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Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms.

Sim MF, Talukder MM, Dennis RJ, O'Rahilly S, Edwardson JM, Rochford JJ - Diabetologia (2013)

Bottom Line: Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts.We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not.Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK.

ABSTRACT

Aims/hypothesis: In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL.

Methods: We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations.

Results: We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not.

Conclusions/interpretation: Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.

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Related in: MedlinePlus

The R275X mutation in seipin leads to loss of protein expression in differentiating adipocyte precursors. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin bearing N-terminal triple-FLAG and C-terminal Myc tags (WT) or identically tagged forms with the point mutations E113X, R138X, R275X or Q391X. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG), Myc (α-Myc) and calnexin (α-Calnexin). (b) Confluent C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days; lysates were separated by SDS-PAGE and immunoblotted for FLAG, Myc and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, E113X, R138X, R275X or Q391X forms of seipin protein in C3H10T1/2 cells. FLAG–seipin bands from replicated blots as shown in (b) were normalised to calnexin expression in the same samples and expressed as means ± SEM, n = 3. *p < 0.05 vs WT. (d) C3H10T1/2 cells were transfected with empty vector (m) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days, and expression of mRNA encoding transfected human seipin (hBSCL2 mRNA) was determined by real-time PCR. Data are normalised to cyclophilin A and expressed as means ± SEM, n = 4. (e) Subconfluent C3H10T1/2 pre-adipocytes were transfected with epitope-tagged WT or mutant forms of seipin, fixed and immunostained for seipin using anti-FLAG antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale, and merged images show overlay of FLAG–seipin (green) and calnexin (red). Scale bars, 10 μm
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Fig1: The R275X mutation in seipin leads to loss of protein expression in differentiating adipocyte precursors. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin bearing N-terminal triple-FLAG and C-terminal Myc tags (WT) or identically tagged forms with the point mutations E113X, R138X, R275X or Q391X. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG), Myc (α-Myc) and calnexin (α-Calnexin). (b) Confluent C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days; lysates were separated by SDS-PAGE and immunoblotted for FLAG, Myc and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, E113X, R138X, R275X or Q391X forms of seipin protein in C3H10T1/2 cells. FLAG–seipin bands from replicated blots as shown in (b) were normalised to calnexin expression in the same samples and expressed as means ± SEM, n = 3. *p < 0.05 vs WT. (d) C3H10T1/2 cells were transfected with empty vector (m) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days, and expression of mRNA encoding transfected human seipin (hBSCL2 mRNA) was determined by real-time PCR. Data are normalised to cyclophilin A and expressed as means ± SEM, n = 4. (e) Subconfluent C3H10T1/2 pre-adipocytes were transfected with epitope-tagged WT or mutant forms of seipin, fixed and immunostained for seipin using anti-FLAG antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale, and merged images show overlay of FLAG–seipin (green) and calnexin (red). Scale bars, 10 μm

Mentions: To examine how premature stop mutations may affect the function of seipin, we generated constructs in which the E113X, R138X, R275X and Q391X pathogenic mutations [2, 3, 21] were introduced by site-directed mutagenesis into the full-length cDNA encoding the long form of seipin bearing an N-terminal triple-FLAG tag and a C-terminal Myc tag. These constructs were transfected into HEK293 cells to ensure maximal transfection efficiency and protein abundance and each resulted in the expression of proteins of the expected sizes when probed with antibodies to detect the triple-FLAG tag at their N-termini (Fig. 1a). As predicted, only wild-type seipin resulted in the expression of a protein that could be detected via the C-terminal Myc tag, confirming that each pathogenic mutation caused the expected premature stop in translation.Fig. 1


Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms.

Sim MF, Talukder MM, Dennis RJ, O'Rahilly S, Edwardson JM, Rochford JJ - Diabetologia (2013)

The R275X mutation in seipin leads to loss of protein expression in differentiating adipocyte precursors. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin bearing N-terminal triple-FLAG and C-terminal Myc tags (WT) or identically tagged forms with the point mutations E113X, R138X, R275X or Q391X. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG), Myc (α-Myc) and calnexin (α-Calnexin). (b) Confluent C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days; lysates were separated by SDS-PAGE and immunoblotted for FLAG, Myc and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, E113X, R138X, R275X or Q391X forms of seipin protein in C3H10T1/2 cells. FLAG–seipin bands from replicated blots as shown in (b) were normalised to calnexin expression in the same samples and expressed as means ± SEM, n = 3. *p < 0.05 vs WT. (d) C3H10T1/2 cells were transfected with empty vector (m) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days, and expression of mRNA encoding transfected human seipin (hBSCL2 mRNA) was determined by real-time PCR. Data are normalised to cyclophilin A and expressed as means ± SEM, n = 4. (e) Subconfluent C3H10T1/2 pre-adipocytes were transfected with epitope-tagged WT or mutant forms of seipin, fixed and immunostained for seipin using anti-FLAG antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale, and merged images show overlay of FLAG–seipin (green) and calnexin (red). Scale bars, 10 μm
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Related In: Results  -  Collection

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Fig1: The R275X mutation in seipin leads to loss of protein expression in differentiating adipocyte precursors. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin bearing N-terminal triple-FLAG and C-terminal Myc tags (WT) or identically tagged forms with the point mutations E113X, R138X, R275X or Q391X. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG), Myc (α-Myc) and calnexin (α-Calnexin). (b) Confluent C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days; lysates were separated by SDS-PAGE and immunoblotted for FLAG, Myc and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, E113X, R138X, R275X or Q391X forms of seipin protein in C3H10T1/2 cells. FLAG–seipin bands from replicated blots as shown in (b) were normalised to calnexin expression in the same samples and expressed as means ± SEM, n = 3. *p < 0.05 vs WT. (d) C3H10T1/2 cells were transfected with empty vector (m) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days, and expression of mRNA encoding transfected human seipin (hBSCL2 mRNA) was determined by real-time PCR. Data are normalised to cyclophilin A and expressed as means ± SEM, n = 4. (e) Subconfluent C3H10T1/2 pre-adipocytes were transfected with epitope-tagged WT or mutant forms of seipin, fixed and immunostained for seipin using anti-FLAG antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale, and merged images show overlay of FLAG–seipin (green) and calnexin (red). Scale bars, 10 μm
Mentions: To examine how premature stop mutations may affect the function of seipin, we generated constructs in which the E113X, R138X, R275X and Q391X pathogenic mutations [2, 3, 21] were introduced by site-directed mutagenesis into the full-length cDNA encoding the long form of seipin bearing an N-terminal triple-FLAG tag and a C-terminal Myc tag. These constructs were transfected into HEK293 cells to ensure maximal transfection efficiency and protein abundance and each resulted in the expression of proteins of the expected sizes when probed with antibodies to detect the triple-FLAG tag at their N-termini (Fig. 1a). As predicted, only wild-type seipin resulted in the expression of a protein that could be detected via the C-terminal Myc tag, confirming that each pathogenic mutation caused the expected premature stop in translation.Fig. 1

Bottom Line: Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts.We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not.Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK.

ABSTRACT

Aims/hypothesis: In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL.

Methods: We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations.

Results: We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not.

Conclusions/interpretation: Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.

Show MeSH
Related in: MedlinePlus