Abstract
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 (polyadenylate-binding protein nuclear 1) gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology. An emerging hypothesis is that a loss of PABPN1 function and/or aberrant protein interactions contribute to pathology in OPMD. Here, we present the first global proteomic analysis of the protein interactions of WT and alanine-expanded PABPN1 in skeletal muscle tissue. These data provide both insight into the function of PABPN1 in muscle and evidence that the alanine expansion alters the protein-protein interactions of PABPN1. We extended this analysis to demonstrate altered complex formation with and loss of function of TDP-43 (TAR DNA-binding protein 43), which we show interacts with alanine-expanded but not WT PABPN1. The results from our study support a model where altered protein interactions with alanine-expanded PABPN1 that lead to loss or gain of function could contribute to pathology in OPMD.
Highlights
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 gene that results in an alanine expansion at the N terminus of the PABPN1 protein
Immunoprecipitation of FLAG-PABPN1 from these electroporated muscles followed by MS allowed for the first comparison of the protein interactions of the WT and OPMD-associated alanine-expanded PABPN1 in skeletal muscle tissue
We examined the possible consequences of TDP-43 interactions with alanine-expanded PABPN1 by employing two additional OPMD mouse models
Summary
Levels of PABPN1 protein are very low in skeletal muscle [15], making biochemical studies of PABPN1 protein interactions in this disease-relevant tissue challenging [29]. Immunoblot analysis of fractions collected from top to bottom of the density gradients shows that TDP-43 in lysate from OPMD model (Ala-17/Ala10) myotubes migrates in heavier fractions when compared with lysate prepared from WT (Ala-10/Ala-10) myoblasts (Fig. 5B) These results imply that soluble TDP-43 exists in larger protein complexes in myotubes expressing Ala-17 PABPN1 than in control myotubes. HMW TDP-43 was not detected in the pellet fraction in control cells that express Ala-10/Ala-10 PABPN1 in either the absence (Ϫ) or the presence (ϩ) of hydrogen peroxide treatment This result demonstrates that TDP-43 is more prone to redox-mediated aggregation in cells obtained from the OPMD Ala-17/ Ala-10 mouse model compared with WT muscle cells. Results from quantitative RT-PCR analysis confirm the increased inclusion of the Sort1 17b cassette exon in myoblasts isolated from the OPMD Ala-17/Ala-10 mouse model (Fig. 6C) These data suggest that TDP-43 function is impaired in muscle cells expressing Ala-17 PABPN1. The increased uptake of both glucose and N-TAP PGRN suggests that the inclusion of the cassette exon in Sort affects the function of the SORT1 protein in muscle cells that express Ala-17 PABPN1
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
