Transfected cells were maintained for 24 hours without selection; cultures were then subjected to G418 selection before infection. Results Salmonella SPI2 effector protein SseF interacts with TIP60 histone acetylase In a search for host proteins that interact with SseF, we conducted a yeast two-hybrid screening  of a human cell cDNA library, using a fusion of the DNA-binding domain of GAL4 and the truncated SseF devoid of transmembrane Tipifarnib cost regions (pZP784, SseFΔ67-106, 161-174, 186-205) as the bait. One clone was identified which encodes the C-terminal 164-546 TIP60 histone acetyltransferase isoform 1 (Fig. 1). There are at least three splice variants of TIP60: TIP60 isoform 1 (iTIP60), TIP60 isoform
2 (TIP60α), and TIP60 isoform 3 (TIP60β). iTIP60 retains the alternatively spliced intron 1 . TIP60β lacks exon 5 . Different isoforms potentially involve
distinct functions in the cells. When tested in the yeast two-hybrid, all three TIP60 isoforms interacted with GAL4BD-SseF chimerical protein (Fig. 1). To determine the region of SseF that is responsible for interacting with TIP60, a series of SseF deletions was constructed and tested in the yeast two-hybrid for their selleck products NU7441 mw ability to interact with TIP60. We found that amino acids 50-66 were sufficient for mediating the SseF and TIP60 interaction (Fig. 1). We observed weak interactions occasionally when confirming the interactions biochemically using purified recombinant proteins. This is not unusual as most wild-type enzymes do not interact strongly with their target molecules. It is also possible Etoposide that the three putative transmembrane regions in SseF are essential
for tight interactions and the fragment devoid of the transmembrane regions has reduced affinity rendering it difficult to detecting the interactions in vitro. Figure 1 Interaction of SseF with TIP60. (A) Plasmids expressing the SseF devoid of putative transmembrane regions fused to the GAL4 binding domain were transformed into yeast strain AH109 expressing a fusion between the GAL4 activation domain and iTIP60164-546 (pZF1). (B) Plasmids expressing the various SseF fragments fused to the GAL4 binding domain were transformed into yeast strain AH109 expressing a fusion between the GAL4 activation domain and different TIP60 isoforms. SipA together with Plastin was used as a positive control. Yeast strains expressing the above plasmid combinations were streaked on SD-Leu-Trp (-LW) or -Leu-Trp-His+15 mM 3-AT media (-LWH). Quantitative β-galactosidase activities were measured from yeast grown in SD-Leu-Trp and expressed in Miller units. SseF increases the histone acetylation activity of TIP60 TIP60 is a multifunctional acetyltransferase involved in many transcriptional regulations by serving as a co-regulator . The interaction of SseF with TIP60 suggested that SseF may serve as the substrate for TIP60-mediated acetylation.