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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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Related in: MedlinePlus

Molecular dynamics snapshots of normal and expanded PABPN1 N-terminal peptides.Conformational behavior of peptides corresponding to the N-terminal segment of PABPN1 variants were studied by molecular dynamics simulation during a time scale of 1000 ps. The figure shows selected snapshots along the molecular dynamics simulation trajectory for wild-type (wt) PABPN1 and expanded variants with 3, 5 and 7 additional alanines. Sequences of the peptides are indicated at the top of the table. Figure prepared with PyMol [52].
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pone-0006418-g005: Molecular dynamics snapshots of normal and expanded PABPN1 N-terminal peptides.Conformational behavior of peptides corresponding to the N-terminal segment of PABPN1 variants were studied by molecular dynamics simulation during a time scale of 1000 ps. The figure shows selected snapshots along the molecular dynamics simulation trajectory for wild-type (wt) PABPN1 and expanded variants with 3, 5 and 7 additional alanines. Sequences of the peptides are indicated at the top of the table. Figure prepared with PyMol [52].

Mentions: Next, we asked how an expansion in alanine residues may affect the natively disordered structure of the N-terminal segment of PABPN1 using molecular dynamics simulations. N-terminal peptides from wtPABPN1 and extended variants, containing 10, 13, 15 and 17 alanine residues, were studied during a time scale of 1000 ps. Figure 5 shows several representative snapshots of the molecular dynamics trajectory for each peptide. These snapshots were selected to demonstrate typical changes of the peptide structure along the simulation. The simulation results reveal that 10-alanine peptides are very flexible, with no preference for any stable secondary structure. As the number of alanines increases there is an increment of structured secondary elements along the molecular dynamics trajectory. The most striking difference is observed with the 15- and 17-alanine peptides, which form a short alpha helical structure. Analyzing the evolution of the hydrodynamic radius during the simulation shows that, in contrast to peptides with 10 or 13 alanines, peptides with 15 or 17 alanines rapidly evolve to a very compact structure characterized by an abrupt decrease of the hydrodynamic radius of the molecule (Fig. 6A). Analysis of the evolution of secondary structure as a function of time in the simulation was determined by analysis of the trajectories with GROMACS [24]. The results show that 10-alanine peptides behave as a very flexible ensemble along the whole simulation with the majority of the residues in coiled structure (Fig. 6). The simulation with 13-alanine peptides starts with a high content of amino acids in coiled conformation, but at 400 ps the system evolved in a different way, with a decrease of the contribution of the coiled structure and an increase in the residues present in bended fragments. This suggests the presence of a more compact conformation but without any predominant secondary structure elements. The simulation with either 15- or 17-alanine peptides starts with a high content of flexible, non-structured regions and evolves to a structured segment mainly constituted by a helical secondary structure. Particularly interesting is the presence of a stable helical fragment in the 17-alanine peptide at the end of the simulation. This helical fragment is composed of 14 residues distributed by a main stretch of 8 amino acids in α-helix structure and also by a small segment of 5 amino acids that forms a typical 5α-helix (Fig. 6B). The 5α-helix is a subtype of helical secondary structure in which each amino acid corresponds to a 87° turn in the helix (i.e., the helix has 4.1 residues per turn), and a translation of 1.15 Å along the helical axis, being more expanded and flexible than a common α-helix [25]. In conclusion, data from molecular simulations suggest that expansions of polyalanine tracts result in transition from a disordered, flexible conformation to a stable helical secondary structure.


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)

Molecular dynamics snapshots of normal and expanded PABPN1 N-terminal peptides.Conformational behavior of peptides corresponding to the N-terminal segment of PABPN1 variants were studied by molecular dynamics simulation during a time scale of 1000 ps. The figure shows selected snapshots along the molecular dynamics simulation trajectory for wild-type (wt) PABPN1 and expanded variants with 3, 5 and 7 additional alanines. Sequences of the peptides are indicated at the top of the table. Figure prepared with PyMol [52].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712759&req=5

pone-0006418-g005: Molecular dynamics snapshots of normal and expanded PABPN1 N-terminal peptides.Conformational behavior of peptides corresponding to the N-terminal segment of PABPN1 variants were studied by molecular dynamics simulation during a time scale of 1000 ps. The figure shows selected snapshots along the molecular dynamics simulation trajectory for wild-type (wt) PABPN1 and expanded variants with 3, 5 and 7 additional alanines. Sequences of the peptides are indicated at the top of the table. Figure prepared with PyMol [52].
Mentions: Next, we asked how an expansion in alanine residues may affect the natively disordered structure of the N-terminal segment of PABPN1 using molecular dynamics simulations. N-terminal peptides from wtPABPN1 and extended variants, containing 10, 13, 15 and 17 alanine residues, were studied during a time scale of 1000 ps. Figure 5 shows several representative snapshots of the molecular dynamics trajectory for each peptide. These snapshots were selected to demonstrate typical changes of the peptide structure along the simulation. The simulation results reveal that 10-alanine peptides are very flexible, with no preference for any stable secondary structure. As the number of alanines increases there is an increment of structured secondary elements along the molecular dynamics trajectory. The most striking difference is observed with the 15- and 17-alanine peptides, which form a short alpha helical structure. Analyzing the evolution of the hydrodynamic radius during the simulation shows that, in contrast to peptides with 10 or 13 alanines, peptides with 15 or 17 alanines rapidly evolve to a very compact structure characterized by an abrupt decrease of the hydrodynamic radius of the molecule (Fig. 6A). Analysis of the evolution of secondary structure as a function of time in the simulation was determined by analysis of the trajectories with GROMACS [24]. The results show that 10-alanine peptides behave as a very flexible ensemble along the whole simulation with the majority of the residues in coiled structure (Fig. 6). The simulation with 13-alanine peptides starts with a high content of amino acids in coiled conformation, but at 400 ps the system evolved in a different way, with a decrease of the contribution of the coiled structure and an increase in the residues present in bended fragments. This suggests the presence of a more compact conformation but without any predominant secondary structure elements. The simulation with either 15- or 17-alanine peptides starts with a high content of flexible, non-structured regions and evolves to a structured segment mainly constituted by a helical secondary structure. Particularly interesting is the presence of a stable helical fragment in the 17-alanine peptide at the end of the simulation. This helical fragment is composed of 14 residues distributed by a main stretch of 8 amino acids in α-helix structure and also by a small segment of 5 amino acids that forms a typical 5α-helix (Fig. 6B). The 5α-helix is a subtype of helical secondary structure in which each amino acid corresponds to a 87° turn in the helix (i.e., the helix has 4.1 residues per turn), and a translation of 1.15 Å along the helical axis, being more expanded and flexible than a common α-helix [25]. In conclusion, data from molecular simulations suggest that expansions of polyalanine tracts result in transition from a disordered, flexible conformation to a stable helical secondary structure.

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