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Hsp70 chaperones and type I PRMTs are sequestered at intranuclear inclusions caused by polyalanine expansions in PABPN1.

Tavanez JP, Bengoechea R, Berciano MT, Lafarga M, Carmo-Fonseca M, Enguita FJ - PLoS ONE (2009)

Bottom Line: Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure.Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones.This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A)-binding protein nuclear 1 (PABPN1) gene causes oculopharyngeal muscular dystrophy (OPMD). To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3) associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.

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Hsp70 accumulates in OPMD intranuclear inclusions and binds with higher affinity to expanded PABPN1 in vitro.(A–F) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. Tryptophan emission fluorescence spectra were recorded in response to the addition of increasing amounts of purified recombinant Hsp70 to purified recombinant normal (wt) and expanded PABPN1 variants. The graph depicts a double reciprocal plot of the increase of fluorescence intensity at the maximum wavelength of PABPN1 variants in response to increasing concentrations of Hsp70. Concentration of PABPN1 variants was 100 µM. (▴) wt-PABPN1, (•) 4Ala-PABPN1 and (▪) 7Ala-PABPN1. RFI, relative fluorescence intensity. The estimated dissociation constants (Kd) for each complex are indicated in the table.
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pone-0006418-g003: Hsp70 accumulates in OPMD intranuclear inclusions and binds with higher affinity to expanded PABPN1 in vitro.(A–F) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. Tryptophan emission fluorescence spectra were recorded in response to the addition of increasing amounts of purified recombinant Hsp70 to purified recombinant normal (wt) and expanded PABPN1 variants. The graph depicts a double reciprocal plot of the increase of fluorescence intensity at the maximum wavelength of PABPN1 variants in response to increasing concentrations of Hsp70. Concentration of PABPN1 variants was 100 µM. (▴) wt-PABPN1, (•) 4Ala-PABPN1 and (▪) 7Ala-PABPN1. RFI, relative fluorescence intensity. The estimated dissociation constants (Kd) for each complex are indicated in the table.

Mentions: Having established that PRMT1 and PRMT3 associate preferentially with expanded PABPN1 and concentrate in OPMD intranuclear inclusions, we next examined the sub-cellular distribution of Hsp70 and Hsp90 in muscle fibers from OPMD patients. Immunofluorescence analysis revealed a clear concentration of Hsp70 in the PABPN1 inclusions (Fig. 3), while antibodies anti-Hsp90 did not stain these structures (not shown). To further characterize the influence of polyalanine extension on the interaction between Hsp70 and PABPN1, we performed steady-state tryptophan fluorescence spectroscopy. PABPN1 has a single tryptophan residue located in the C-terminal region that can be used to monitor changes in the amino acid environment caused by protein-protein interactions. Tryptophan emission fluorescence spectra were recorded in response to the addition of increasing amounts of purified recombinant Hsp70 to purified recombinant normal and expanded PABPN1 variants. Contribution of Hsp70 to the fluorescence spectra was subtracted in all the cases. For each PABPN1/Hsp70 complex, the relative dissociation constant (Kd) was calculated (Fig. 3). The results show that, compared to normal PABPN1, the interaction with Hsp70 is stronger for the expanded protein with 14 alanines and even stronger for the expanded variant with 17 alanines (Fig. 3). This suggests that the number of residues in the polyalanine tract of PABPN1 correlates with the binding affinity to Hsp70.


Hsp70 chaperones and type I PRMTs are sequestered at intranuclear inclusions caused by polyalanine expansions in PABPN1.

Tavanez JP, Bengoechea R, Berciano MT, Lafarga M, Carmo-Fonseca M, Enguita FJ - PLoS ONE (2009)

Hsp70 accumulates in OPMD intranuclear inclusions and binds with higher affinity to expanded PABPN1 in vitro.(A–F) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. Tryptophan emission fluorescence spectra were recorded in response to the addition of increasing amounts of purified recombinant Hsp70 to purified recombinant normal (wt) and expanded PABPN1 variants. The graph depicts a double reciprocal plot of the increase of fluorescence intensity at the maximum wavelength of PABPN1 variants in response to increasing concentrations of Hsp70. Concentration of PABPN1 variants was 100 µM. (▴) wt-PABPN1, (•) 4Ala-PABPN1 and (▪) 7Ala-PABPN1. RFI, relative fluorescence intensity. The estimated dissociation constants (Kd) for each complex are indicated in the table.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712759&req=5

pone-0006418-g003: Hsp70 accumulates in OPMD intranuclear inclusions and binds with higher affinity to expanded PABPN1 in vitro.(A–F) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. Tryptophan emission fluorescence spectra were recorded in response to the addition of increasing amounts of purified recombinant Hsp70 to purified recombinant normal (wt) and expanded PABPN1 variants. The graph depicts a double reciprocal plot of the increase of fluorescence intensity at the maximum wavelength of PABPN1 variants in response to increasing concentrations of Hsp70. Concentration of PABPN1 variants was 100 µM. (▴) wt-PABPN1, (•) 4Ala-PABPN1 and (▪) 7Ala-PABPN1. RFI, relative fluorescence intensity. The estimated dissociation constants (Kd) for each complex are indicated in the table.
Mentions: Having established that PRMT1 and PRMT3 associate preferentially with expanded PABPN1 and concentrate in OPMD intranuclear inclusions, we next examined the sub-cellular distribution of Hsp70 and Hsp90 in muscle fibers from OPMD patients. Immunofluorescence analysis revealed a clear concentration of Hsp70 in the PABPN1 inclusions (Fig. 3), while antibodies anti-Hsp90 did not stain these structures (not shown). To further characterize the influence of polyalanine extension on the interaction between Hsp70 and PABPN1, we performed steady-state tryptophan fluorescence spectroscopy. PABPN1 has a single tryptophan residue located in the C-terminal region that can be used to monitor changes in the amino acid environment caused by protein-protein interactions. Tryptophan emission fluorescence spectra were recorded in response to the addition of increasing amounts of purified recombinant Hsp70 to purified recombinant normal and expanded PABPN1 variants. Contribution of Hsp70 to the fluorescence spectra was subtracted in all the cases. For each PABPN1/Hsp70 complex, the relative dissociation constant (Kd) was calculated (Fig. 3). The results show that, compared to normal PABPN1, the interaction with Hsp70 is stronger for the expanded protein with 14 alanines and even stronger for the expanded variant with 17 alanines (Fig. 3). This suggests that the number of residues in the polyalanine tract of PABPN1 correlates with the binding affinity to Hsp70.

Bottom Line: Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure.Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones.This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A)-binding protein nuclear 1 (PABPN1) gene causes oculopharyngeal muscular dystrophy (OPMD). To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3) associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.

Show MeSH
Related in: MedlinePlus