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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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Cholesterol binding by erlins. (A) Structures of cholesterol and related compounds used. (B) Analysis of purified recombinant erlin-1 with cholesterol binding and competition. Cholesterol binding curve (left), competition of 5 µM of radiolabeled cholesterol by 50 µM of the indicated nonlabeled compounds (middle), and analysis of purified erlin-1-V5 by SDS-PAGE and Coomassie blue staining (right). Shown is representative material eluted from anti-V5 beads (left lane) and control IgG beads (right lane). Densitometry established >90% purity for erlin-1-V5. Error bars indicate standard deviations. (C) Binding and competition of cholesterol to the erlin complex from mouse liver microsomal membranes. Left and middle panels are the same as in B. (right) SDS-PAGE with silver staining of material eluted from anti–erlin-2 beads (left lane) and control beads (right lane). The bracketed gel areas were analyzed by mass spectrometry. Erlin-1 (14.2% sequence coverage) and erlin-2 (18.2% sequence coverage) were found only in the anti–erlin-2 sample.
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fig4: Cholesterol binding by erlins. (A) Structures of cholesterol and related compounds used. (B) Analysis of purified recombinant erlin-1 with cholesterol binding and competition. Cholesterol binding curve (left), competition of 5 µM of radiolabeled cholesterol by 50 µM of the indicated nonlabeled compounds (middle), and analysis of purified erlin-1-V5 by SDS-PAGE and Coomassie blue staining (right). Shown is representative material eluted from anti-V5 beads (left lane) and control IgG beads (right lane). Densitometry established >90% purity for erlin-1-V5. Error bars indicate standard deviations. (C) Binding and competition of cholesterol to the erlin complex from mouse liver microsomal membranes. Left and middle panels are the same as in B. (right) SDS-PAGE with silver staining of material eluted from anti–erlin-2 beads (left lane) and control beads (right lane). The bracketed gel areas were analyzed by mass spectrometry. Erlin-1 (14.2% sequence coverage) and erlin-2 (18.2% sequence coverage) were found only in the anti–erlin-2 sample.

Mentions: Because SREBP activation is modulated by binding of cholesterol to Scap (Goldstein et al., 2006), and because erlin interactions with the SREBP machinery appear to be sensitive to changes in cholesterol (Fig. 3 A), we examined whether cholesterol binds to erlins (Fig. 4; see Materials and methods). We found that cholesterol bound to recombinant erlin-1 with an apparent Kd of 13.2 µM and approximately six molecules of cholesterol per erlin monomer at saturation (Fig. 4 B). The binding curve fit best to a sigmoidal function (r2 = 0.98) with a Hill coefficient of 4.05, indicating a high degree of binding cooperativity. The binding of radiolabeled cholesterol to erlin-1 was specific, as it was strongly diminished by a 10-fold excess of unlabeled cholesterol, epicholesterol, or lanosterol, but was not substantially reduced by progesterone, 25-hydroxycholesterol, β-sitosterol, or squalene (Fig. 4 B). We also found that the native erlin-1/2 complex isolated from murine liver microsomal membranes saturably bound cholesterol (Fig. 4 C; apparent Kd = 13.4 µM; approximately four molecules of cholesterol bound per molecule of erlin monomer at saturation). Here the binding curve was best described by a sigmoidal function (r2 = 0.91) with a Hill coefficient of 3.47. Competition assays showed sterol binding specificity similar to recombinant erlin-1 (Fig. 4 C). Supporting our results, erlin-2 was specifically labeled with a photoreactive cholesterol analogue in a proteome-wide screen (Hulce et al., 2013). Cholesterol binding by erlins may occur outside the lipid bilayer as suggested for NPC1 (Infante et al., 2008a) and Scap (Motamed et al., 2011) or may involve a segment of erlins with close bilayer interaction.


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

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

Cholesterol binding by erlins. (A) Structures of cholesterol and related compounds used. (B) Analysis of purified recombinant erlin-1 with cholesterol binding and competition. Cholesterol binding curve (left), competition of 5 µM of radiolabeled cholesterol by 50 µM of the indicated nonlabeled compounds (middle), and analysis of purified erlin-1-V5 by SDS-PAGE and Coomassie blue staining (right). Shown is representative material eluted from anti-V5 beads (left lane) and control IgG beads (right lane). Densitometry established >90% purity for erlin-1-V5. Error bars indicate standard deviations. (C) Binding and competition of cholesterol to the erlin complex from mouse liver microsomal membranes. Left and middle panels are the same as in B. (right) SDS-PAGE with silver staining of material eluted from anti–erlin-2 beads (left lane) and control beads (right lane). The bracketed gel areas were analyzed by mass spectrometry. Erlin-1 (14.2% sequence coverage) and erlin-2 (18.2% sequence coverage) were found only in the anti–erlin-2 sample.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824017&req=5

fig4: Cholesterol binding by erlins. (A) Structures of cholesterol and related compounds used. (B) Analysis of purified recombinant erlin-1 with cholesterol binding and competition. Cholesterol binding curve (left), competition of 5 µM of radiolabeled cholesterol by 50 µM of the indicated nonlabeled compounds (middle), and analysis of purified erlin-1-V5 by SDS-PAGE and Coomassie blue staining (right). Shown is representative material eluted from anti-V5 beads (left lane) and control IgG beads (right lane). Densitometry established >90% purity for erlin-1-V5. Error bars indicate standard deviations. (C) Binding and competition of cholesterol to the erlin complex from mouse liver microsomal membranes. Left and middle panels are the same as in B. (right) SDS-PAGE with silver staining of material eluted from anti–erlin-2 beads (left lane) and control beads (right lane). The bracketed gel areas were analyzed by mass spectrometry. Erlin-1 (14.2% sequence coverage) and erlin-2 (18.2% sequence coverage) were found only in the anti–erlin-2 sample.
Mentions: Because SREBP activation is modulated by binding of cholesterol to Scap (Goldstein et al., 2006), and because erlin interactions with the SREBP machinery appear to be sensitive to changes in cholesterol (Fig. 3 A), we examined whether cholesterol binds to erlins (Fig. 4; see Materials and methods). We found that cholesterol bound to recombinant erlin-1 with an apparent Kd of 13.2 µM and approximately six molecules of cholesterol per erlin monomer at saturation (Fig. 4 B). The binding curve fit best to a sigmoidal function (r2 = 0.98) with a Hill coefficient of 4.05, indicating a high degree of binding cooperativity. The binding of radiolabeled cholesterol to erlin-1 was specific, as it was strongly diminished by a 10-fold excess of unlabeled cholesterol, epicholesterol, or lanosterol, but was not substantially reduced by progesterone, 25-hydroxycholesterol, β-sitosterol, or squalene (Fig. 4 B). We also found that the native erlin-1/2 complex isolated from murine liver microsomal membranes saturably bound cholesterol (Fig. 4 C; apparent Kd = 13.4 µM; approximately four molecules of cholesterol bound per molecule of erlin monomer at saturation). Here the binding curve was best described by a sigmoidal function (r2 = 0.91) with a Hill coefficient of 3.47. Competition assays showed sterol binding specificity similar to recombinant erlin-1 (Fig. 4 C). Supporting our results, erlin-2 was specifically labeled with a photoreactive cholesterol analogue in a proteome-wide screen (Hulce et al., 2013). Cholesterol binding by erlins may occur outside the lipid bilayer as suggested for NPC1 (Infante et al., 2008a) and Scap (Motamed et al., 2011) or may involve a segment of erlins with close bilayer interaction.

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