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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 pathogenic point mutations T78A, L91P and A212P do not affect the capacity of seipin to bind the PA phosphatase lipin 1. (a) HEK293 cells were transfected with empty vector (−), FLAG-tagged long form of wild-type seipin (WT) or with identically tagged T78A, L91P, A212P forms of seipin in the absence or presence of lipin 1β. Cell lysates or anti-FLAG immunoprecipitates were immunoblotted for seipin using anti (α)-FLAG antibodies or lipin 1β using and anti-lipin 1 antibodies, as indicated. Lysates were also probed for calnexin as a loading control. (b) The binding of lipin 1β to WT or mutant forms of seipin was quantified in replicate immunoblots (n = 3), normalised to expression levels and expressed as a fold of that observed with wild-type seipin. Data shown are means ± SEM
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Fig3: The pathogenic point mutations T78A, L91P and A212P do not affect the capacity of seipin to bind the PA phosphatase lipin 1. (a) HEK293 cells were transfected with empty vector (−), FLAG-tagged long form of wild-type seipin (WT) or with identically tagged T78A, L91P, A212P forms of seipin in the absence or presence of lipin 1β. Cell lysates or anti-FLAG immunoprecipitates were immunoblotted for seipin using anti (α)-FLAG antibodies or lipin 1β using and anti-lipin 1 antibodies, as indicated. Lysates were also probed for calnexin as a loading control. (b) The binding of lipin 1β to WT or mutant forms of seipin was quantified in replicate immunoblots (n = 3), normalised to expression levels and expressed as a fold of that observed with wild-type seipin. Data shown are means ± SEM

Mentions: We have recently demonstrated that seipin can bind to the PA phosphatase lipin 1, including endogenous lipin 1 in differentiating cultured adipocytes. This interaction appears to be important for appropriately targeting this pro-adipogenic enzyme to the ER in differentiating pre-adipocytes [17]. We also showed that the premature stop mutants of seipin, E113X, R138X and R275X, appeared unable to bind lipin 1. In addition, the ability of Q391X seipin to bind lipin 1 is significantly reduced [17]. Similar data are shown in ESM Fig. 2. We therefore asked whether the point mutations T78A, L91P and A212P influence lipin 1 binding to seipin. As shown in Fig. 3a, b, we observed no decrease in the ability of seipin to co-immunoprecipitate lipin 1 (quantified data are shown in Fig. 3b). Hence, while the failure to target lipin 1 appropriately offers a potential pathogenic mechanism for the premature stop mutants of seipin, it appears unlikely to explain the failure of adipocyte development in cells expressing the T78A, L91P and A212P mutant forms of seipin.Fig. 3


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 the capacity of seipin to bind the PA phosphatase lipin 1. (a) HEK293 cells were transfected with empty vector (−), FLAG-tagged long form of wild-type seipin (WT) or with identically tagged T78A, L91P, A212P forms of seipin in the absence or presence of lipin 1β. Cell lysates or anti-FLAG immunoprecipitates were immunoblotted for seipin using anti (α)-FLAG antibodies or lipin 1β using and anti-lipin 1 antibodies, as indicated. Lysates were also probed for calnexin as a loading control. (b) The binding of lipin 1β to WT or mutant forms of seipin was quantified in replicate immunoblots (n = 3), normalised to expression levels and expressed as a fold of that observed with wild-type seipin. Data shown are means ± SEM
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: The pathogenic point mutations T78A, L91P and A212P do not affect the capacity of seipin to bind the PA phosphatase lipin 1. (a) HEK293 cells were transfected with empty vector (−), FLAG-tagged long form of wild-type seipin (WT) or with identically tagged T78A, L91P, A212P forms of seipin in the absence or presence of lipin 1β. Cell lysates or anti-FLAG immunoprecipitates were immunoblotted for seipin using anti (α)-FLAG antibodies or lipin 1β using and anti-lipin 1 antibodies, as indicated. Lysates were also probed for calnexin as a loading control. (b) The binding of lipin 1β to WT or mutant forms of seipin was quantified in replicate immunoblots (n = 3), normalised to expression levels and expressed as a fold of that observed with wild-type seipin. Data shown are means ± SEM
Mentions: We have recently demonstrated that seipin can bind to the PA phosphatase lipin 1, including endogenous lipin 1 in differentiating cultured adipocytes. This interaction appears to be important for appropriately targeting this pro-adipogenic enzyme to the ER in differentiating pre-adipocytes [17]. We also showed that the premature stop mutants of seipin, E113X, R138X and R275X, appeared unable to bind lipin 1. In addition, the ability of Q391X seipin to bind lipin 1 is significantly reduced [17]. Similar data are shown in ESM Fig. 2. We therefore asked whether the point mutations T78A, L91P and A212P influence lipin 1 binding to seipin. As shown in Fig. 3a, b, we observed no decrease in the ability of seipin to co-immunoprecipitate lipin 1 (quantified data are shown in Fig. 3b). Hence, while the failure to target lipin 1 appropriately offers a potential pathogenic mechanism for the premature stop mutants of seipin, it appears unlikely to explain the failure of adipocyte development in cells expressing the T78A, L91P and A212P mutant forms of seipin.Fig. 3

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