Journal: The Journal of Biological Chemistry
Article Title: Physiological and pathophysiological characteristics of ataxin-3 isoforms
Figure Lengend Snippet: A, volcano plots comparing the interaction of normal ataxin-3 isoforms. HEK 293T cells were transfected with pN-SF-TAP-ataxin-3 isoforms and grown in SILAC medium for 72 h. Ataxin-3 was purified by the Strep-tag. Isoform-specific purifications were combined afterward and concentrated by precipitation before the identification of co-precipitated proteins by MS. Volcano plots show a direct comparison between the p value of interactors of two isoforms (ataxin-3c versus 3aL, ataxin-3c versus 3aS, and ataxin-3aL versus 3aS) and the difference between the isoforms. The plots are divided into four quadrants (small scheme). The bottom two show interactors with insignificant differences (light gray open circle, p > 0.05); the top ones show interactors that interact significantly more strongly with one isoform, depending on the side. Although significant, interactors with p < 0.05 were only considered stronger or weaker upon a difference of at least ±1. Although sharing numerous interactions (light gray open and filled circles), ataxin-3 isoforms show divergent interactions with different proteins. Partners interacting more strongly with ataxin-3c (black filled circles), ataxin-3aL (blue filled circles), and ataxin-3aS (yellow filled circles) could be identified. Ataxin-3c and ataxin-3a show more differences in their interaction than ataxin-3aL and ataxin-3aS. B, Venn diagram comparing proteins that interact more strongly with ataxin-3aL and ataxin-3aS compared with ataxin-3c. 65 interaction partners interact more strongly with ataxin-3aS than ataxin-3c. 38 partners have a stronger interaction with both ataxin-3aS and ataxin-3aL, whereas seven partners have a stronger interaction with ataxin-3aL. A GO annotation of these proteins and analysis of KEGG pathways revealed that these interactors take part in different pathways, whereas proteins associated with Huntington's disease and Parkinson's disease can be found in all three groups. C, interaction network for proteins showing a stronger binding to ataxin-3c. Ataxin-3c shows a common interaction with HR23B and NGLY1 as well as UBR2 and HNRNPL, specifying its role in the ERAD pathway of glycosylated proteins.
Article Snippet: Vectors for ataxin-3 interaction partners pcDNA3-V5-hHR23A and pcDNA3-V5-hHR23B were a gift from Steven Grossman (Addgene plasmid 13054) ( 100 ), and pcDNA3.1(−)-FLAG-UBR2 was a gift from Alexander Varshavsky (Addgene plasmid 50573).
Techniques: Transfection, Purification, Strep-tag, Binding Assay