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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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PRMT1 and PRMT3 accumulate in OPMD intranuclear inclusions.(A–O) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. (P–Q) From each patient (OPMD1 and OPMD2), approximately 60 nuclei labelled with anti-PRMT1 antibody were analyzed. The mean fluorescence intensity measured in the nucleoplasm is higher in nuclei devoid of inclusions compared to nuclei with inclusions (P). The mean fluorescence intensity measured in the nuclear inclusions is approximately two-fold higher than the intensity measured in the nucleoplasm of nuclei with inclusions (Q). Quantitative measurements of fluorescence intensity were performed with Image J software and expressed in arbitrary units (AU). Results are presented as mean fluorescence intensity±SE; p values (Student's t-test) are indicated.
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pone-0006418-g002: PRMT1 and PRMT3 accumulate in OPMD intranuclear inclusions.(A–O) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. (P–Q) From each patient (OPMD1 and OPMD2), approximately 60 nuclei labelled with anti-PRMT1 antibody were analyzed. The mean fluorescence intensity measured in the nucleoplasm is higher in nuclei devoid of inclusions compared to nuclei with inclusions (P). The mean fluorescence intensity measured in the nuclear inclusions is approximately two-fold higher than the intensity measured in the nucleoplasm of nuclei with inclusions (Q). Quantitative measurements of fluorescence intensity were performed with Image J software and expressed in arbitrary units (AU). Results are presented as mean fluorescence intensity±SE; p values (Student's t-test) are indicated.

Mentions: As the major pathological hallmark of OPMD consists of intranuclear inclusions formed by deposition of PABPN1 fibrils, we next analyzed the sub-cellular distribution of proteins identified as preferentially associated with expanded PABPN1. Immunofluorescence was performed on muscle fibers from two OPMD patients that have one normal PABPN1 allele (GCG6) and one expanded allele (GCG11) coding for a protein with a polyalanine tract of 15 residues. Typical intranuclear inclusions were detected using anti-PABPN1 antibodies (Fig. 2A, D, G, J, M), and double-labelling experiments reveal that both PRMT1 (Fig. 2B, C) and PRMT3 (Fig. E, F) are concentrated in the inclusions. In contrast to PRMT1 and PRMT3, which are protein arginine methyltransferases involved in the modification of PABPN1 [11], [12], PRMT2, which does not methylate PABPN1, is not detected in the inclusions (Fig. 2H, I). The intranuclear inclusions are intensely stained by anti-peptide antibodies that specifically recognize asymmetrically dimethylated PABPN1 (Fig. 2K, L), but do not react with antibodies specific to unmethylated PABPN1 (Fig. 2N, O). These results indicate that pathological aggregates of PABPN1 consist of methylated protein and accumulate PRMT1 and PRMT3.


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)

PRMT1 and PRMT3 accumulate in OPMD intranuclear inclusions.(A–O) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. (P–Q) From each patient (OPMD1 and OPMD2), approximately 60 nuclei labelled with anti-PRMT1 antibody were analyzed. The mean fluorescence intensity measured in the nucleoplasm is higher in nuclei devoid of inclusions compared to nuclei with inclusions (P). The mean fluorescence intensity measured in the nuclear inclusions is approximately two-fold higher than the intensity measured in the nucleoplasm of nuclei with inclusions (Q). Quantitative measurements of fluorescence intensity were performed with Image J software and expressed in arbitrary units (AU). Results are presented as mean fluorescence intensity±SE; p values (Student's t-test) are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712759&req=5

pone-0006418-g002: PRMT1 and PRMT3 accumulate in OPMD intranuclear inclusions.(A–O) Immunofluorescence was performed on biopsy samples of quadriceps muscle from two OPMD patients using the indicated antibodies. Scale bar = 5 µm. (P–Q) From each patient (OPMD1 and OPMD2), approximately 60 nuclei labelled with anti-PRMT1 antibody were analyzed. The mean fluorescence intensity measured in the nucleoplasm is higher in nuclei devoid of inclusions compared to nuclei with inclusions (P). The mean fluorescence intensity measured in the nuclear inclusions is approximately two-fold higher than the intensity measured in the nucleoplasm of nuclei with inclusions (Q). Quantitative measurements of fluorescence intensity were performed with Image J software and expressed in arbitrary units (AU). Results are presented as mean fluorescence intensity±SE; p values (Student's t-test) are indicated.
Mentions: As the major pathological hallmark of OPMD consists of intranuclear inclusions formed by deposition of PABPN1 fibrils, we next analyzed the sub-cellular distribution of proteins identified as preferentially associated with expanded PABPN1. Immunofluorescence was performed on muscle fibers from two OPMD patients that have one normal PABPN1 allele (GCG6) and one expanded allele (GCG11) coding for a protein with a polyalanine tract of 15 residues. Typical intranuclear inclusions were detected using anti-PABPN1 antibodies (Fig. 2A, D, G, J, M), and double-labelling experiments reveal that both PRMT1 (Fig. 2B, C) and PRMT3 (Fig. E, F) are concentrated in the inclusions. In contrast to PRMT1 and PRMT3, which are protein arginine methyltransferases involved in the modification of PABPN1 [11], [12], PRMT2, which does not methylate PABPN1, is not detected in the inclusions (Fig. 2H, I). The intranuclear inclusions are intensely stained by anti-peptide antibodies that specifically recognize asymmetrically dimethylated PABPN1 (Fig. 2K, L), but do not react with antibodies specific to unmethylated PABPN1 (Fig. 2N, O). These results indicate that pathological aggregates of PABPN1 consist of methylated protein and accumulate PRMT1 and PRMT3.

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