Limits...
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.

Show MeSH

Related in: MedlinePlus

The pathogenic point mutations T78A, L91P and A212P do not affect seipin protein expression, but L91P and A212P seipin partially mislocalise to the nuclear envelope. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin with N-terminal triple-FLAG and C-terminal Myc tags (WT) or with identically tagged T78A, L91P or A212P forms. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG) and calnexin (α-Calnexin). (b) C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Lysates were separated by SDS-PAGE and immunoblotted for FLAG and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, T78A, L91P or A212P 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) 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 and were fixed and immunostained for seipin using anti-Myc antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale and merged images show overlay of Myc–seipin (green) and calnexin (red). Scale bars, 10 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3824349&req=5

Fig2: The pathogenic point mutations T78A, L91P and A212P do not affect seipin protein expression, but L91P and A212P seipin partially mislocalise to the nuclear envelope. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin with N-terminal triple-FLAG and C-terminal Myc tags (WT) or with identically tagged T78A, L91P or A212P forms. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG) and calnexin (α-Calnexin). (b) C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Lysates were separated by SDS-PAGE and immunoblotted for FLAG and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, T78A, L91P or A212P 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) 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 and were fixed and immunostained for seipin using anti-Myc antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale and merged images show overlay of Myc–seipin (green) and calnexin (red). Scale bars, 10 μm

Mentions: We next examined the effect of the T78A, L91P and A212P mutations on seipin expression. When each of these constructs was transfected into either HEK293 cells (Fig. 2a) or C3H10T1/2 cells (Fig. 2b), seipin was robustly expressed. However, we observed that the L91P and A212P mutations did result in a modest reduction in protein expression when compared with wild-type or T78A seipin (Fig. 2c). This was not the result of a significant effect of these mutations on mRNA expression levels (Fig. 2d).Fig. 2


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 pathogenic point mutations T78A, L91P and A212P do not affect seipin protein expression, but L91P and A212P seipin partially mislocalise to the nuclear envelope. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin with N-terminal triple-FLAG and C-terminal Myc tags (WT) or with identically tagged T78A, L91P or A212P forms. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG) and calnexin (α-Calnexin). (b) C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Lysates were separated by SDS-PAGE and immunoblotted for FLAG and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, T78A, L91P or A212P 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) 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 and were fixed and immunostained for seipin using anti-Myc antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale and merged images show overlay of Myc–seipin (green) and calnexin (red). Scale bars, 10 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824349&req=5

Fig2: The pathogenic point mutations T78A, L91P and A212P do not affect seipin protein expression, but L91P and A212P seipin partially mislocalise to the nuclear envelope. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin with N-terminal triple-FLAG and C-terminal Myc tags (WT) or with identically tagged T78A, L91P or A212P forms. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG) and calnexin (α-Calnexin). (b) C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Lysates were separated by SDS-PAGE and immunoblotted for FLAG and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, T78A, L91P or A212P 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) 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 and were fixed and immunostained for seipin using anti-Myc antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale and merged images show overlay of Myc–seipin (green) and calnexin (red). Scale bars, 10 μm
Mentions: We next examined the effect of the T78A, L91P and A212P mutations on seipin expression. When each of these constructs was transfected into either HEK293 cells (Fig. 2a) or C3H10T1/2 cells (Fig. 2b), seipin was robustly expressed. However, we observed that the L91P and A212P mutations did result in a modest reduction in protein expression when compared with wild-type or T78A seipin (Fig. 2c). This was not the result of a significant effect of these mutations on mRNA expression levels (Fig. 2d).Fig. 2

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