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Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Huber MD, Vesely PW, Datta K, Gerace L - J. Cell Biol. (2013)

Bottom Line: Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins.Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery.We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

ABSTRACT
Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

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Validation of SREBP activation with erlin depletion. (A) SREBP-2 processing. HEK293 cells were transfected with FLAG-SREBP-2 and siRNA, and membrane (membr.) and nuclear extract (nuc. ex.) fractions were analyzed by Western blotting. Lipid-depleted control, second lane. Membrane fractions showing SREBP-2 precursor (P-SREBP-2; ∼120 kD; top) and nuclear extracts showing the processed form of SREBP-2 (N-SREBP-2; ∼60 kD; bottom). N-SREBP-2/P-SREBP-2 ratios, relative to the control, are indicated. (B) Nuclear accumulation of SREPB-2. HeLa cells were examined by immunofluorescence with an antibody to the N-terminal domain of endogenous SREBP-2 and scored for nuclear SREBP-2 enrichment (nuclear/cytoplasmic ratio >1.8; see Fig. S2). P-values for all comparisons to control ≤1.2 × 10−6; n = 3. (C) Inhibition of SREBP target gene activation with fatostatin. Sets of siRNA-transfected HeLa cultures were treated with either vehicle (DMSO) or fatostatin. Lipid-depleted control, second lane. Values shown are average ratios of relative mRNA levels in fatostatin- versus DMSO-treated samples. P-values for all comparisons to control <0.027; n = 3. (D) Insig turnover in erlin-depleted cells. HEK293 cells transfected with Insig-1-Myc and siRNAs were treated with cycloheximide (CHX) for the times indicated (and, where specified, with MG132) and analyzed by Western blotting. Shown are Western blots (top panels) and corresponding Ponceau S–stained membranes (Ponc.; bottom panels) of a typical experiment. Insig-1 signals were quantified, normalized to protein loaded, and plotted to determine t1/2 values for Insig-1 turnover. (right) Closed circles, control; open circles, erlin depleted. P-values for si-erlin versus si-control data points at 60–180 min are <0.003; n = 4. Error bars indicate standard deviations.
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fig2: Validation of SREBP activation with erlin depletion. (A) SREBP-2 processing. HEK293 cells were transfected with FLAG-SREBP-2 and siRNA, and membrane (membr.) and nuclear extract (nuc. ex.) fractions were analyzed by Western blotting. Lipid-depleted control, second lane. Membrane fractions showing SREBP-2 precursor (P-SREBP-2; ∼120 kD; top) and nuclear extracts showing the processed form of SREBP-2 (N-SREBP-2; ∼60 kD; bottom). N-SREBP-2/P-SREBP-2 ratios, relative to the control, are indicated. (B) Nuclear accumulation of SREPB-2. HeLa cells were examined by immunofluorescence with an antibody to the N-terminal domain of endogenous SREBP-2 and scored for nuclear SREBP-2 enrichment (nuclear/cytoplasmic ratio >1.8; see Fig. S2). P-values for all comparisons to control ≤1.2 × 10−6; n = 3. (C) Inhibition of SREBP target gene activation with fatostatin. Sets of siRNA-transfected HeLa cultures were treated with either vehicle (DMSO) or fatostatin. Lipid-depleted control, second lane. Values shown are average ratios of relative mRNA levels in fatostatin- versus DMSO-treated samples. P-values for all comparisons to control <0.027; n = 3. (D) Insig turnover in erlin-depleted cells. HEK293 cells transfected with Insig-1-Myc and siRNAs were treated with cycloheximide (CHX) for the times indicated (and, where specified, with MG132) and analyzed by Western blotting. Shown are Western blots (top panels) and corresponding Ponceau S–stained membranes (Ponc.; bottom panels) of a typical experiment. Insig-1 signals were quantified, normalized to protein loaded, and plotted to determine t1/2 values for Insig-1 turnover. (right) Closed circles, control; open circles, erlin depleted. P-values for si-erlin versus si-control data points at 60–180 min are <0.003; n = 4. Error bars indicate standard deviations.

Mentions: We next investigated whether the activation of SREBP target genes by erlin silencing under cholesterol sufficiency involves the canonical pathway that is normally induced by cholesterol depletion (Goldstein et al., 2006; Fig. 2). First, we found that silencing of erlin-1 and/or erlin-2 led to the appearance of proteolytically cleaved SREBP-2 in nuclear extracts, as seen with LD (Fig. 2 A). Correspondingly, SREBP-2 accumulated in the nucleus (Fig. 2 B and Fig. S2). Second, we determined that SREBP target gene activation with erlin silencing involves Scap. Fatostatin, a small molecule that binds to Scap and inhibits ER to Golgi transport of SREBP (Kamisuki et al., 2009), reduced SREBP target gene activation to similar extents for both erlin knockdown and LD (Fig. 2 C). Finally, knockdown of erlins increased the rate of proteasomal degradation of Insig-1 (Fig. 2 D), similar to the destabilization of Insig induced by cholesterol depletion (Gong et al., 2006). Together these data indicate that activation of SREBP target genes with erlin silencing occurs by the canonical Scap-mediated pathway. This implies that erlins inhibit the production of cholesterol by restraining SREBP activation. These cholesterol-limiting effects could be amplified by the ability of erlin-2 to promote ERAD of HMGR (Jo et al., 2011).


Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Huber MD, Vesely PW, Datta K, Gerace L - J. Cell Biol. (2013)

Validation of SREBP activation with erlin depletion. (A) SREBP-2 processing. HEK293 cells were transfected with FLAG-SREBP-2 and siRNA, and membrane (membr.) and nuclear extract (nuc. ex.) fractions were analyzed by Western blotting. Lipid-depleted control, second lane. Membrane fractions showing SREBP-2 precursor (P-SREBP-2; ∼120 kD; top) and nuclear extracts showing the processed form of SREBP-2 (N-SREBP-2; ∼60 kD; bottom). N-SREBP-2/P-SREBP-2 ratios, relative to the control, are indicated. (B) Nuclear accumulation of SREPB-2. HeLa cells were examined by immunofluorescence with an antibody to the N-terminal domain of endogenous SREBP-2 and scored for nuclear SREBP-2 enrichment (nuclear/cytoplasmic ratio >1.8; see Fig. S2). P-values for all comparisons to control ≤1.2 × 10−6; n = 3. (C) Inhibition of SREBP target gene activation with fatostatin. Sets of siRNA-transfected HeLa cultures were treated with either vehicle (DMSO) or fatostatin. Lipid-depleted control, second lane. Values shown are average ratios of relative mRNA levels in fatostatin- versus DMSO-treated samples. P-values for all comparisons to control <0.027; n = 3. (D) Insig turnover in erlin-depleted cells. HEK293 cells transfected with Insig-1-Myc and siRNAs were treated with cycloheximide (CHX) for the times indicated (and, where specified, with MG132) and analyzed by Western blotting. Shown are Western blots (top panels) and corresponding Ponceau S–stained membranes (Ponc.; bottom panels) of a typical experiment. Insig-1 signals were quantified, normalized to protein loaded, and plotted to determine t1/2 values for Insig-1 turnover. (right) Closed circles, control; open circles, erlin depleted. P-values for si-erlin versus si-control data points at 60–180 min are <0.003; n = 4. Error bars indicate standard deviations.
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fig2: Validation of SREBP activation with erlin depletion. (A) SREBP-2 processing. HEK293 cells were transfected with FLAG-SREBP-2 and siRNA, and membrane (membr.) and nuclear extract (nuc. ex.) fractions were analyzed by Western blotting. Lipid-depleted control, second lane. Membrane fractions showing SREBP-2 precursor (P-SREBP-2; ∼120 kD; top) and nuclear extracts showing the processed form of SREBP-2 (N-SREBP-2; ∼60 kD; bottom). N-SREBP-2/P-SREBP-2 ratios, relative to the control, are indicated. (B) Nuclear accumulation of SREPB-2. HeLa cells were examined by immunofluorescence with an antibody to the N-terminal domain of endogenous SREBP-2 and scored for nuclear SREBP-2 enrichment (nuclear/cytoplasmic ratio >1.8; see Fig. S2). P-values for all comparisons to control ≤1.2 × 10−6; n = 3. (C) Inhibition of SREBP target gene activation with fatostatin. Sets of siRNA-transfected HeLa cultures were treated with either vehicle (DMSO) or fatostatin. Lipid-depleted control, second lane. Values shown are average ratios of relative mRNA levels in fatostatin- versus DMSO-treated samples. P-values for all comparisons to control <0.027; n = 3. (D) Insig turnover in erlin-depleted cells. HEK293 cells transfected with Insig-1-Myc and siRNAs were treated with cycloheximide (CHX) for the times indicated (and, where specified, with MG132) and analyzed by Western blotting. Shown are Western blots (top panels) and corresponding Ponceau S–stained membranes (Ponc.; bottom panels) of a typical experiment. Insig-1 signals were quantified, normalized to protein loaded, and plotted to determine t1/2 values for Insig-1 turnover. (right) Closed circles, control; open circles, erlin depleted. P-values for si-erlin versus si-control data points at 60–180 min are <0.003; n = 4. Error bars indicate standard deviations.
Mentions: We next investigated whether the activation of SREBP target genes by erlin silencing under cholesterol sufficiency involves the canonical pathway that is normally induced by cholesterol depletion (Goldstein et al., 2006; Fig. 2). First, we found that silencing of erlin-1 and/or erlin-2 led to the appearance of proteolytically cleaved SREBP-2 in nuclear extracts, as seen with LD (Fig. 2 A). Correspondingly, SREBP-2 accumulated in the nucleus (Fig. 2 B and Fig. S2). Second, we determined that SREBP target gene activation with erlin silencing involves Scap. Fatostatin, a small molecule that binds to Scap and inhibits ER to Golgi transport of SREBP (Kamisuki et al., 2009), reduced SREBP target gene activation to similar extents for both erlin knockdown and LD (Fig. 2 C). Finally, knockdown of erlins increased the rate of proteasomal degradation of Insig-1 (Fig. 2 D), similar to the destabilization of Insig induced by cholesterol depletion (Gong et al., 2006). Together these data indicate that activation of SREBP target genes with erlin silencing occurs by the canonical Scap-mediated pathway. This implies that erlins inhibit the production of cholesterol by restraining SREBP activation. These cholesterol-limiting effects could be amplified by the ability of erlin-2 to promote ERAD of HMGR (Jo et al., 2011).

Bottom Line: Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins.Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery.We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

ABSTRACT
Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

Show MeSH