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A yeast model for polyalanine-expansion aggregation and toxicity.

Konopka CA, Locke MN, Gallagher PS, Pham N, Hart MP, Walker CJ, Gitler AD, Gardner RG - Mol. Biol. Cell (2011)

Bottom Line: Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts.In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A.Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, USA.

ABSTRACT
Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by using yeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

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PolyA-expanded Pab1 inclusions are distinct from TDP-43 and α-syn inclusions. Cells coexpressing Pab117A-GFP or Pab120A-GFP with either TDP43-DsRed (A) or α-syn-mCherry (B) were imaged by epifluorescence microscopy. Bars = 2 μm.
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Figure 7: PolyA-expanded Pab1 inclusions are distinct from TDP-43 and α-syn inclusions. Cells coexpressing Pab117A-GFP or Pab120A-GFP with either TDP43-DsRed (A) or α-syn-mCherry (B) were imaged by epifluorescence microscopy. Bars = 2 μm.

Mentions: We also wanted to determine whether polyA-expanded Pab1 inclusions were different from inclusions of TAR DNA-binding protein 43 (TDP-43), which has been implicated in amyotrophic lateral sclerosis (Chen-Plotkin et al., 2010), and α-syn, which forms inclusions in Parkinson's disease (Lucking and Brice, 2000). In general, Pab117A-GFP and Pab120A-GFP inclusions and TDP-43–DsRed inclusions were distinct with little colocalization (Figure 7A), although we did observe limited colocalization with Pab1 polyA-expansion inclusions and TDP-43 at a low frequency. When we did observe colocalization, Pab120A-GFP and TDP-43–DsRed inclusions overlapped more than Pab117A-GFP and TDP-43–DsRed inclusions, which could be due to differences in the kinetics of inclusion formation. TDP-43 forms inclusions as early as 3 h after induction (Johnson et al., 2008), and Pab120A forms visible inclusions faster than Pab117A (Figure 3B). We did not detect any colocalization of Pab117A-GFP or Pab120A-GFP with α-syn–mCherry (Figure 7B), indicating that these inclusions are distinct.


A yeast model for polyalanine-expansion aggregation and toxicity.

Konopka CA, Locke MN, Gallagher PS, Pham N, Hart MP, Walker CJ, Gitler AD, Gardner RG - Mol. Biol. Cell (2011)

PolyA-expanded Pab1 inclusions are distinct from TDP-43 and α-syn inclusions. Cells coexpressing Pab117A-GFP or Pab120A-GFP with either TDP43-DsRed (A) or α-syn-mCherry (B) were imaged by epifluorescence microscopy. Bars = 2 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113764&req=5

Figure 7: PolyA-expanded Pab1 inclusions are distinct from TDP-43 and α-syn inclusions. Cells coexpressing Pab117A-GFP or Pab120A-GFP with either TDP43-DsRed (A) or α-syn-mCherry (B) were imaged by epifluorescence microscopy. Bars = 2 μm.
Mentions: We also wanted to determine whether polyA-expanded Pab1 inclusions were different from inclusions of TAR DNA-binding protein 43 (TDP-43), which has been implicated in amyotrophic lateral sclerosis (Chen-Plotkin et al., 2010), and α-syn, which forms inclusions in Parkinson's disease (Lucking and Brice, 2000). In general, Pab117A-GFP and Pab120A-GFP inclusions and TDP-43–DsRed inclusions were distinct with little colocalization (Figure 7A), although we did observe limited colocalization with Pab1 polyA-expansion inclusions and TDP-43 at a low frequency. When we did observe colocalization, Pab120A-GFP and TDP-43–DsRed inclusions overlapped more than Pab117A-GFP and TDP-43–DsRed inclusions, which could be due to differences in the kinetics of inclusion formation. TDP-43 forms inclusions as early as 3 h after induction (Johnson et al., 2008), and Pab120A forms visible inclusions faster than Pab117A (Figure 3B). We did not detect any colocalization of Pab117A-GFP or Pab120A-GFP with α-syn–mCherry (Figure 7B), indicating that these inclusions are distinct.

Bottom Line: Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts.In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A.Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, USA.

ABSTRACT
Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by using yeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

Show MeSH
Related in: MedlinePlus