regulated by Ral GTPases that links extracellular cholesterol signals to the intracellular trafficking machinery controlling LDLR turnover.”

Pharmacological inhibition of CTSA activity increases hepatic LDLR function.

“Pharmacological inhibition of CTSA activity increases hepatic LDLR function and improves cholesterol clearance, offering a potential new therapeutic strategy for hypercholesterolaemia and cardiovascular disease.”

Ldlr and Srebp2 mRNA levels were significantly reduced in liver tissues from HCD-fed mice at all time points compared with NCD-fed controls.

“Ldlr and Srebp2 mRNA levels were significantly reduced in liver tissues from HCD-fed mice at all time points compared with NCD-fed controls (Extended Data Fig. 1a-c).”

Overexpression of either wild-type or constitutively active GTP-bound RalA mutant (G23V) reduced LDLR protein levels in HepG2 cells.

“Moreover, overexpression of either wild-type or the constitutively active GTP-bound RalA mutant (G23V) reduced LDLR protein levels in HepG2 cells, whereas the dominant-negative GDP-bound mutant (S28N) had no effect (Fig. 1f), indicating that RalA suppresses LDLR protein levels in a GTP-dependent manner.”

Constitutive activation of Ral via RalGAPB deletion in hepatocytes reduces LDLR levels.

“Constitutive activation of Ral via RalGAPB deletion or overexpression of constitutively active Ral mutants in hepatocytes reduces LDLR levels and impairs cholesterol clearance.”

LDLR protein levels progressively declined at 12 and 20 weeks of HCD feeding.

“By contrast, LDLR protein levels progressively declined at 12 and 20 weeks (Fig. 1a, Extended Data Fig. 1d).”

Pharmacological inhibition of CTSA activity improves cholesterol clearance.

“Pharmacological inhibition of CTSA activity increases hepatic LDLR function and improves cholesterol clearance, offering a potential new therapeutic strategy for hypercholesterolaemia and cardiovascular disease.”

Both RalA and RalB were markedly activated in primary hepatocytes from RalGAPB HKO mice.

“Both RalA and RalB were markedly activated in primary hepatocytes from these mice (Extended Data Fig. 1i).”

LDLR is degraded by the lysosomal protease cathepsin A (CTSA).

“where LDLR is degraded by the lysosomal protease cathepsin A (CTSA).”

LDL supplementation under LPDS conditions further enhanced the reduction in LDLR protein levels in RalGAPB HKO hepatocytes.

“Primary hepatocytes isolated from RalGAPB HKO mice showed a significant reduction in LDLR protein levels compared with RalGAPB f/f controls, and this reduction was further enhanced by LDL supplementation under lipoprotein-deficient serum (LPDS) conditions (Fig. 1d,e).”

Ral activation limits CTSA secretion.

“Ral activation directs CTSA towards lysosomes for maturation while limiting its secretion, further promoting LDLR degradation in lysosomes.”

GFP-LDLR co-immunoprecipitated with HA-tagged CTSA in HEK293T cells.

“GFP-LDLR co-immunoprecipitated with human influenza hemagglutinin (HA)-tagged CTSA in HEK293T cells (Fig. 4d)”

Other canonical RAS downstream pathways, including MEK-ERK and PI3K-AKT, did not show sustained changes after 20 weeks of HCD feeding.

“Other canonical RAS downstream pathways, including MEK-ERK and PI3K-AKT, did not show sustained changes after 20 weeks of HCD feeding (Extended Data Fig. 1n).”

Hepatic LDLR protein levels were reduced in both RalA(G23V) and RalB(G23V) overexpression groups.

“Hepatic LDLR protein levels were reduced in both RalA(G23V) and RalB(G23V) overexpression groups (Fig. 2m,n), whereas Ldlr mRNA remained unchanged (Fig. 2o).”

An interaction between endogenous LDLR and RalA was detected in HepG2 hepatocytes.

“To investigate whether Ral proteins physically interact with LDLR, we performed co-immunoprecipitation assays and detected an interaction between endogenous LDLR and RalA in HepG2 hepatocytes (Extended Data Fig. 5a).”

Plasma total cholesterol levels were elevated in mice overexpressing RalA(G23V) or RalB(G23V).

“Plasma total cholesterol (Fig. 2k) and VLDL/LDL-C levels (Fig. 2l) were elevated, whereas HDL-C levels were unchanged (Extended Data Fig. 3s).”

REPS1 is a binding partner of RalBP1.

“REPS1 was validated as a binding partner of RalBP1 (Extended Data Fig. 6c).”

Simultaneous knockout of CTSA and RalGAPB restored plasma membrane LDLR abundance compared with RalGAPB-KO alone.

“plasma membrane LDLR abundance (Extended Data Fig. 7m,n)”

Variant rs72974722 is strongly associated with LDL-C levels (P = 5.01 × 10 -23 ).

“Variant rs72974722 is strongly associated with LDL-C levels ( P = 5.01 × 10 -23 ) (Fig. 3v)”

Immunofluorescence staining confirmed CTSA localization to lysosomes.

“Immunofluorescence staining confirmed CTSA localization to lysosomes (Extended Data Fig. 7a).”

RalGAPB-KO did not alter PCSK9-induced LDLR degradation.

“Furthermore, treatment with a PCSK9 antagonist failed to restore LDLR expression in RalGAPB-deficient hepatocytes (Extended Data Fig. 4r), and RalGAPB-KO did not alter PCSK9-induced LDLR degradation (Extended Data Fig. 4s).”

HDL-C levels were unchanged in mice overexpressing RalA(G23V) or RalB(G23V).

“Plasma total cholesterol (Fig. 2k) and VLDL/LDL-C levels (Fig. 2l) were elevated, whereas HDL-C levels were unchanged (Extended Data Fig. 3s).”

Primary hepatocytes isolated from CTSA deletion mice had a prolonged LDLR half-life.

“Primary hepatocytes isolated from CTSA deletion mice had a prolonged LDLR half-life (Fig. 4m,n).”

Treatment with the serine protease inhibitor AEBSF blocked LDLR downregulation in RalGAPB-KO cells.

“Consistently, treatment with the serine protease inhibitor AEBSF blocked LDLR downregulation in RalGAPB-KO cells (Extended Data Fig. 7l).”

Immunofluorescence analysis revealed reduced LDLR levels in recycling endosomes, as indicated by decreased co-localization with RAB11.

“Immunofluorescence analysis revealed reduced LDLR levels in recycling endosomes, as indicated by decreased co-localization with RAB11 (Fig. 3n,o).”

Overexpression of the constitutively active RalA(G23V) mutant reduced the interaction between SNX17 and LDLR.

“We found that overexpression of the constitutively active RalA(G23V) mutant reduced the interaction between SNX17 and LDLR (Fig. 3m).”

Ldlr mRNA remained unchanged in RalA(G23V) and RalB(G23V) overexpression groups.

“Hepatic LDLR protein levels were reduced in both RalA(G23V) and RalB(G23V) overexpression groups (Fig. 2m,n), whereas Ldlr mRNA remained unchanged (Fig. 2o).”

RalA(G23V) preferentially associated with several well-known components of the exocyst complex.

“RalA(G23V) preferentially associated with several well-known components of the exocyst complex 28 (Table 2).”

Other cholesterol-related receptors, including LXRα, LRP1 and transferrin receptor (TFR), showed no notable differences in RalGAPB HKO livers.

“Other cholesterol-related receptors, including LXRα, LRP1 and transferrin receptor (TFR) showed no notable differences (Fig. 2f).”

Under lysosomal blockade with BafA1, RalGAPB-KO enhanced LDL internalization.

“Under lysosomal blockade with BafA1, RalGAPB-KO reduced cell surface LDLR and enhanced LDL internalization (Fig. 3p,q), supporting increased LDLR endocytosis upon Ral activation.”

RalGAPB deletion led to reduced liver total cholesterol levels.

“Notably, RalGAPB deletion led to elevated plasma total cholesterol and reduced liver total cholesterol levels (Fig. 2a,b), with no significant change in biliary cholesterol (Fig. 2c), implying impaired hepatic cholesterol uptake rather than altered cholesterol excretion.”

Lipoprotein profiling revealed a marked increase in VLDL/LDL-C fractions in RalGAPB HKO mice.

“Lipoprotein profiling revealed a marked increase in VLDL/LDL-C fractions in RalGAPB HKO mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unchanged (Fig. 2d).”

Simultaneous knockout of CTSA and RalGAPB restored LDL uptake compared with RalGAPB-KO alone.

“and LDL uptake (Extended Data Fig. 7o,p) compared with RalGAPB-KO alone.”

Treatment of HepG2 cells with Pan-RAS-IN-1 restored LDLR levels reduced by cholesterol exposure.

“Treatment of HepG2 cells with Pan-RAS-IN-1, a pan-RAS inhibitor (RASi) that disrupts RAS-effector interactions 21 , prevented cholesterol-induced RAS activation, suppressed RalA and RalB activities (Fig. 1h), and restored LDLR levels reduced by cholesterol exposure (Extended Data Fig. 2b), suggesting that cholesterol promotes Ral activation through RAS signalling.”

LDL uptake was diminished in RalGAPB-deficient cells.

“LDL uptake was diminished in RalGAPB-deficient cells (Fig. 3b,c).”

Treatment of HepG2 cells with Pan-RAS-IN-1 prevented cholesterol-induced RAS activation.

“Treatment of HepG2 cells with Pan-RAS-IN-1, a pan-RAS inhibitor (RASi) that disrupts RAS-effector interactions 21 , prevented cholesterol-induced RAS activation, suppressed RalA and RalB activities (Fig. 1h), and restored LDLR levels reduced by cholesterol exposure (Extended Data Fig. 2b), suggesting that cholesterol promotes Ral activation through RAS signalling.”

RAS activity was elevated in cholesterol-loaded HepG2 cells.

“RAS activity was chronically elevated in the livers of HCD-fed mice at 12 and 20 weeks (Fig. 1g) and in cholesterol-loaded HepG2 cells (Extended Data Fig. 2a).”

Secreted CTSA levels were reduced in plasma from RalGAPB-KO mice.

“secreted CTSA levels were reduced in plasma from RalGAPB-KO mice (Extended Data Fig. 7h)”

LDLR degraded more rapidly in RalGAPB-deficient cells.

“Cycloheximide (CHX) chase assays revealed that LDLR degraded more rapidly in RalGAPB-deficient cells (Fig. 3a and Extended Data Fig. 4a).”

RalB overexpression in HEK293T cells, which lack endogenous PCSK9, significantly reduced GFP-LDLR protein levels.

“Importantly, RalB overexpression in HEK293T cells, which lack endogenous PCSK9, still significantly reduced green fluorescent protein (GFP)-LDLR protein levels (Extended Data Fig. 4q), supporting a PCSK9-independent effect of RalB on LDLR abundance.”

The interaction between GFP-tagged LDLR and FLAG-tagged RalA was further enhanced following cholesterol or LDL treatment.

“Interestingly, the interaction between GFP-tagged LDLR and FLAG-tagged RalA was further enhanced following cholesterol or LDL treatment (Fig. 3k), suggesting that cholesterol loading promotes RalA-LDLR association.”

RalA and CTSA co-localize in lysosomes.

“this interaction was further supported by co-localization of RalA and CTSA in lysosomes (Extended Data Fig. 7c).”

LDLR protein levels were significantly reduced in RalGAPB HKO livers.

“Further analysis showed that LDLR protein levels were significantly reduced in RalGAPB HKO livers (Fig. 2f), whereas Ldlr mRNA levels remained unchanged (Fig. 2g).”

Variant rs72974722 is associated with REPS1 expression in human liver (P = 1.45 × 10 -10 ).

“and REPS1 expression in human liver ( P = 1.45 × 10 -10 ) (Fig. 3w).”

Ldlr mRNA levels remained unchanged in RalGAPB HKO livers.

“Further analysis showed that LDLR protein levels were significantly reduced in RalGAPB HKO livers (Fig. 2f), whereas Ldlr mRNA levels remained unchanged (Fig. 2g).”

At the protein level, RGL2 and RGL3 remained unchanged between NCD- and HCD-fed mice.

“At the protein level, RGL2 and RGL3 remained unchanged, whereas RGL1 was decreased (Extended Data Fig. 1m).”

Overexpression of RalBP1 significantly reduces GFP-LDLR expression.

“Interestingly, we found that overexpression of RalBP1 significantly reduced GFP-LDLR expression”

RalGAPB deletion led to elevated plasma total cholesterol levels.

“Notably, RalGAPB deletion led to elevated plasma total cholesterol and reduced liver total cholesterol levels (Fig. 2a,b), with no significant change in biliary cholesterol (Fig. 2c), implying impaired hepatic cholesterol uptake rather than altered cholesterol excretion.”

Plasma PCSK9 levels were elevated in RalGAPB HKO mice.

“However, plasma PCSK9 levels were elevated (Extended Data Fig. 3c).”

Secreted CTSA levels were reduced in supernatants from RalGAPB-KO AML12 cells.

“and in supernatants from RalGAPB-KO AML12 cells (Extended Data Fig. 7i,j).”

RAS activity was chronically elevated in the livers of HCD-fed mice at 12 and 20 weeks.

“RAS activity was chronically elevated in the livers of HCD-fed mice at 12 and 20 weeks (Fig. 1g) and in cholesterol-loaded HepG2 cells (Extended Data Fig. 2a).”

Formal co-localization analysis identified strong evidence of co-localization between genetically determined hepatic REPS1 expression and LDL-C (PP.H4 = 0.823).

“Formal co-localization analysis identified strong evidence of co-localization between genetically determined hepatic REPS1 expression and both LDL-C (PP.H4 = 0.823) and total cholesterol levels (PP.H4 = 0.837).”

Plasma total cholesterol levels were significantly reduced in CTSA deletion mice.

“whereas plasma total cholesterol (Fig. 4k) and VLDL/LDL-C levels (Fig. 4l) were significantly reduced in CTSA deletion mice.”

Among proteins enriched in RalA(G23V) pulldowns were the Ral effector RalBP1, the endocytic protein REPS1, the clathrin adaptor PICALM and several components of the AP-2 complex.

“Among the proteins enriched in RalA(G23V) pulldowns were the Ral effector RalBP1, the endocytic protein REPS1, the clathrin adaptor PICALM and several components of the AP-2 complex (Table 2).”

Overexpression of REPS1 decreases LDLR expression.

“Overexpression of REPS1 decreased LDLR expression (Fig. 3t)”

Formal co-localization analysis identified strong evidence of co-localization between genetically determined hepatic REPS1 expression and total cholesterol levels (PP.H4 = 0.837).

“Formal co-localization analysis identified strong evidence of co-localization between genetically determined hepatic REPS1 expression and both LDL-C (PP.H4 = 0.823) and total cholesterol levels (PP.H4 = 0.837).”

Lysosomal LDLR signals were enhanced in RalGAPB HKO hepatocytes.

“By contrast, lysosomal LDLR signals were enhanced, as shown by increased co-localization of LDLR with LysoTracker, accompanied by a reduction in total cellular LDLR fluorescence intensity in RalGAPB HKO hepatocytes (Fig. 3f,g).”

Treatment of HepG2 cells with Pan-RAS-IN-1 suppressed RalA and RalB activities.

“Treatment of HepG2 cells with Pan-RAS-IN-1, a pan-RAS inhibitor (RASi) that disrupts RAS-effector interactions 21 , prevented cholesterol-induced RAS activation, suppressed RalA and RalB activities (Fig. 1h), and restored LDLR levels reduced by cholesterol exposure (Extended Data Fig. 2b), suggesting that cholesterol promotes Ral activation through RAS signalling.”

RGL1 protein levels were decreased in livers from HCD-fed mice.

“At the protein level, RGL2 and RGL3 remained unchanged, whereas RGL1 was decreased (Extended Data Fig. 1m).”

Under lysosomal blockade with BafA1, RalGAPB-KO reduced cell surface LDLR.

“Under lysosomal blockade with BafA1, RalGAPB-KO reduced cell surface LDLR and enhanced LDL internalization (Fig. 3p,q), supporting increased LDLR endocytosis upon Ral activation.”

Total cellular LDLR fluorescence intensity was reduced in RalGAPB HKO hepatocytes.

“By contrast, lysosomal LDLR signals were enhanced, as shown by increased co-localization of LDLR with LysoTracker, accompanied by a reduction in total cellular LDLR fluorescence intensity in RalGAPB HKO hepatocytes (Fig. 3f,g).”

Expression levels of key components of the CCC-WASH complex were comparable in livers from C57BL/6J mice fed either an NCD or HCD for 20 weeks.

“The expression levels of key components of this complex were comparable in livers from C57BL/6J mice fed either an NCD or HCD for 20 weeks (Extended Data Fig. 5g-i).”

shRNA-mediated knockdown of RalBP1 restores LDLR expression in RalGAPB-KO AML12 cells.

“Conversely, short-hairpin RNA (shRNA)-mediated knockdown of RalBP1 restored LDLR expression in RalGAPB-KO AML12 cells (Extended Data Fig. 6b).”

Treatment with a PCSK9 antagonist failed to restore LDLR expression in RalGAPB-deficient hepatocytes.

“Furthermore, treatment with a PCSK9 antagonist failed to restore LDLR expression in RalGAPB-deficient hepatocytes (Extended Data Fig. 4r), and RalGAPB-KO did not alter PCSK9-induced LDLR degradation (Extended Data Fig. 4s).”

Treatment with the lysosomal inhibitor bafilomycin A1 restored LDLR protein levels in RalGAPB-deficient primary hepatocytes.

“Treatment with the lysosomal inhibitor bafilomycin A1 (BafA1) restored LDLR protein levels in RalGAPB-deficient primary hepatocytes (Fig. 3h,i).”

Plasma VLDL/LDL-C levels were significantly reduced in CTSA deletion mice.

“whereas plasma total cholesterol (Fig. 4k) and VLDL/LDL-C levels (Fig. 4l) were significantly reduced in CTSA deletion mice.”

Deletion of the LDLR cytosolic C-terminal tail (C-del) or mutation of the NPxY motif (NPxY-4A) markedly reduced the interaction between LDLR and RalA.

“Deletion of the LDLR cytosolic C-terminal tail (C-del) or mutation of the NPxY motif (NPxY-4A) markedly reduced the interaction between LDLR and RalA (Fig. 3l), indicating that the LDLR cytosolic tail and NPxY motif are required for efficient RalA-LDLR complex formation.”

Cholesterol did not alter KRAS localization to the plasma membrane.

“Further analysis showed that cholesterol did not alter KRAS localization to the plasma membrane (Extended Data Fig. 2c-e), but enhanced the interaction between KRAS and RGL2, a RalGEF downstream of RAS (Fig. 1i).”

Knocking down SEC5 or SEC8 in AML12 cells did not alter LDLR protein levels.

“Knocking down SEC5 or SEC8 in AML12 cells did not alter LDLR protein levels (Extended Data Fig. 6a), suggesting that exocyst-complex-mediated exocytosis is unlikely to account for Ral-induced LDLR degradation.”

REPS1 knockdown restores LDLR protein expression in RalGAPB-KO AML12 cells.

“whereas REPS1 knockdown restored LDLR protein expression in RalGAPB-KO AML12 cells (Fig. 3u and Extended Data Fig. 6h).”

Cathepsin A was identified as a potential interactor with RalA-WT and RalA(G23V).

“Cathepsin A-a lysosomal serine protease involved in lysosomal protein degradation 30,31 -was identified as a potential interactor with RalA-WT and RalA(G23V) (Table 2).”

Co-localization of LDLR with LysoTracker was increased in RalGAPB HKO hepatocytes.

“By contrast, lysosomal LDLR signals were enhanced, as shown by increased co-localization of LDLR with LysoTracker, accompanied by a reduction in total cellular LDLR fluorescence intensity in RalGAPB HKO hepatocytes (Fig. 3f,g).”

Precursor CTSA (CTSA-pre) is more abundant during short-term HCD feeding.

“Precursor CTSA (CTSA-pre) was more abundant during short-term HCD feeding (Extended Data Fig. 7d,e)”

Plasma membrane-associated LDLR was markedly reduced in RalGAPB-deficient primary hepatocytes.

“Plasma membrane-associated LDLR was markedly reduced in RalGAPB-deficient primary hepatocytes (Fig. 3d,e).”

Plasma triglyceride (TG) levels were comparable between RalGAPB f/f and RalGAPB HKO mice.

“Plasma triglyceride (TG) levels were comparable between groups (Extended Data Fig. 3b).”

REPS1 protein expression is elevated in livers overexpressing RalA(G23V) or RalB(G23V).

“as well as in livers overexpressing RalA(G23V) or RalB(G23V) (Extended Data Fig. 6f,g).”

Rare predicted loss-of-function (pLoF) variants in KRAS were nominally associated with lower apolipoprotein B (APOB) levels (β = -0.00531, P = 0.0242).

“Using an orthogonal human genetic approach based on exome sequencing 24 , rare predicted loss-of-function (pLoF) variants in KRAS were also nominally associated with lower apolipoprotein B (APOB) levels ( β = -0.00531, P = 0.0242), further supporting a role for KRAS in human cholesterol metabolism (Table 1).”

Lysosomal proteins and other lysosome-targeted membrane proteins remained unchanged upon CTSA overexpression.

“whereas lysosomal proteins and other lysosome-targeted membrane proteins remained unchanged (Extended Data Fig. 7k).”

Expression of the Ral-binding domain (RBD) of RalBP1 alone reproduces the reduction in GFP-LDLR expression caused by RalBP1 overexpression.

“and this effect was reproduced by expression of the Ral-binding domain (RBD) of RalBP1 alone (Fig. 3r).”

Cholesterol enhanced the interaction between KRAS and RGL2.

“Further analysis showed that cholesterol did not alter KRAS localization to the plasma membrane (Extended Data Fig. 2c-e), but enhanced the interaction between KRAS and RGL2, a RalGEF downstream of RAS (Fig. 1i).”

Primary hepatocytes isolated from control and RalGAPB HKO mice secreted comparable levels of PCSK9 into the culture medium.

“Primary hepatocytes isolated from control and RalGAPB HKO mice secreted comparable levels of PCSK9 into the culture medium (Extended Data Fig. 4o,p).”

Simultaneous knockout of CTSA and RalGAPB by CRISPR-Cas9 restored LDLR protein levels compared with RalGAPB-KO alone.

“Simultaneous knockout of CTSA and RalGAPB by CRISPRCas9 restored LDLR protein levels (Fig. 4h)”

DiI-LDL clearance was significantly impaired in RalGAPB HKO mice.

“DiI-LDL (1′-dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine-labelled low-density lipoprotein) clearance was significantly impaired in RalGAPB HKO mice (Fig. 2e).”

HDL-C remained unchanged in RalGAPB HKO mice.

“Lipoprotein profiling revealed a marked increase in VLDL/LDL-C fractions in RalGAPB HKO mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unchanged (Fig. 2d).”

REPS1 protein expression is elevated in RalGAPB-KO hepatocytes and livers.

“REPS1 protein expression was elevated in RalGAPB-KO hepatocytes and livers (Fig. 3s and Extended Data Fig. 6d,e)”

These findings were recapitulated in RalGAPB-knockout (KO) AML12 cells generated using CRISPR-Cas9.

“These findings were recapitulated in RalGAPB-knockout (KO) AML12 cells generated using CRISPR-Cas9 (Extended Data Fig. 4e-n).”

Knockdown of SNX17 alone promoted LDLR degradation.

“In line with these observations, knockdown of SNX17 alone promoted LDLR degradation, supporting its critical role in receptor recycling (Extended Data Fig. 5e,f).”

RalGAPB deletion caused no significant change in biliary cholesterol.

“Notably, RalGAPB deletion led to elevated plasma total cholesterol and reduced liver total cholesterol levels (Fig. 2a,b), with no significant change in biliary cholesterol (Fig. 2c), implying impaired hepatic cholesterol uptake rather than altered cholesterol excretion.”

Hepatic precursor CTSA levels were not altered in RalGAPB HKO mice.

“Although hepatic precursor CTSA levels were not altered in RalGAPB HKO mice (Extended Data Fig. 7g)”

rs75667995 was also a strong eQTL for hepatic KRAS expression (P = 1 × 10^-5).

“This variant was also a strong eQTL for hepatic KRAS expression ( P = 1 × 10 -5 ) (Fig. 1m).”

Variant rs62441842 is associated with hepatic REPS1 expression (P = 5.5 × 10 -11 ).

“as well as hepatic REPS1 expression ( P = 5.5 × 10 -11 ) (Extended Data Fig. 6j).”

At the KRAS locus, a strong signal of association with altered serum cholesterol levels was identified, with rs75667995 nominated as the lead causal variant (P = 3.16 × 10^-20).

“At the KRAS locus, a strong signal of association with altered serum cholesterol levels was identified, with rs75667995 nominated as the lead causal variant ( P = 3.16 × 10 -20 ) (Fig. 1l).”

VLDL/LDL-C levels were elevated in mice overexpressing RalA(G23V) or RalB(G23V).

“Plasma total cholesterol (Fig. 2k) and VLDL/LDL-C levels (Fig. 2l) were elevated, whereas HDL-C levels were unchanged (Extended Data Fig. 3s).”

In RalGAPB-KO cells, knockdown of SNX17 did not further reduce LDLR levels.

“Moreover, in RalGAPB-KO cells, which had reduced LDLR due to increased Ral activity, knockdown of SNX17 did not further reduce LDLR levels (Extended Data Fig. 5f), indicating that both manipulations act by affecting the same recycling-degradation pathway, with Ral activation exerting the dominant effect.”

CTSAi restored plasma membrane LDLR abundance in RalGAPB HKO primary hepatocytes.

“Furthermore, CTSAi restored plasma membrane LDLR abundance in RalGAPB HKO primary hepatocytes (Fig. 5p,q).”

RBC8 treatment reduced Ral activity in HepG2 cells.

“accompanied by reduced Ral activity (Extended Data Fig. 8b).”

RalBP1 interacts with REPS1.

“The interaction between the two proteins was detected by Western blots.”

Plasma VLDL/LDL-C and total cholesterol levels were significantly reduced by RBC8 treatment.

“whereas plasma VLDL/LDL-C and total cholesterol levels were significantly reduced (Fig. 5j and Extended Data Fig. 8n).”

RBC8 treatment slowed LDLR degradation.

“slowed LDLR degradation (Fig. 5b,c)”

Cholesterol-induced RAS activation suppresses LDLR in hepatocytes.

“Cholesterol-induced RAS activation suppresses LDLR in hepatocytes.”

Hepatic LDLR protein expression was elevated by RBC8 treatment.

“hepatic LDLR protein expression was elevated (Fig. 5h,i).”

Cholesterol exposure activates Ral GTPase to decrease LDLR protein expression in hepatocytes.

“Cholesterol exposure activates Ral GTPase to decrease LDLR protein expression in hepatocytes.”

Ral activation correlated well with RAS activity in both in vivo and in vitro settings.

“however, Ral activation correlated well with RAS activity in both in vivo and in vitro settings”

CTSA showed a significant gene-level association with cholesterol (P = 8.71 × 10 -4 ).

“and cholesterol ( P = 8.71 × 10 -4 ) (Table 3).”

CTSA deletion increased plasma membrane LDLR abundance in primary hepatocytes.

“CTSA deletion increased plasma membrane LDLR abundance (Fig. 4o,p)”

CTSA showed a significant gene-level association with coronary artery disease (CAD) in the European cardiogram-based meta-analysis (P = 5.97 × 10 -4 ).

“CTSA also showed a significant gene-level association with coronary artery disease (CAD) in the European cardiogram-based meta-analysis ( P = 5.97 × 10 -4 ) (Table 3)”

CTSAi treatment normalized plasma VLDL/LDL-C in RalGAPB HKO mice to those observed in RalGAPB f/f mice.

“normalized plasma VLDL/LDL-C (Fig. 5t)”

Knockdown of exocyst components had no effect on the control of LDLR levels by active Ral.

“Although we identified exocyst components in our proteomic studies examining the RalA interactome, knockdown of these proteins had no effect on the control of LDLR levels by active Ral.”

CTSA deletion restored the cholesterol-induced reduction in plasma membrane LDLR abundance in primary hepatocytes.

“Importantly, CTSA deletion restored the cholesterol-induced reduction in plasma membrane LDLR abundance in primary hepatocytes (Fig. 4s,t).”

Blockade of RAS activity prevented cholesterol-induced activation of Ral.

“and blockade of RAS activity prevented cholesterol-induced activation of Ral.”

RBC8 treatment reduced Ldlr mRNA in vivo.

“Despite a reduction in Ldlr mRNA (Extended Data Fig. 8j)”

RBC8 treatment markedly increased LDLR protein levels in HepG2 cells.

“RBC8 treatment markedly increased LDLR protein levels (Fig. 5a)”

RBC8 treatment increased cell surface LDLR abundance in HepG2 cells.

“increased cell surface LDLR abundance (Fig. 5d,e)”

RBC8 did not alter REPS1 protein expression.

“Interestingly, RBC8 did not alter REPS1 protein expression (Fig. 5a)”

RBC8 treatment reduced mature CTSA expression.

“RBC8 treatment reduced mature CTSA expression (Fig. 5a)”

HA-CTSA interacts with GTPγS- or GDP-loaded FLAG-RalA.

“showing the interaction between HA-CTSA and GTPγS- or GDP-loaded FLAG-RalA.”

Activation of Ral in vivo impairs cholesterol homeostasis by reducing hepatic LDLR protein levels.

“Activation of Ral in vivo impairs cholesterol homeostasis by reducing hepatic LDLR protein levels.”

CTSAi did not affect HDL-C.

“without affecting HDL-C (Extended Data Fig. 8t).”

CTSA deletion increased LDL uptake in primary hepatocytes.

“and LDL uptake (Fig. 4q,r) in primary hepatocytes.”

RalA interacts with LDLR in HepG2 cells.

“The interaction between RalA and LDLR was detected by Western blot.”

CTSAi treatment normalized total cholesterol levels in RalGAPB HKO mice to those observed in RalGAPB f/f mice.

“and total cholesterol levels (Extended Data Fig. 8u) to those observed in RalGAPB f/f mice”

In primary mouse hepatocytes, CTSAi increased LDLR protein levels.

“In primary mouse hepatocytes, CTSAi increased LDLR protein levels (Extended Data Fig. 8o,p)”

RBC8 treatment enhanced LDL uptake in HepG2 cells.

“and enhanced LDL uptake in HepG2 cells (Fig. 5f,g).”

CTSAi reduced plasma VLDL/LDL-C by approximately 40%.

“and reduced plasma VLDL/LDL-C by approximately 40% (Fig. 5o)”

RBC8 treatment suppressed Ral activity in the liver.

“RBC8 treatment suppressed Ral activity in the liver (Extended Data Fig. 8e)”

CTSAi restored LDLR protein expression after cholesterol loading in primary mouse hepatocytes.

“and restored LDLR protein expression after cholesterol loading (Fig. 5k,l).”

CTSA showed a significant gene-level association with LDL-C (P = 4.60 × 10 -7 ).

“LDL-C ( P = 4.60 × 10 -7 )”

CTSAi increased hepatic LDLR protein levels in HCD-fed mice.

“CTSAi increased hepatic LDLR protein levels (Fig. 5m,n)”

RBC8 did not alter LDLR internalization.

“or LDLR internalization (Extended Data Fig. 8c,d)”

HDL-C remained unchanged with CTSAi treatment.

“whereas HDL-C remained unchanged (Extended Data Fig. 8v).”

CTSAi enhanced plasma membrane localization of LDLR in primary human hepatocytes.

“Consistently, CTSAi enhanced plasma membrane localization of LDLR (Extended Data Fig. 8q,r)”

CTSAi restored LDLR levels under cholesterol-loading conditions in primary human hepatocytes.

“and restored LDLR levels under cholesterol-loading conditions (Extended Data Fig. 8s) in primary human hepatocytes.”

GFP-LDLR interacts with FLAG-RalB (including its mutants) in HEK293T cells.

“The interactions between GFP-LDLR and FLAG-RalB (including its mutants) were confirmed by Western blot.”

CTSAi treatment normalized hepatic LDLR protein levels in livers from RalGAPB HKO mice.

“CTSAi treatment normalized hepatic LDLR protein levels in livers from RalGAPB HKO mice (Fig. 5r,s)”

RBC8 treatment decreased Ldlr transcription in HepG2 cells.

“despite a decrease in Ldlr transcription (Extended Data Fig. 8a)”

Co-immunoprecipitation and pulldown assays support a functional interaction between Ral proteins and LDLR.

“Although co-immunoprecipitation and pulldown assays support a functional interaction between Ral proteins and LDLR”

Under dietary cholesterol stress, Ral proteins become chronically activated by RAS.

“Under dietary cholesterol stress, Ral proteins become chronically activated by RAS.”

Our findings reveal a Ral-dependent signalling pathway as a key regulator of LDLR turnover and cholesterol homeostasis.

“Together, our findings reveal a Ral-dependent signalling pathway as a key regulator of LDLR turnover and cholesterol homeostasis.”

Chronic dietary cholesterol inhibits LDLR recycling.

“Chronic dietary cholesterol activates the Ral proteins by increasing RAS activity, routing LDLR to lysosomes for degradation and inhibiting its recycling independently of transcriptional regulation or PCSK9.”

Chronic dietary cholesterol activates Ral proteins by increasing RAS activity.

“Chronic dietary cholesterol activates the Ral proteins by increasing RAS activity, routing LDLR to lysosomes for degradation and inhibiting its recycling independently of transcriptional regulation or PCSK9.”

Chronic dietary cholesterol reduces LDLR independently of transcriptional regulation or PCSK9.

“Chronic dietary cholesterol activates the Ral proteins by increasing RAS activity, routing LDLR to lysosomes for degradation and inhibiting its recycling independently of transcriptional regulation or PCSK9.”

Ral engages the endocytic RalBP1-REPS1 complex to promote LDLR lysosomal routing.

“Ral engages the endocytic RalBP1-REPS1 complex to promote LDLR internalization and lysosomal routing, where LDLR is degraded by the lysosomal protease cathepsin A (CTSA).”

Prolonged cholesterol exposure activates Ral proteins to reduce LDLR abundance through post-translational mechanisms in hepatocytes.

“Taken together, these data demonstrate that prolonged cholesterol exposure activates Ral proteins to reduce LDLR abundance through post-translational mechanisms in hepatocytes.”

Ral engages the endocytic RalBP1-REPS1 complex to promote LDLR internalization.

“Ral engages the endocytic RalBP1-REPS1 complex to promote LDLR internalization and lysosomal routing, where LDLR is degraded by the lysosomal protease cathepsin A (CTSA).”

RalA suppresses LDLR protein levels in a GTP-dependent manner.

“Moreover, overexpression of either wild-type or the constitutively active GTP-bound RalA mutant (G23V) reduced LDLR protein levels in HepG2 cells, whereas the dominant-negative GDP-bound mutant (S28N) had no effect (Fig. 1f), indicating that RalA suppresses LDLR protein levels in a GTP-dependent manner.”

Chronic dietary cholesterol routes LDLR to lysosomes for degradation.

“Chronic dietary cholesterol activates the Ral proteins by increasing RAS activity, routing LDLR to lysosomes for degradation and inhibiting its recycling independently of transcriptional regulation or PCSK9.”

Ral activation further promotes LDLR degradation in lysosomes.

“Ral activation directs CTSA towards lysosomes for maturation while limiting its secretion, further promoting LDLR degradation in lysosomes.”

These findings provide human genetic support for a role of hepatic RAS signalling in cholesterol metabolism.

“These findings provide human genetic support for a role of hepatic RAS signalling in cholesterol metabolism.”

Higher plasma PCSK9 in RalGAPB HKO mice is probably due to reduced LDLR-mediated clearance, not increased PCSK9 production.

“As the clearance of circulating PCSK9 is largely mediated by LDLR-dependent uptake 25 , higher plasma PCSK9 in RalGAPB HKO mice is probably due to reduced LDLR-mediated clearance, not increased PCSK9 production.”

Cholesterol loading promotes RalA-LDLR association.

“Interestingly, the interaction between GFP-tagged LDLR and FLAG-tagged RalA was further enhanced following cholesterol or LDL treatment (Fig. 3k), suggesting that cholesterol loading promotes RalA-LDLR association.”

The LDLR cytosolic tail and NPxY motif are required for efficient RalA-LDLR complex formation.

“Deletion of the LDLR cytosolic C-terminal tail (C-del) or mutation of the NPxY motif (NPxY-4A) markedly reduced the interaction between LDLR and RalA (Fig. 3l), indicating that the LDLR cytosolic tail and NPxY motif are required for efficient RalA-LDLR complex formation.”

Activated RalA may engage the RalBP1- or REPS1-dependent endocytic machinery to promote LDLR endocytosis.

“These interactions suggest that activated RalA may engage the RalBP1- or REPS1-dependent endocytic machinery to promote LDLR endocytosis.”

The effects of Ral activation on LDLR trafficking are more directly linked to disruption of SNX17-dependent recycling than to the CCC-WASH pathway.

“Although these findings do not exclude a contribution of the CCC-WASH pathway, they suggest that the effects of Ral activation on LDLR trafficking are more directly linked to disruption of SNX17-dependent recycling.”

RalB reduces LDLR abundance independently of PCSK9.

“Importantly, RalB overexpression in HEK293T cells, which lack endogenous PCSK9, still significantly reduced green fluorescent protein (GFP)-LDLR protein levels (Extended Data Fig. 4q), supporting a PCSK9-independent effect of RalB on LDLR abundance.”

SNX17 plays a critical role in receptor recycling.

“In line with these observations, knockdown of SNX17 alone promoted LDLR degradation, supporting its critical role in receptor recycling (Extended Data Fig. 5e,f).”

Enhanced lysosomal CTSA processing occurs during prolonged cholesterol exposure and Ral activation.

“These findings suggest enhanced lysosomal CTSA processing during prolonged cholesterol exposure and Ral activation.”

RalGAPB deletion implies impaired hepatic cholesterol uptake rather than altered cholesterol excretion.

“Notably, RalGAPB deletion led to elevated plasma total cholesterol and reduced liver total cholesterol levels (Fig. 2a,b), with no significant change in biliary cholesterol (Fig. 2c), implying impaired hepatic cholesterol uptake rather than altered cholesterol excretion.”

Ral activation increases LDLR endocytosis.

“Under lysosomal blockade with BafA1, RalGAPB-KO reduced cell surface LDLR and enhanced LDL internalization (Fig. 3p,q), supporting increased LDLR endocytosis upon Ral activation.”

Ral activation may interfere with SNX17 association with LDLR at the NPxY motif, providing a mechanistic basis for reduced LDLR recycling and enhanced lysosomal degradation.

“These findings support the notion that Ral activation may interfere with SNX17 association with LDLR at the NPxY motif, providing a mechanistic basis for reduced LDLR recycling and enhanced lysosomal degradation.”

KRAS plays a role in human cholesterol metabolism.

“Using an orthogonal human genetic approach based on exome sequencing 24 , rare predicted loss-of-function (pLoF) variants in KRAS were also nominally associated with lower apolipoprotein B (APOB) levels ( β = -0.00531, P = 0.0242), further supporting a role for KRAS in human cholesterol metabolism (Table 1).”

Ral activation by chronic cholesterol exposure is not driven by major changes in RalGAP or RalGEF expression.

“These results suggest that Ral activation by chronic cholesterol exposure is not driven by major changes in RalGAP or RalGEF expression.”

Ral-mediated LDLR degradation occurs independently of PCSK9.

“These findings indicate that Ral-mediated LDLR degradation occurs independently of PCSK9.”

The same genetic variants that alter hepatic REPS1 expression also modulate circulating lipid levels.

“These data indicate that the same genetic variants that alter hepatic REPS1 expression also modulate circulating lipid levels.”

Cholesterol promotes Ral activation through RAS signalling.

“Treatment of HepG2 cells with Pan-RAS-IN-1, a pan-RAS inhibitor (RASi) that disrupts RAS-effector interactions 21 , prevented cholesterol-induced RAS activation, suppressed RalA and RalB activities (Fig. 1h), and restored LDLR levels reduced by cholesterol exposure (Extended Data Fig. 2b), suggesting that cholesterol promotes Ral activation through RAS signalling.”

Exocyst-complex-mediated exocytosis is unlikely to account for Ral-induced LDLR degradation.

“Knocking down SEC5 or SEC8 in AML12 cells did not alter LDLR protein levels (Extended Data Fig. 6a), suggesting that exocyst-complex-mediated exocytosis is unlikely to account for Ral-induced LDLR degradation.”

Insulin signalling is unlikely to contribute to the observed Ral activation under these conditions.

“Although insulin can inhibit RalGAP through AKT-mediated phosphorylation in adipocytes 19 , hepatic AKT phosphorylation and plasma insulin levels did not differ between NCD- and HCD-fed mice (Extended Data Fig. 1n,o), indicating that insulin signalling is unlikely to contribute to the observed Ral activation under these conditions.”

The RAS-RGL2-Ral pathway plays a role in cholesterol-induced LDLR downregulation.

“Furthermore, overexpression of RGL2 or KRAS reduced LDLR protein levels (Fig. 1j,k and Extended Data Fig. 2f,g), supporting a role for the RAS-RGL2-Ral pathway in cholesterol-induced LDLR downregulation.”

RAS recruits RalGEFs to promote Ral activation.

“Although hepatic RalGEF levels were not changed by HCD, these proteins can drive Ral activation through a GTPase cascade downstream of RAS, in which RAS recruits RalGEFs to promote Ral activation 20 .”

• cites [20] Wolthuis, R. M., Zwartkruis, F., Moen, T. C. & Bos, J. L. Ras-dependent activation of the small GTPase Ral. Curr. Biol. 8, 471–474 (1998). sourceNot checked — no full text of the source

High dietary cholesterol activates RAS.

“High dietary cholesterol activates RAS, which promotes Ral activation through RalGEFs.”

Prolonged cholesterol exposure leads to sustained activation of Ral.

“Prolonged cholesterol exposure leads to sustained activation of Ral”

REPS1 contributes to Ral-driven LDLR trafficking.

“REPS1 contributes to Ral-driven LDLR trafficking”

The Ral-RalBP1-REPS1 axis promotes LDLR endocytosis and degradation.

“The Ral-RalBP1-REPS1 axis promotes LDLR endocytosis and degradation.”

CTSA drives lysosomal LDLR degradation.

“CTSA then drives lysosomal LDLR degradation.”

Ral can control endocytosis when localized to the plasma membrane via its effector RalBP1, as is the case for LDLR trafficking to lysosomes.

“but also control endocytosis when localized to the plasma membrane via its effector RalBP1, as is the case for LDLR trafficking to lysosomes.”

RAS promotes Ral activation through RalGEFs.

“High dietary cholesterol activates RAS, which promotes Ral activation through RalGEFs.”

Chronic Ral activation promotes LDLR degradation in hepatocytes.

“By contrast, chronic Ral activation has detrimental effects, including promoting LDLR degradation in hepatocytes”

REPS1 genetic variants correlate with human cholesterol levels.

“and its genetic variants correlate with human cholesterol levels.”

Basal Ral activity is dispensable for LDLR internalization.

“indicating that basal Ral activity is dispensable for LDLR internalization.”

Cathepsin A mediates the lysosomal degradation of LDLR.

“Cathepsin A mediates the lysosomal degradation of LDLR, making it a pivotal effector in this pathway.”

Activated Ral interacts with RalBP1.

“Activated Ral interacts with RalBP1, which serves as a scaffold to recruit REPS1 and other endocytic components, coordinating efficient internalization of LDLR from the plasma membrane.”

The absence of LDLR peptides in the mass spectrometry dataset suggests that the Ral-LDLR interaction may be indirect, transient or highly dynamic.

“the absence of LDLR peptides in our mass spectrometry dataset suggests that this interaction may be indirect, transient or highly dynamic.”

The increase in Ral activity produced by HCD seems to be unrelated to insulin levels or RalGAP inactivation.

“the increase in activity produced by HCD seems to be unrelated to insulin levels or RalGAP inactivation”

Sustained Ral activation is a key modulator of LDLR stability and cholesterol homeostasis.

“These findings highlight the specificity and physiological relevance of sustained Ral activation as a key modulator of LDLR stability and cholesterol homeostasis.”

Sustained Ral activation promotes LDLR degradation through a new post-translational regulatory mechanism that operates independently of canonical LDLR degradation pathways.

“which in turn promotes LDLR degradation through a new post-translational regulatory mechanism that operates independently of canonical LDLR degradation pathways.”

RalBP1 serves as a scaffold to recruit REPS1 and other endocytic components, coordinating efficient internalization of LDLR from the plasma membrane.

“Activated Ral interacts with RalBP1, which serves as a scaffold to recruit REPS1 and other endocytic components, coordinating efficient internalization of LDLR from the plasma membrane.”

CTSA acts as a key downstream effector of Ral-mediated LDLR degradation.

“These results confirm that CTSA acts as a key downstream effector of Ral-mediated LDLR degradation”

Ral activation bypasses PCSK9 to promote lysosomal LDLR degradation.

“Ral activation bypasses PCSK9 to promote lysosomal LDLR degradation.”

High dietary cholesterol activates a Ral-dependent pathway to regulate LDLR trafficking and degradation in hepatocytes.

“Schematic model illustrating how high dietary cholesterol activates a Ral-dependent pathway to regulate LDLR trafficking and degradation in hepatocytes.”

Ral protein function depends crucially on activation kinetics.

“We note that Ral protein function depends crucially on activation kinetics.”