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MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

Tomé S, Manley K, Simard JP, Clark GW, Slean MM, Swami M, Shelbourne PF, Tillier ER, Monckton DG, Messer A, Pearson CE - PLoS Genet. (2013)

Bottom Line: The CAG stabilization was as dramatic as genetic deficiency of Msh2.B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability.Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

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Related in: MedlinePlus

Structural and sequence analysis of MSH3.A: Multiple sequence alignment of MSH3. Jalview created visualization [120] using the first 500 amino acids of the mouse B6 MSH3 (NP_034959.2). Conservation values and consensus sequence are based on alignment of S. cerevisiae Msh3p, E. coli MutS and 17 mammalian MSH3 homologs; values range from 0–9, where 0 is lowest and 9 is the highest. Protein interacting domains indicated pertain to those regions of the human MSH3 protein. This panel only shows an abbreviated set of the species of MSH3 sequence, the full set analysed is shown in Figure S5. B: MSH3 variant within β-turn. The T321I variant occurs within a Type I β-turn, as determined by specific backbone turn angles [117], [118] from the human MSH3 structure (3THW_B). Top left: hMSH3 tube diagram of Cα atoms of β-turn (blue), i+2 (T) residue (red) and additional three residues on N- and C-terminal ends (green). Bottom left table shows the β-turn propensity is relatively strong throughout MutS/MSH3 homologs, while the CBy variant (Isoleucine at i+2 position) is extremely disfavored (table bottom left) [69]. Right: Ball and stick diagram of contact sites of Asp (D) and Thr (T) residues in β-turn with residues 194 and 214 respectively. Line diagram of Thr (T) hydroxyl group contact with neighbouring Threonine residue at position 365. The absence of the Threonine hydroxyl group may be important to stabilizing the β-turn itself, and/or may change the conformation of the turn, potentially disrupting distant contacts important for proper protein folding. MSH3 visualizations created using PyMol (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).
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pgen-1003280-g005: Structural and sequence analysis of MSH3.A: Multiple sequence alignment of MSH3. Jalview created visualization [120] using the first 500 amino acids of the mouse B6 MSH3 (NP_034959.2). Conservation values and consensus sequence are based on alignment of S. cerevisiae Msh3p, E. coli MutS and 17 mammalian MSH3 homologs; values range from 0–9, where 0 is lowest and 9 is the highest. Protein interacting domains indicated pertain to those regions of the human MSH3 protein. This panel only shows an abbreviated set of the species of MSH3 sequence, the full set analysed is shown in Figure S5. B: MSH3 variant within β-turn. The T321I variant occurs within a Type I β-turn, as determined by specific backbone turn angles [117], [118] from the human MSH3 structure (3THW_B). Top left: hMSH3 tube diagram of Cα atoms of β-turn (blue), i+2 (T) residue (red) and additional three residues on N- and C-terminal ends (green). Bottom left table shows the β-turn propensity is relatively strong throughout MutS/MSH3 homologs, while the CBy variant (Isoleucine at i+2 position) is extremely disfavored (table bottom left) [69]. Right: Ball and stick diagram of contact sites of Asp (D) and Thr (T) residues in β-turn with residues 194 and 214 respectively. Line diagram of Thr (T) hydroxyl group contact with neighbouring Threonine residue at position 365. The absence of the Threonine hydroxyl group may be important to stabilizing the β-turn itself, and/or may change the conformation of the turn, potentially disrupting distant contacts important for proper protein folding. MSH3 visualizations created using PyMol (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).

Mentions: In order to uncover potential amino acid changes, which could be contributing to loss of MSH3 protein expression in the CBy variants, we have examined both sequence and structural features of MSH3 homologs. Sequence alignment has revealed that most of the B6-CBy variants are well conserved, but occur where amino acid changes are not predicted to have physiochemical consequences, or occur within poorly conserved regions, suggesting those regions minimally contribute to structure/function of the protein (Figure 5A). One exception is the T321I variant, which is conserved in 16/17 of the mammalian homologs and yeast. Further, in this one exception (in both giant panda and yeast), the Threonine is replaced by physiochemically-similar Serine, so that a hydroxyl group at this position is observed to be highly conserved (Figure 5A and Figure S6). Importantly, the T321 variant occurs within a Type I β-Turn (Figure 5B), where Isoleucine is extremely unfavoured (Figure 5B) [69]. Despite the large evolutionary distance, a Type I β-Turn also occurs in E. coli MutS (Figure 5B) [70], suggesting the importance of this region to overall function. β-Turns are thought to be crucial to the protein folding process [71], [72], where they may direct nucleation of secondary structure elements towards hydrophobic collapse [73]. The change of Threonine to disfavoured Isoleucine at the ‘i+2’ site within the turn of MSH3 may disrupt the β-Turn, representing a significant barrier to protein folding, potentially leading to proteolysis. The full effect of the T321I change upon MSH3 protein stability may require some of the other amino acid changes, which will require experimental assessment.


MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

Tomé S, Manley K, Simard JP, Clark GW, Slean MM, Swami M, Shelbourne PF, Tillier ER, Monckton DG, Messer A, Pearson CE - PLoS Genet. (2013)

Structural and sequence analysis of MSH3.A: Multiple sequence alignment of MSH3. Jalview created visualization [120] using the first 500 amino acids of the mouse B6 MSH3 (NP_034959.2). Conservation values and consensus sequence are based on alignment of S. cerevisiae Msh3p, E. coli MutS and 17 mammalian MSH3 homologs; values range from 0–9, where 0 is lowest and 9 is the highest. Protein interacting domains indicated pertain to those regions of the human MSH3 protein. This panel only shows an abbreviated set of the species of MSH3 sequence, the full set analysed is shown in Figure S5. B: MSH3 variant within β-turn. The T321I variant occurs within a Type I β-turn, as determined by specific backbone turn angles [117], [118] from the human MSH3 structure (3THW_B). Top left: hMSH3 tube diagram of Cα atoms of β-turn (blue), i+2 (T) residue (red) and additional three residues on N- and C-terminal ends (green). Bottom left table shows the β-turn propensity is relatively strong throughout MutS/MSH3 homologs, while the CBy variant (Isoleucine at i+2 position) is extremely disfavored (table bottom left) [69]. Right: Ball and stick diagram of contact sites of Asp (D) and Thr (T) residues in β-turn with residues 194 and 214 respectively. Line diagram of Thr (T) hydroxyl group contact with neighbouring Threonine residue at position 365. The absence of the Threonine hydroxyl group may be important to stabilizing the β-turn itself, and/or may change the conformation of the turn, potentially disrupting distant contacts important for proper protein folding. MSH3 visualizations created using PyMol (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585117&req=5

pgen-1003280-g005: Structural and sequence analysis of MSH3.A: Multiple sequence alignment of MSH3. Jalview created visualization [120] using the first 500 amino acids of the mouse B6 MSH3 (NP_034959.2). Conservation values and consensus sequence are based on alignment of S. cerevisiae Msh3p, E. coli MutS and 17 mammalian MSH3 homologs; values range from 0–9, where 0 is lowest and 9 is the highest. Protein interacting domains indicated pertain to those regions of the human MSH3 protein. This panel only shows an abbreviated set of the species of MSH3 sequence, the full set analysed is shown in Figure S5. B: MSH3 variant within β-turn. The T321I variant occurs within a Type I β-turn, as determined by specific backbone turn angles [117], [118] from the human MSH3 structure (3THW_B). Top left: hMSH3 tube diagram of Cα atoms of β-turn (blue), i+2 (T) residue (red) and additional three residues on N- and C-terminal ends (green). Bottom left table shows the β-turn propensity is relatively strong throughout MutS/MSH3 homologs, while the CBy variant (Isoleucine at i+2 position) is extremely disfavored (table bottom left) [69]. Right: Ball and stick diagram of contact sites of Asp (D) and Thr (T) residues in β-turn with residues 194 and 214 respectively. Line diagram of Thr (T) hydroxyl group contact with neighbouring Threonine residue at position 365. The absence of the Threonine hydroxyl group may be important to stabilizing the β-turn itself, and/or may change the conformation of the turn, potentially disrupting distant contacts important for proper protein folding. MSH3 visualizations created using PyMol (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).
Mentions: In order to uncover potential amino acid changes, which could be contributing to loss of MSH3 protein expression in the CBy variants, we have examined both sequence and structural features of MSH3 homologs. Sequence alignment has revealed that most of the B6-CBy variants are well conserved, but occur where amino acid changes are not predicted to have physiochemical consequences, or occur within poorly conserved regions, suggesting those regions minimally contribute to structure/function of the protein (Figure 5A). One exception is the T321I variant, which is conserved in 16/17 of the mammalian homologs and yeast. Further, in this one exception (in both giant panda and yeast), the Threonine is replaced by physiochemically-similar Serine, so that a hydroxyl group at this position is observed to be highly conserved (Figure 5A and Figure S6). Importantly, the T321 variant occurs within a Type I β-Turn (Figure 5B), where Isoleucine is extremely unfavoured (Figure 5B) [69]. Despite the large evolutionary distance, a Type I β-Turn also occurs in E. coli MutS (Figure 5B) [70], suggesting the importance of this region to overall function. β-Turns are thought to be crucial to the protein folding process [71], [72], where they may direct nucleation of secondary structure elements towards hydrophobic collapse [73]. The change of Threonine to disfavoured Isoleucine at the ‘i+2’ site within the turn of MSH3 may disrupt the β-Turn, representing a significant barrier to protein folding, potentially leading to proteolysis. The full effect of the T321I change upon MSH3 protein stability may require some of the other amino acid changes, which will require experimental assessment.

Bottom Line: The CAG stabilization was as dramatic as genetic deficiency of Msh2.B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability.Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

Show MeSH
Related in: MedlinePlus