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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

Wild-type human seipin and T78A mutant seipin form high-molecular-mass oligomers, while L91P seipin and A212P seipin are unable to do so. (a–d) tsA 201 cells were transfected with the long form of wild-type seipin with an N-terminal triple-FLAG tag and a C-terminal Myc tag (a) or with the T78A (b), A212P (c) or L91P (d) mutant forms of this protein. Isolated proteins were subjected to AFM imaging. Scale bar, 200 nm; shade-height scale, 0–5 nm. (e–h) Galleries of zoomed images of wild-type seipin (e) and the T78A (f), A212P (g) and L91P (h) mutants. Scale bar, 20 nm; shade-height scale, 0–5 nm. (i–l) Frequency distributions of molecular volumes of wild-type (i), T78A (j), A212P (k) and L91P (l) seipin. The curves indicate the fitted Gaussian functions
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Fig4: Wild-type human seipin and T78A mutant seipin form high-molecular-mass oligomers, while L91P seipin and A212P seipin are unable to do so. (a–d) tsA 201 cells were transfected with the long form of wild-type seipin with an N-terminal triple-FLAG tag and a C-terminal Myc tag (a) or with the T78A (b), A212P (c) or L91P (d) mutant forms of this protein. Isolated proteins were subjected to AFM imaging. Scale bar, 200 nm; shade-height scale, 0–5 nm. (e–h) Galleries of zoomed images of wild-type seipin (e) and the T78A (f), A212P (g) and L91P (h) mutants. Scale bar, 20 nm; shade-height scale, 0–5 nm. (i–l) Frequency distributions of molecular volumes of wild-type (i), T78A (j), A212P (k) and L91P (l) seipin. The curves indicate the fitted Gaussian functions

Mentions: The yeast orthologue of seipin, Fld1p, has been reported to form a homo-oligomer of nine subunits in a circular arrangement [7]. Moreover, analysis of a mutant form of Fld1p carrying the single amino acid substitution G228P, which mimics A212P in the human protein, suggested that this mutation abrogated the ability of seipin to form this oligomeric structure. To date, no studies have examined whether human seipin also forms high-order oligomers or whether pathogenic point mutations affect homo-oligomerisation. To study the architecture of human seipin, we selected AFM. AFM is a scanning probe technique that provides nanometre-scale resolution without the requirement for extensive sample preparation [26]. FLAG-tagged wild-type seipin and the T78A, A212P and L91P mutants were immunopurified and the isolated protein was adsorbed to a mica substrate and imaged using tapping-mode AFM in air. Figure 4a–d shows representative low-magnification images of the four seipin constructs. Wild-type seipin (Fig. 4a) and the T78A mutant (Fig. 4b) appeared as large particles of uniform size. In contrast, the A212P mutant (Fig. 4c) and the L91P mutant (Fig. 4d) appeared as noticeably smaller and less homogenous particles. The galleries of zoomed images of particles shown in Fig. 4e–h confirm the difference in size between the two pairs of constructs; that is, wild-type (Fig. 4e) and T78A (Fig. 4f) vs A212P (Fig. 4g) and L91P (Fig. 4h). Frequency distributions of volumes of the particles, calculated using Equation 1, had peaks at 2,394 ± 36 (mean ± SEM) nm3 (n = 100) for wild-type seipin (Fig. 4i), 2,396 ± 22 nm3 (n = 100) for the T78A mutant (Fig. 4j), 753 ± 20 nm3 (n = 100) for the A212P mutant (Fig. 4k) and 807 ± 33 nm3 (n = 100) for the L91P mutant (Fig. 4l).Fig. 4


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)

Wild-type human seipin and T78A mutant seipin form high-molecular-mass oligomers, while L91P seipin and A212P seipin are unable to do so. (a–d) tsA 201 cells were transfected with the long form of wild-type seipin with an N-terminal triple-FLAG tag and a C-terminal Myc tag (a) or with the T78A (b), A212P (c) or L91P (d) mutant forms of this protein. Isolated proteins were subjected to AFM imaging. Scale bar, 200 nm; shade-height scale, 0–5 nm. (e–h) Galleries of zoomed images of wild-type seipin (e) and the T78A (f), A212P (g) and L91P (h) mutants. Scale bar, 20 nm; shade-height scale, 0–5 nm. (i–l) Frequency distributions of molecular volumes of wild-type (i), T78A (j), A212P (k) and L91P (l) seipin. The curves indicate the fitted Gaussian functions
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Related In: Results  -  Collection

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Fig4: Wild-type human seipin and T78A mutant seipin form high-molecular-mass oligomers, while L91P seipin and A212P seipin are unable to do so. (a–d) tsA 201 cells were transfected with the long form of wild-type seipin with an N-terminal triple-FLAG tag and a C-terminal Myc tag (a) or with the T78A (b), A212P (c) or L91P (d) mutant forms of this protein. Isolated proteins were subjected to AFM imaging. Scale bar, 200 nm; shade-height scale, 0–5 nm. (e–h) Galleries of zoomed images of wild-type seipin (e) and the T78A (f), A212P (g) and L91P (h) mutants. Scale bar, 20 nm; shade-height scale, 0–5 nm. (i–l) Frequency distributions of molecular volumes of wild-type (i), T78A (j), A212P (k) and L91P (l) seipin. The curves indicate the fitted Gaussian functions
Mentions: The yeast orthologue of seipin, Fld1p, has been reported to form a homo-oligomer of nine subunits in a circular arrangement [7]. Moreover, analysis of a mutant form of Fld1p carrying the single amino acid substitution G228P, which mimics A212P in the human protein, suggested that this mutation abrogated the ability of seipin to form this oligomeric structure. To date, no studies have examined whether human seipin also forms high-order oligomers or whether pathogenic point mutations affect homo-oligomerisation. To study the architecture of human seipin, we selected AFM. AFM is a scanning probe technique that provides nanometre-scale resolution without the requirement for extensive sample preparation [26]. FLAG-tagged wild-type seipin and the T78A, A212P and L91P mutants were immunopurified and the isolated protein was adsorbed to a mica substrate and imaged using tapping-mode AFM in air. Figure 4a–d shows representative low-magnification images of the four seipin constructs. Wild-type seipin (Fig. 4a) and the T78A mutant (Fig. 4b) appeared as large particles of uniform size. In contrast, the A212P mutant (Fig. 4c) and the L91P mutant (Fig. 4d) appeared as noticeably smaller and less homogenous particles. The galleries of zoomed images of particles shown in Fig. 4e–h confirm the difference in size between the two pairs of constructs; that is, wild-type (Fig. 4e) and T78A (Fig. 4f) vs A212P (Fig. 4g) and L91P (Fig. 4h). Frequency distributions of volumes of the particles, calculated using Equation 1, had peaks at 2,394 ± 36 (mean ± SEM) nm3 (n = 100) for wild-type seipin (Fig. 4i), 2,396 ± 22 nm3 (n = 100) for the T78A mutant (Fig. 4j), 753 ± 20 nm3 (n = 100) for the A212P mutant (Fig. 4k) and 807 ± 33 nm3 (n = 100) for the L91P mutant (Fig. 4l).Fig. 4

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