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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

Both wild-type human seipin and T78A mutant seipin form dodecamers. (a) AFM images of wild-type seipin either alone or after incubation with anti-Myc or anti-HA antibodies. (b) AFM images of T78A mutant seipin either alone or after incubation with either anti-Myc or anti-V5 antibodies. Arrows indicate multiply decorated seipin complexes. Scale bar, 100 nm; shade-height scale, 0–5 nm. (c, d) Gallery of zoomed images showing multiply decorated wild-type (c) or T78A mutant seipin (d). Scale bar, 20 nm; shade-height scale, 0–5 nm. (e, f) Frequency distributions of angles between pairs of antibodies bound to either wild-type (e) or T78A mutant seipin (f). The curves indicate the fitted Gaussian functions
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Fig5: Both wild-type human seipin and T78A mutant seipin form dodecamers. (a) AFM images of wild-type seipin either alone or after incubation with anti-Myc or anti-HA antibodies. (b) AFM images of T78A mutant seipin either alone or after incubation with either anti-Myc or anti-V5 antibodies. Arrows indicate multiply decorated seipin complexes. Scale bar, 100 nm; shade-height scale, 0–5 nm. (c, d) Gallery of zoomed images showing multiply decorated wild-type (c) or T78A mutant seipin (d). Scale bar, 20 nm; shade-height scale, 0–5 nm. (e, f) Frequency distributions of angles between pairs of antibodies bound to either wild-type (e) or T78A mutant seipin (f). The curves indicate the fitted Gaussian functions

Mentions: It is well known that measurement of the molecular volumes of proteins by AFM is complicated by factors such as the convolution introduced by the geometry of the scanning tip. Hence, the measured volume is not by itself a reliable indicator of the stoichiometry of the seipin complex. To conclusively establish the stoichiometry of the wild-type and T78A seipin complexes, they were imaged either after incubation with anti-Myc antibodies, which should decorate the C-terminal Myc epitope tag present on each subunit, or anti-HA or anti-V5 antibodies as negative controls. Representative low-magnification AFM images of wild-type seipin are shown in Fig. 5a. In the absence of antibodies, the large particles previously shown in Fig. 4a were again seen (Fig. 5a). After incubation with anti-Myc antibodies, the large particles became decorated by multiple smaller particles (antibodies, indicated by arrows) and took on a rosette-like appearance (Fig. 5a). Many unbound antibodies were also seen on the mica substrate. In contrast, after incubation with anti-HA antibodies, there was very little binding of antibodies to the seipin complexes (Fig. 5a). Quantification of antibody binding revealed that 88% of the seipin complexes were decorated by anti-Myc antibodies (usually multiply), whereas only 18% of the complexes were decorated by anti-HA antibodies (usually singly). A similar antibody decoration profile was seen with the T78A seipin mutant (Fig. 5b). In this case 83% of the seipin particles were decorated by anti-Myc antibodies, compared with 21% with anti-V5 antibodies. Galleries of zoomed images of antibody-decorated wild-type and T78A mutant seipin complexes are shown in Fig. 5c and Fig. 5d, respectively.Fig. 5


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)

Both wild-type human seipin and T78A mutant seipin form dodecamers. (a) AFM images of wild-type seipin either alone or after incubation with anti-Myc or anti-HA antibodies. (b) AFM images of T78A mutant seipin either alone or after incubation with either anti-Myc or anti-V5 antibodies. Arrows indicate multiply decorated seipin complexes. Scale bar, 100 nm; shade-height scale, 0–5 nm. (c, d) Gallery of zoomed images showing multiply decorated wild-type (c) or T78A mutant seipin (d). Scale bar, 20 nm; shade-height scale, 0–5 nm. (e, f) Frequency distributions of angles between pairs of antibodies bound to either wild-type (e) or T78A mutant seipin (f). The curves indicate the fitted Gaussian functions
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Related In: Results  -  Collection

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Fig5: Both wild-type human seipin and T78A mutant seipin form dodecamers. (a) AFM images of wild-type seipin either alone or after incubation with anti-Myc or anti-HA antibodies. (b) AFM images of T78A mutant seipin either alone or after incubation with either anti-Myc or anti-V5 antibodies. Arrows indicate multiply decorated seipin complexes. Scale bar, 100 nm; shade-height scale, 0–5 nm. (c, d) Gallery of zoomed images showing multiply decorated wild-type (c) or T78A mutant seipin (d). Scale bar, 20 nm; shade-height scale, 0–5 nm. (e, f) Frequency distributions of angles between pairs of antibodies bound to either wild-type (e) or T78A mutant seipin (f). The curves indicate the fitted Gaussian functions
Mentions: It is well known that measurement of the molecular volumes of proteins by AFM is complicated by factors such as the convolution introduced by the geometry of the scanning tip. Hence, the measured volume is not by itself a reliable indicator of the stoichiometry of the seipin complex. To conclusively establish the stoichiometry of the wild-type and T78A seipin complexes, they were imaged either after incubation with anti-Myc antibodies, which should decorate the C-terminal Myc epitope tag present on each subunit, or anti-HA or anti-V5 antibodies as negative controls. Representative low-magnification AFM images of wild-type seipin are shown in Fig. 5a. In the absence of antibodies, the large particles previously shown in Fig. 4a were again seen (Fig. 5a). After incubation with anti-Myc antibodies, the large particles became decorated by multiple smaller particles (antibodies, indicated by arrows) and took on a rosette-like appearance (Fig. 5a). Many unbound antibodies were also seen on the mica substrate. In contrast, after incubation with anti-HA antibodies, there was very little binding of antibodies to the seipin complexes (Fig. 5a). Quantification of antibody binding revealed that 88% of the seipin complexes were decorated by anti-Myc antibodies (usually multiply), whereas only 18% of the complexes were decorated by anti-HA antibodies (usually singly). A similar antibody decoration profile was seen with the T78A seipin mutant (Fig. 5b). In this case 83% of the seipin particles were decorated by anti-Myc antibodies, compared with 21% with anti-V5 antibodies. Galleries of zoomed images of antibody-decorated wild-type and T78A mutant seipin complexes are shown in Fig. 5c and Fig. 5d, respectively.Fig. 5

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