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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

Seipin forms a triple complex with AGPAT2 and lipin 1. (A) Seipin-Myc, FLAG-AGPAT2 and HA-lipin 1α were co-expressed in tsA 201 cells. A detergent extract of the cells was subjected to sequential anti-FLAG (IP1) and anti-Myc (IP2) immunoaffinity chromatography steps. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-Myc (seipin; top panels), anti-FLAG (AGPAT2; centre panels) or anti-lipin 1 (bottom panels) antibodies. The positions of the three proteins are indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Control experiment in which three batches of cells separately expressing either seipin-Myc, FLAG-AGPAT2 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-Myc (top panel), anti-FLAG (centre panel) or anti-lipin 1 (bottom panel) antibodies. The positions of seipin, AGPAT2 and lipin 1 are indicated at the left, and molecular mass markers (kDa) are shown at the right. (C) Low-magnification AFM image of proteins isolated using sequential immunoaffinity chromatography, as in (A). The arrowhead indicates a large particle (seipin) decorated by two differently-sized smaller particles (AGPAT2 and lipin 1). Scale bar, 100 nm; height scale, 0–2 nm. (D) Gallery of zoomed images showing seipin particles decorated by two differently-sized smaller particles. Scale bar, 25 nm; height scale, 0–2 nm. (E) Frequency distribution of volumes of the smaller particles (AGPAT2 and lipin 1). The curve indicates the fitted Gaussian functions. The peaks of the distribution (±SEM) are indicated. (F) Frequency distribution of the larger (seipin) particles. (G) Frequency distribution of angles between pairs of bound smaller particles. (H) tsA 201 cells were co-transfected with FLAG-AGPAT2 and HA-lipin 1α in the absence or presence of wild-type seipin (WT) or seipin lacking either the cytoplasmic C-terminus (ΔCT) or the first transmembrane domain (ΔTM1). FLAG-AGPAT2 was immunoprecipitated and lysates or immunoprecipitates (IP) were immunoblotted to determine the levels of FLAG-AGPAT2 and associated Myc-seipin or lipin 1α using anti-FLAG, anti-Myc and anti-lipin 1 antibodies, as indicated. (I) The levels of lipin 1α associated with immunoprecipitated AGPAT2 in the absence or presence of wild-type or mutant seipin proteins were determined in three independent experiments. Data shown are means ± SEM of co-precipitated lipin 1α normalized to levels of co-precipitated AGPAT2. ** indicates difference from levels of lipin 1 associated with AGPAT2 in the presence of WT seipin (p < 0.01).
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fig5: Seipin forms a triple complex with AGPAT2 and lipin 1. (A) Seipin-Myc, FLAG-AGPAT2 and HA-lipin 1α were co-expressed in tsA 201 cells. A detergent extract of the cells was subjected to sequential anti-FLAG (IP1) and anti-Myc (IP2) immunoaffinity chromatography steps. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-Myc (seipin; top panels), anti-FLAG (AGPAT2; centre panels) or anti-lipin 1 (bottom panels) antibodies. The positions of the three proteins are indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Control experiment in which three batches of cells separately expressing either seipin-Myc, FLAG-AGPAT2 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-Myc (top panel), anti-FLAG (centre panel) or anti-lipin 1 (bottom panel) antibodies. The positions of seipin, AGPAT2 and lipin 1 are indicated at the left, and molecular mass markers (kDa) are shown at the right. (C) Low-magnification AFM image of proteins isolated using sequential immunoaffinity chromatography, as in (A). The arrowhead indicates a large particle (seipin) decorated by two differently-sized smaller particles (AGPAT2 and lipin 1). Scale bar, 100 nm; height scale, 0–2 nm. (D) Gallery of zoomed images showing seipin particles decorated by two differently-sized smaller particles. Scale bar, 25 nm; height scale, 0–2 nm. (E) Frequency distribution of volumes of the smaller particles (AGPAT2 and lipin 1). The curve indicates the fitted Gaussian functions. The peaks of the distribution (±SEM) are indicated. (F) Frequency distribution of the larger (seipin) particles. (G) Frequency distribution of angles between pairs of bound smaller particles. (H) tsA 201 cells were co-transfected with FLAG-AGPAT2 and HA-lipin 1α in the absence or presence of wild-type seipin (WT) or seipin lacking either the cytoplasmic C-terminus (ΔCT) or the first transmembrane domain (ΔTM1). FLAG-AGPAT2 was immunoprecipitated and lysates or immunoprecipitates (IP) were immunoblotted to determine the levels of FLAG-AGPAT2 and associated Myc-seipin or lipin 1α using anti-FLAG, anti-Myc and anti-lipin 1 antibodies, as indicated. (I) The levels of lipin 1α associated with immunoprecipitated AGPAT2 in the absence or presence of wild-type or mutant seipin proteins were determined in three independent experiments. Data shown are means ± SEM of co-precipitated lipin 1α normalized to levels of co-precipitated AGPAT2. ** indicates difference from levels of lipin 1 associated with AGPAT2 in the presence of WT seipin (p < 0.01).

Mentions: The significantly different molecular volumes of AGPAT2 and lipin 1 allowed us to use AFM to assess whether seipin dodecamers might simultaneously bind both AGPAT2 and lipin 1. Both seipin-Myc and lipin 1 could be detected in immunoprecipitates of FLAG-AGPAT2 from cells co-transfected with seipin-Myc, FLAG-AGPAT2 and HA-lipin 1α (Figure 5A, IP1). After elution of bound proteins a second, anti-Myc immunoprecipitation was performed to enrich for seipin-Myc-associated AGPAT2. Both FLAG-AGPAT2 and HA-lipin 1α were detected in this second eluate (Figure 5A, IP2), indicating a three-way interaction between seipin, AGPAT2 and lipin 1. Importantly, an anti-Myc IP did not precipitate either FLAG-AGPAT2 or HA-lipin 1α when singly transfected cells were mixed prior to lysis (Figure 5B). AFM analysis of the sequentially purified complexes revealed multiple seipin dodecamers doubly decorated by a large and a small peripheral particle, consistent with the binding of both AGPAT2 and lipin 1 (Figure 5C). Zoomed images are shown in Figure 5D. We found that 10.1% (50/496) of seipin particles were doubly decorated by AGPAT2 plus lipin 1. Volume analysis of the peripheral particles revealed two peaks, at 64 ± 2 nm3, consistent with the volume of AGPAT2, and 292 ± 7 nm3, consistent with the volume of lipin 1 (n = 132; Figure 5E). The core of the complex had a molecular volume of 2435 ± 29 nm3 (n = 66), consistent with that of seipin dodecamers (Figure 5F). Together these data provide strong evidence that both lipin 1 and AGPAT2 can associate with the same seipin oligomer. The distribution of angles between the seipin-associated lipin and AGPAT2 particles had a peak at 78 ± 8° (n = 50; Figure 5G), suggesting that there may be some preference for a selective distribution of these proteins around the seipin oligomer. Interestingly, this arrangement would imply that AGPAT2 and lipin 1 could not be simultaneously bound by the A212P and L91P pathogenic mutants of seipin which we have previously shown do not appropriately form dodecamers and instead typically assemble into tetramers [24]


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)

Seipin forms a triple complex with AGPAT2 and lipin 1. (A) Seipin-Myc, FLAG-AGPAT2 and HA-lipin 1α were co-expressed in tsA 201 cells. A detergent extract of the cells was subjected to sequential anti-FLAG (IP1) and anti-Myc (IP2) immunoaffinity chromatography steps. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-Myc (seipin; top panels), anti-FLAG (AGPAT2; centre panels) or anti-lipin 1 (bottom panels) antibodies. The positions of the three proteins are indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Control experiment in which three batches of cells separately expressing either seipin-Myc, FLAG-AGPAT2 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-Myc (top panel), anti-FLAG (centre panel) or anti-lipin 1 (bottom panel) antibodies. The positions of seipin, AGPAT2 and lipin 1 are indicated at the left, and molecular mass markers (kDa) are shown at the right. (C) Low-magnification AFM image of proteins isolated using sequential immunoaffinity chromatography, as in (A). The arrowhead indicates a large particle (seipin) decorated by two differently-sized smaller particles (AGPAT2 and lipin 1). Scale bar, 100 nm; height scale, 0–2 nm. (D) Gallery of zoomed images showing seipin particles decorated by two differently-sized smaller particles. Scale bar, 25 nm; height scale, 0–2 nm. (E) Frequency distribution of volumes of the smaller particles (AGPAT2 and lipin 1). The curve indicates the fitted Gaussian functions. The peaks of the distribution (±SEM) are indicated. (F) Frequency distribution of the larger (seipin) particles. (G) Frequency distribution of angles between pairs of bound smaller particles. (H) tsA 201 cells were co-transfected with FLAG-AGPAT2 and HA-lipin 1α in the absence or presence of wild-type seipin (WT) or seipin lacking either the cytoplasmic C-terminus (ΔCT) or the first transmembrane domain (ΔTM1). FLAG-AGPAT2 was immunoprecipitated and lysates or immunoprecipitates (IP) were immunoblotted to determine the levels of FLAG-AGPAT2 and associated Myc-seipin or lipin 1α using anti-FLAG, anti-Myc and anti-lipin 1 antibodies, as indicated. (I) The levels of lipin 1α associated with immunoprecipitated AGPAT2 in the absence or presence of wild-type or mutant seipin proteins were determined in three independent experiments. Data shown are means ± SEM of co-precipitated lipin 1α normalized to levels of co-precipitated AGPAT2. ** indicates difference from levels of lipin 1 associated with AGPAT2 in the presence of WT seipin (p < 0.01).
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fig5: Seipin forms a triple complex with AGPAT2 and lipin 1. (A) Seipin-Myc, FLAG-AGPAT2 and HA-lipin 1α were co-expressed in tsA 201 cells. A detergent extract of the cells was subjected to sequential anti-FLAG (IP1) and anti-Myc (IP2) immunoaffinity chromatography steps. Isolated protein was analyzed by SDS-PAGE followed by immunoblotting using either anti-Myc (seipin; top panels), anti-FLAG (AGPAT2; centre panels) or anti-lipin 1 (bottom panels) antibodies. The positions of the three proteins are indicated at the left, and molecular mass markers (kDa) are shown at the right. (B) Control experiment in which three batches of cells separately expressing either seipin-Myc, FLAG-AGPAT2 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-Myc (top panel), anti-FLAG (centre panel) or anti-lipin 1 (bottom panel) antibodies. The positions of seipin, AGPAT2 and lipin 1 are indicated at the left, and molecular mass markers (kDa) are shown at the right. (C) Low-magnification AFM image of proteins isolated using sequential immunoaffinity chromatography, as in (A). The arrowhead indicates a large particle (seipin) decorated by two differently-sized smaller particles (AGPAT2 and lipin 1). Scale bar, 100 nm; height scale, 0–2 nm. (D) Gallery of zoomed images showing seipin particles decorated by two differently-sized smaller particles. Scale bar, 25 nm; height scale, 0–2 nm. (E) Frequency distribution of volumes of the smaller particles (AGPAT2 and lipin 1). The curve indicates the fitted Gaussian functions. The peaks of the distribution (±SEM) are indicated. (F) Frequency distribution of the larger (seipin) particles. (G) Frequency distribution of angles between pairs of bound smaller particles. (H) tsA 201 cells were co-transfected with FLAG-AGPAT2 and HA-lipin 1α in the absence or presence of wild-type seipin (WT) or seipin lacking either the cytoplasmic C-terminus (ΔCT) or the first transmembrane domain (ΔTM1). FLAG-AGPAT2 was immunoprecipitated and lysates or immunoprecipitates (IP) were immunoblotted to determine the levels of FLAG-AGPAT2 and associated Myc-seipin or lipin 1α using anti-FLAG, anti-Myc and anti-lipin 1 antibodies, as indicated. (I) The levels of lipin 1α associated with immunoprecipitated AGPAT2 in the absence or presence of wild-type or mutant seipin proteins were determined in three independent experiments. Data shown are means ± SEM of co-precipitated lipin 1α normalized to levels of co-precipitated AGPAT2. ** indicates difference from levels of lipin 1 associated with AGPAT2 in the presence of WT seipin (p < 0.01).
Mentions: The significantly different molecular volumes of AGPAT2 and lipin 1 allowed us to use AFM to assess whether seipin dodecamers might simultaneously bind both AGPAT2 and lipin 1. Both seipin-Myc and lipin 1 could be detected in immunoprecipitates of FLAG-AGPAT2 from cells co-transfected with seipin-Myc, FLAG-AGPAT2 and HA-lipin 1α (Figure 5A, IP1). After elution of bound proteins a second, anti-Myc immunoprecipitation was performed to enrich for seipin-Myc-associated AGPAT2. Both FLAG-AGPAT2 and HA-lipin 1α were detected in this second eluate (Figure 5A, IP2), indicating a three-way interaction between seipin, AGPAT2 and lipin 1. Importantly, an anti-Myc IP did not precipitate either FLAG-AGPAT2 or HA-lipin 1α when singly transfected cells were mixed prior to lysis (Figure 5B). AFM analysis of the sequentially purified complexes revealed multiple seipin dodecamers doubly decorated by a large and a small peripheral particle, consistent with the binding of both AGPAT2 and lipin 1 (Figure 5C). Zoomed images are shown in Figure 5D. We found that 10.1% (50/496) of seipin particles were doubly decorated by AGPAT2 plus lipin 1. Volume analysis of the peripheral particles revealed two peaks, at 64 ± 2 nm3, consistent with the volume of AGPAT2, and 292 ± 7 nm3, consistent with the volume of lipin 1 (n = 132; Figure 5E). The core of the complex had a molecular volume of 2435 ± 29 nm3 (n = 66), consistent with that of seipin dodecamers (Figure 5F). Together these data provide strong evidence that both lipin 1 and AGPAT2 can associate with the same seipin oligomer. The distribution of angles between the seipin-associated lipin and AGPAT2 particles had a peak at 78 ± 8° (n = 50; Figure 5G), suggesting that there may be some preference for a selective distribution of these proteins around the seipin oligomer. Interestingly, this arrangement would imply that AGPAT2 and lipin 1 could not be simultaneously bound by the A212P and L91P pathogenic mutants of seipin which we have previously shown do not appropriately form dodecamers and instead typically assemble into tetramers [24]

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