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Seipin oligomers can interact directly with AGPAT2 and lipin 1, physically scaffolding critical regulators of adipogenesis.

Talukder MM, Sim MF, O'Rahilly S, Edwardson JM, Rochford JJ - Mol Metab (2015)

Bottom Line: Our study provides the first direct molecular link between seipin and AGPAT2, two proteins whose disruption causes CGL.Moreover, it provides the first example of an interaction between seipin and another protein that causally influences a key aspect of adipogenesis.Together our data suggest that the critical role of seipin in adipogenesis may involve its capacity to juxtapose important regulators of this process in a multi-protein complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.

ABSTRACT

Objective: Disruption of the genes encoding either seipin or 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) causes severe congenital generalized lipodystrophy (CGL) in humans. However, the function of seipin in adipogenesis remains poorly defined. We demonstrated recently that seipin can bind the key adipogenic phosphatidic acid (PA) phosphatase lipin 1 and that seipin forms stable dodecamers. As AGPAT2 generates PA, the substrate for lipin 1, we investigated whether seipin might bind both enzymes of this lipid biosynthetic pathway, which is required for adipogenesis to occur.

Methods: We employed co-immunoprecipitation and immunofluorescence methods to determine whether seipin can interact with AGPAT2 and the consequences of this in developing adipocytes. Atomic force microscopy was used to determine whether these interactions involved direct association of the proteins and to define the molecular architecture of these complexes.

Results: Our data reveal that seipin can bind AGPAT2 during adipogenesis and that stabilizing this interaction during adipogenesis can increase the nuclear accumulation of PPARγ. Both AGPAT2 and lipin 1 can directly associate with seipin dodecamers, and a single seipin complex can simultaneously bind both AGPAT2 and lipin with a defined orientation.

Conclusions: Our study provides the first direct molecular link between seipin and AGPAT2, two proteins whose disruption causes CGL. Moreover, it provides the first example of an interaction between seipin and another protein that causally influences a key aspect of adipogenesis. Together our data suggest that the critical role of seipin in adipogenesis may involve its capacity to juxtapose important regulators of this process in a multi-protein complex.

No MeSH data available.


Related in: MedlinePlus

AFM analysis of lipin 1 and its interaction with seipin. (A) HA-lipin 1α was expressed in tsA 201 cells and isolated using anti-HA immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by either silver staining (left panel), or immunoblotting using either an anti-lipin 1 antibody (center panel) or an anti-HA antibody (right panel). The position of lipin 1 is indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Gallery of zoomed AFM images showing individual isolated lipin 1α particles. Scale bar, 25 nm; height scale, 0–1 nm. (C) Frequency distribution of volumes of the lipin 1α particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (D) FLAG-seipin-Myc and HA-lipin 1α were co-expressed in tsA 201 cells and proteins were isolated using anti-Myc immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-FLAG (left panel) or anti-lipin 1 (right panel) antibodies. Molecular mass markers (kDa) are shown at the right. (E) Control experiment in which two batches of cells separately expressing either FLAG-seipin-Myc or HA-lipin 1α were mixed immediately before solubilization, followed by anti-Myc immunoaffinity chromatography. Samples of both total cell lysate and immunoprecipitate were immunoblotted using either anti-FLAG (top panel) or anti-lipin 1 (bottom panel) antibodies. (F) Low-magnification AFM image of isolated proteins. The arrowhead indicates a large particle (seipin) decorated by two smaller particles (lipin 1α). Scale bar, 100 nm; height scale, 0–2 nm. (G) Gallery of zoomed images showing seipin particles decorated by either one (top panels) or two (bottom panels) lipin 1 particles. Scale bar, 25 nm; height scale, 0–2 nm. (H) Frequency distribution of volumes of the smaller (lipin 1) particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (I) Frequency distribution of volumes of the larger (seipin) particles. (J) Frequency distribution of angles between pairs of bound lipin 1 particles.
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fig4: AFM analysis of lipin 1 and its interaction with seipin. (A) HA-lipin 1α was expressed in tsA 201 cells and isolated using anti-HA immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by either silver staining (left panel), or immunoblotting using either an anti-lipin 1 antibody (center panel) or an anti-HA antibody (right panel). The position of lipin 1 is indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Gallery of zoomed AFM images showing individual isolated lipin 1α particles. Scale bar, 25 nm; height scale, 0–1 nm. (C) Frequency distribution of volumes of the lipin 1α particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (D) FLAG-seipin-Myc and HA-lipin 1α were co-expressed in tsA 201 cells and proteins were isolated using anti-Myc immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-FLAG (left panel) or anti-lipin 1 (right panel) antibodies. Molecular mass markers (kDa) are shown at the right. (E) Control experiment in which two batches of cells separately expressing either FLAG-seipin-Myc or HA-lipin 1α were mixed immediately before solubilization, followed by anti-Myc immunoaffinity chromatography. Samples of both total cell lysate and immunoprecipitate were immunoblotted using either anti-FLAG (top panel) or anti-lipin 1 (bottom panel) antibodies. (F) Low-magnification AFM image of isolated proteins. The arrowhead indicates a large particle (seipin) decorated by two smaller particles (lipin 1α). Scale bar, 100 nm; height scale, 0–2 nm. (G) Gallery of zoomed images showing seipin particles decorated by either one (top panels) or two (bottom panels) lipin 1 particles. Scale bar, 25 nm; height scale, 0–2 nm. (H) Frequency distribution of volumes of the smaller (lipin 1) particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (I) Frequency distribution of volumes of the larger (seipin) particles. (J) Frequency distribution of angles between pairs of bound lipin 1 particles.

Mentions: We next examined the association of lipin 1 with seipin that we have reported previously. HA-tagged lipin 1α isolated from transfected tsA 201 cells (Figure 4A) had a peak volume of 246 ± 16 nm3 (n = 100; Figure 4B,C), close to the expected volume of 227 nm3, and to the volume previously reported by others on the basis of AFM analysis [29]. FLAG-seipin-Myc and HA-lipin 1α could be co-isolated by anti-Myc immunoaffinity chromatography from co-transfected cells (Figure 4D), but no association was seen when individually transfected cells were mixed prior to lysis (Figure 4E). AFM imaging of the isolated proteins revealed large particles decorated by smaller peripheral particles (Figure 4F). Whilst single decoration events were the most common, double decorations were also observed. We found that 9.0% (50/557) of seipin particles were doubly decorated by lipin 1. Zoomed images of representative complexes are shown in Figure 4G. The peak molecular volume of the peripheral particles was 208 ± 7 nm3 (n = 116; Figure 4H), whilst the core particles had a peak volume of 2178 ± 12 nm3 (n = 58; Figure 4I). These sizes indicate that the complexes consist of seipin dodecamers with associated lipin 1 monomers. The distribution of angles between pairs of bound lipin 1 molecules had a peak at 114°±3° (n = 50; Figure 4J), suggesting that lipin 1 may preferentially bind to seipin subunits separated by two subunits in the dodecamers, although other configurations are clearly possible. Importantly, this analysis reveals that lipin 1 can bind directly to seipin dodecamers, and confirms that this interaction is specific and organized.


Seipin oligomers can interact directly with AGPAT2 and lipin 1, physically scaffolding critical regulators of adipogenesis.

Talukder MM, Sim MF, O'Rahilly S, Edwardson JM, Rochford JJ - Mol Metab (2015)

AFM analysis of lipin 1 and its interaction with seipin. (A) HA-lipin 1α was expressed in tsA 201 cells and isolated using anti-HA immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by either silver staining (left panel), or immunoblotting using either an anti-lipin 1 antibody (center panel) or an anti-HA antibody (right panel). The position of lipin 1 is indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Gallery of zoomed AFM images showing individual isolated lipin 1α particles. Scale bar, 25 nm; height scale, 0–1 nm. (C) Frequency distribution of volumes of the lipin 1α particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (D) FLAG-seipin-Myc and HA-lipin 1α were co-expressed in tsA 201 cells and proteins were isolated using anti-Myc immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-FLAG (left panel) or anti-lipin 1 (right panel) antibodies. Molecular mass markers (kDa) are shown at the right. (E) Control experiment in which two batches of cells separately expressing either FLAG-seipin-Myc or HA-lipin 1α were mixed immediately before solubilization, followed by anti-Myc immunoaffinity chromatography. Samples of both total cell lysate and immunoprecipitate were immunoblotted using either anti-FLAG (top panel) or anti-lipin 1 (bottom panel) antibodies. (F) Low-magnification AFM image of isolated proteins. The arrowhead indicates a large particle (seipin) decorated by two smaller particles (lipin 1α). Scale bar, 100 nm; height scale, 0–2 nm. (G) Gallery of zoomed images showing seipin particles decorated by either one (top panels) or two (bottom panels) lipin 1 particles. Scale bar, 25 nm; height scale, 0–2 nm. (H) Frequency distribution of volumes of the smaller (lipin 1) particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (I) Frequency distribution of volumes of the larger (seipin) particles. (J) Frequency distribution of angles between pairs of bound lipin 1 particles.
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fig4: AFM analysis of lipin 1 and its interaction with seipin. (A) HA-lipin 1α was expressed in tsA 201 cells and isolated using anti-HA immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by either silver staining (left panel), or immunoblotting using either an anti-lipin 1 antibody (center panel) or an anti-HA antibody (right panel). The position of lipin 1 is indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Gallery of zoomed AFM images showing individual isolated lipin 1α particles. Scale bar, 25 nm; height scale, 0–1 nm. (C) Frequency distribution of volumes of the lipin 1α particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (D) FLAG-seipin-Myc and HA-lipin 1α were co-expressed in tsA 201 cells and proteins were isolated using anti-Myc immunoaffinity chromatography. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-FLAG (left panel) or anti-lipin 1 (right panel) antibodies. Molecular mass markers (kDa) are shown at the right. (E) Control experiment in which two batches of cells separately expressing either FLAG-seipin-Myc or HA-lipin 1α were mixed immediately before solubilization, followed by anti-Myc immunoaffinity chromatography. Samples of both total cell lysate and immunoprecipitate were immunoblotted using either anti-FLAG (top panel) or anti-lipin 1 (bottom panel) antibodies. (F) Low-magnification AFM image of isolated proteins. The arrowhead indicates a large particle (seipin) decorated by two smaller particles (lipin 1α). Scale bar, 100 nm; height scale, 0–2 nm. (G) Gallery of zoomed images showing seipin particles decorated by either one (top panels) or two (bottom panels) lipin 1 particles. Scale bar, 25 nm; height scale, 0–2 nm. (H) Frequency distribution of volumes of the smaller (lipin 1) particles. The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (I) Frequency distribution of volumes of the larger (seipin) particles. (J) Frequency distribution of angles between pairs of bound lipin 1 particles.
Mentions: We next examined the association of lipin 1 with seipin that we have reported previously. HA-tagged lipin 1α isolated from transfected tsA 201 cells (Figure 4A) had a peak volume of 246 ± 16 nm3 (n = 100; Figure 4B,C), close to the expected volume of 227 nm3, and to the volume previously reported by others on the basis of AFM analysis [29]. FLAG-seipin-Myc and HA-lipin 1α could be co-isolated by anti-Myc immunoaffinity chromatography from co-transfected cells (Figure 4D), but no association was seen when individually transfected cells were mixed prior to lysis (Figure 4E). AFM imaging of the isolated proteins revealed large particles decorated by smaller peripheral particles (Figure 4F). Whilst single decoration events were the most common, double decorations were also observed. We found that 9.0% (50/557) of seipin particles were doubly decorated by lipin 1. Zoomed images of representative complexes are shown in Figure 4G. The peak molecular volume of the peripheral particles was 208 ± 7 nm3 (n = 116; Figure 4H), whilst the core particles had a peak volume of 2178 ± 12 nm3 (n = 58; Figure 4I). These sizes indicate that the complexes consist of seipin dodecamers with associated lipin 1 monomers. The distribution of angles between pairs of bound lipin 1 molecules had a peak at 114°±3° (n = 50; Figure 4J), suggesting that lipin 1 may preferentially bind to seipin subunits separated by two subunits in the dodecamers, although other configurations are clearly possible. Importantly, this analysis reveals that lipin 1 can bind directly to seipin dodecamers, and confirms that this interaction is specific and organized.

Bottom Line: Our study provides the first direct molecular link between seipin and AGPAT2, two proteins whose disruption causes CGL.Moreover, it provides the first example of an interaction between seipin and another protein that causally influences a key aspect of adipogenesis.Together our data suggest that the critical role of seipin in adipogenesis may involve its capacity to juxtapose important regulators of this process in a multi-protein complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.

ABSTRACT

Objective: Disruption of the genes encoding either seipin or 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) causes severe congenital generalized lipodystrophy (CGL) in humans. However, the function of seipin in adipogenesis remains poorly defined. We demonstrated recently that seipin can bind the key adipogenic phosphatidic acid (PA) phosphatase lipin 1 and that seipin forms stable dodecamers. As AGPAT2 generates PA, the substrate for lipin 1, we investigated whether seipin might bind both enzymes of this lipid biosynthetic pathway, which is required for adipogenesis to occur.

Methods: We employed co-immunoprecipitation and immunofluorescence methods to determine whether seipin can interact with AGPAT2 and the consequences of this in developing adipocytes. Atomic force microscopy was used to determine whether these interactions involved direct association of the proteins and to define the molecular architecture of these complexes.

Results: Our data reveal that seipin can bind AGPAT2 during adipogenesis and that stabilizing this interaction during adipogenesis can increase the nuclear accumulation of PPARγ. Both AGPAT2 and lipin 1 can directly associate with seipin dodecamers, and a single seipin complex can simultaneously bind both AGPAT2 and lipin with a defined orientation.

Conclusions: Our study provides the first direct molecular link between seipin and AGPAT2, two proteins whose disruption causes CGL. Moreover, it provides the first example of an interaction between seipin and another protein that causally influences a key aspect of adipogenesis. Together our data suggest that the critical role of seipin in adipogenesis may involve its capacity to juxtapose important regulators of this process in a multi-protein complex.

No MeSH data available.


Related in: MedlinePlus