The transglutaminase 2 interactome in HUVECs suggests its participation in an RNA-binding protein network
Human transglutaminase 2 (TG2) is a multifunctional protein that exhibits various protein-modifying catalytic and protein-protein interaction properties. HUVECs express high levels of TG2, and identification of its interacting partners may provide mechanistic insight into its role in endothelial functions such as adhesion, migration, angiogenesis, and NO homeostasis. For this purpose, two approaches were employed. Firstly, bacterially expressed, site-specifically biotinylated recombinant TG2 was mixed with HUVEC extract to isolate interacting partners by affinity chromatography. Secondly, endogenous TG2 was silenced, and to ensure high antibody affinity, a triple Flag-tagged transgenic TG2-expressing HUVEC line was created, allowing stable binding to anti-Flag antibody-coated agarose for isolating TG2-associated protein complexes. Altogether, 170 and 356 TG2-associated proteins were identified, respectively, with 86 proteins overlapping between the two approaches. The most enriched GO Molecular Functions of cellularly assembled TG2-interacting proteins confirm TG2s involvement in cell-cell and cell-extracellular matrix communication, adhesion, cytoskeleton organisation, and exosome secretion. Stabilising the catalytically inactive closed TG2 conformation reduced the number of TG2 interactors, whereas stabilising its open form increased the number of associated membrane transporters. Significant enrichment of RNA-binding proteins associated with TG2 was observed in all experiments, and a 42% overlap between the TG2 interactome and previously identified RNA-binding proteins in HUVEC was noted. Considering the recently recognised RNA-binding ability of TG2, our results suggest that TG2 participates in post-transcriptional regulations as a central hub within the network of RNA-binding proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/672361v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@169329corg.highwire.dtl.DTLVardef@179d5d4org.highwire.dtl.DTLVardef@1c58f9aorg.highwire.dtl.DTLVardef@156def0_HPS_FORMAT_FIGEXP M_FIG Cellularly assembled TG2-associated proteins were isolated and identified from endogenous TG2-silenced (eTG2-KD) and transgenic, high-affinity antibody, triple Flag-tagged TG2 (3F-TG2) expressing HUVECs under untreated conditions and after NC9 or GTP{gamma}S inhibitor treatments, which stabilise its open or closed conformation, respectively. TG2 interactome analysis suggests that it acts as a central hub in an RNA-binding protein (RBP) network. C_FIG