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Intracellular complexes of the early-onset torsion dystonia-associated AAA+ ATPase TorsinA.

Li H, Wu HC, Liu Z, Zacchi LF, Brodsky JL, Zolkiewski M - Springerplus (2014)

Bottom Line: We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer.Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein.Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506 USA.

ABSTRACT
A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.

No MeSH data available.


Related in: MedlinePlus

BN-PAGE analysis of torsinA complexes. Full-length human torsinA (WT) or the dystonia-linked torsinAΔE protein (ΔE) was expressed in HEK293 (A) and CHO (B) cells. Production of the torsinA variants was confirmed by SDS-PAGE followed by immunoblotting with anti-torsinA antibodies (lower panels) using untransfected cells as a control (C). The cell lysates were separated on BN-PAGE followed by immunoblotting (upper panels). For BN-PAGE, the migration positions of the native-electrophoresis standards are indicated. The migration position of β-amylase (200 kDa) is indicated with an arrow. Protein migration in BN-PAGE can reflect other biophysical properties, besides the molecular weight, so the molecular weight determination is only approximate. The figure shows a representative result from two independent experiments.
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Fig1: BN-PAGE analysis of torsinA complexes. Full-length human torsinA (WT) or the dystonia-linked torsinAΔE protein (ΔE) was expressed in HEK293 (A) and CHO (B) cells. Production of the torsinA variants was confirmed by SDS-PAGE followed by immunoblotting with anti-torsinA antibodies (lower panels) using untransfected cells as a control (C). The cell lysates were separated on BN-PAGE followed by immunoblotting (upper panels). For BN-PAGE, the migration positions of the native-electrophoresis standards are indicated. The migration position of β-amylase (200 kDa) is indicated with an arrow. Protein migration in BN-PAGE can reflect other biophysical properties, besides the molecular weight, so the molecular weight determination is only approximate. The figure shows a representative result from two independent experiments.

Mentions: To determine whether human torsinA and the dystonia-linked torsinAΔE variant oligomerize in the cell, we expressed each protein in two cell lines, HEK293 and CHO cells. After preparation of cell lysates in dodecylmaltoside, BN-PAGE and immunoblotting with an anti-torsinA antibody was used to observe the distribution of the torsinA-containing species (Figure 1). Both stably transfected cell lines produced comparable amounts of torsinA and torsinAΔE (Figure 1A, B, lower panels). In addition to some monomeric torsinA and torsinAΔE (shown by the bands below 66 kDa), BN-PAGE detected a single major immunoreactive species migrating close to the 200-kDa complex of β-amylase, but slower than the 242-kDa protein standard (Figure 1A, B, upper panels). The migration of the torsinA oligomer in BN-PAGE is consistent with that of a homohexamer (predicted molecular weight 216 kDa) and is consistent with the formation of a species of similar size in BN-PAGE using lysates prepared from U2OS cells (Vander Heyden et al. 2009). It cannot be excluded, however, that the detected species corresponds to a hetero-oligomer containing torsinA and other components, such as the torsinA binding partners LAP1 and LULL1 (Goodchild and Dauer 2005; Zhao et al. 2013; Sosa et al. 2014). We also found that the deletion of Glu302 in torsinA apparently destabilizes the oligomeric species (Figure 1A, B, upper panels). This result suggests that the dystonia-linked torsinA variant may be defective in either self-association or interactions with other proteins. Indeed, the efficiency of torsinAΔE interaction with LAP1 and LULL1 is compromised relative to the wild type protein (Naismith et al. 2009; Zhao et al. 2013). In contrast, the dystonia-linked torsinAΔE variant shows an enhanced binding affinity for nesprin (Nery et al. 2008). Thus, the apparent loss of the detected oligomeric species in the torsinAΔE producing cells (Figure 1) suggests that the observed torsinA complex does not include nesprin. Nevertheless, the data presented in Figure 1 indicate that the EOTD-associated mutation has a profound effect on oligomer and/or complex formation, and we propose that this defect might impact the protein’s function and disease presentation.Figure 1


Intracellular complexes of the early-onset torsion dystonia-associated AAA+ ATPase TorsinA.

Li H, Wu HC, Liu Z, Zacchi LF, Brodsky JL, Zolkiewski M - Springerplus (2014)

BN-PAGE analysis of torsinA complexes. Full-length human torsinA (WT) or the dystonia-linked torsinAΔE protein (ΔE) was expressed in HEK293 (A) and CHO (B) cells. Production of the torsinA variants was confirmed by SDS-PAGE followed by immunoblotting with anti-torsinA antibodies (lower panels) using untransfected cells as a control (C). The cell lysates were separated on BN-PAGE followed by immunoblotting (upper panels). For BN-PAGE, the migration positions of the native-electrophoresis standards are indicated. The migration position of β-amylase (200 kDa) is indicated with an arrow. Protein migration in BN-PAGE can reflect other biophysical properties, besides the molecular weight, so the molecular weight determination is only approximate. The figure shows a representative result from two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320221&req=5

Fig1: BN-PAGE analysis of torsinA complexes. Full-length human torsinA (WT) or the dystonia-linked torsinAΔE protein (ΔE) was expressed in HEK293 (A) and CHO (B) cells. Production of the torsinA variants was confirmed by SDS-PAGE followed by immunoblotting with anti-torsinA antibodies (lower panels) using untransfected cells as a control (C). The cell lysates were separated on BN-PAGE followed by immunoblotting (upper panels). For BN-PAGE, the migration positions of the native-electrophoresis standards are indicated. The migration position of β-amylase (200 kDa) is indicated with an arrow. Protein migration in BN-PAGE can reflect other biophysical properties, besides the molecular weight, so the molecular weight determination is only approximate. The figure shows a representative result from two independent experiments.
Mentions: To determine whether human torsinA and the dystonia-linked torsinAΔE variant oligomerize in the cell, we expressed each protein in two cell lines, HEK293 and CHO cells. After preparation of cell lysates in dodecylmaltoside, BN-PAGE and immunoblotting with an anti-torsinA antibody was used to observe the distribution of the torsinA-containing species (Figure 1). Both stably transfected cell lines produced comparable amounts of torsinA and torsinAΔE (Figure 1A, B, lower panels). In addition to some monomeric torsinA and torsinAΔE (shown by the bands below 66 kDa), BN-PAGE detected a single major immunoreactive species migrating close to the 200-kDa complex of β-amylase, but slower than the 242-kDa protein standard (Figure 1A, B, upper panels). The migration of the torsinA oligomer in BN-PAGE is consistent with that of a homohexamer (predicted molecular weight 216 kDa) and is consistent with the formation of a species of similar size in BN-PAGE using lysates prepared from U2OS cells (Vander Heyden et al. 2009). It cannot be excluded, however, that the detected species corresponds to a hetero-oligomer containing torsinA and other components, such as the torsinA binding partners LAP1 and LULL1 (Goodchild and Dauer 2005; Zhao et al. 2013; Sosa et al. 2014). We also found that the deletion of Glu302 in torsinA apparently destabilizes the oligomeric species (Figure 1A, B, upper panels). This result suggests that the dystonia-linked torsinA variant may be defective in either self-association or interactions with other proteins. Indeed, the efficiency of torsinAΔE interaction with LAP1 and LULL1 is compromised relative to the wild type protein (Naismith et al. 2009; Zhao et al. 2013). In contrast, the dystonia-linked torsinAΔE variant shows an enhanced binding affinity for nesprin (Nery et al. 2008). Thus, the apparent loss of the detected oligomeric species in the torsinAΔE producing cells (Figure 1) suggests that the observed torsinA complex does not include nesprin. Nevertheless, the data presented in Figure 1 indicate that the EOTD-associated mutation has a profound effect on oligomer and/or complex formation, and we propose that this defect might impact the protein’s function and disease presentation.Figure 1

Bottom Line: We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer.Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein.Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506 USA.

ABSTRACT
A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.

No MeSH data available.


Related in: MedlinePlus