Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein

Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein–protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST 4QN ) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST 4QN as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein–protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST 4QN tag could improve the reproducibility and reliability of GST pulldown experiments. Mycobacteria: Regulatory and signalling molecules that promote inflammation A small RNA molecule which controls the expression of genes, and cell signaling molecules called cytokines are associated with the damaging inflammation caused by mycobacteria. Mycobacterial infections, which can affect most organs of the body, and are often resistant to most available drugs, are emerging as a serious global health concern. Patients with suppressed immunity are at particular risk. Increased production of a specific microRNA and of a variety of cytokines during mycobacterial infection has been demonstrated by Hyeon Ji Kim and colleagues at Chungnam National University, Daejon, and co-workers from other institutions in South Korea. Their findings come from comparing peripheral blood mononuclear cells in patients with Mycobacteroides abscessus lung infections with those from healthy controls. The researchers found similar increased microRNA and cytokine production in the lungs of infected mice.