Our innate immune system is an important part of the defense against harmful pathogens, but it is a double-edged sword. Inflammation is a systemic response that recruits specialized immune cells and signaling molecules to the site of infection. Extensive inflammation, however, can lead to local tissue damage and is a hallmark of many autoimmune diseases. To prevent excessive inflammation, cells have developed mechanisms to keep these responses in check. TTP is an RNA-binding protein that plays an important role in attenuating the inflammatory response by helping to degrade mRNAs that encode for pro-inflammatory molecules. "The fascinating part about the regulation of TTP is that it is activated with the inflammatory response," says group leader Gijs Versteeg. "In these early stages, it binds to its targets but does not degrade them. At later stages TTP becomes active and at the same time a target for degradation." While it has previously been shown that phosphorylation of TTP plays an important role in regulation of the initial inflammatory response, details of how TTP activity is controlled thereafter have been missing.
In her PhD project, first author of the study Sara Scinicariello set out to understand more about how TTP is regulated, which ultimately results in its degradation. In collaboration with Johannes Zuber, the team performed a genetic screen and identified the ubiquitin E3 ligase HUWE1 as a previously unrecognized regulator of TTP. They were able to show that it mediates the degradation of TTP in later stages of the immune response, dependent on de-phosphorylation of residues in its C-terminus. TTP is degraded by the proteasome, a cellular quality control mechanism that removes unneeded proteins. The proteasome relies primarily on ubiquitination, a chemical tag that marks proteins for degradation. "It was previously described that TTP was degraded by a mechanism independent of ubiquitination. Our results now show that this is not the case and that TTP is heavily ubiquitinated and that this ubiquitination is required for its degradation," explains Gijs Versteeg.
The scientists now aim to gain more detailed insight into the interplay between HUWE1-mediated ubiquitination and TTP phosphorylation. More than 10% of TTP residues have been reported to be modified by phosphorylation, making TTP one of the most heavily phosphorylated proteins in the cell. "There is an intricate timing mechanism at work that carries TTP through the inflammatory response from inactive, to active, and eventually to its degradation," says Gijs Versteeg. "It will be interesting to elucidate how the 30+ phosphorylation sites in TTP contribute to this process."
Sara Scinicariello, Adrian Soderholm, Markus Schäfer, Alexandra Shulkina, Irene Schwartz, Kathrin Hacker, Rebeca Gogova, Robert Kalis, Kimon Froussios, Valentina Budroni, Annika Bestehorn, Tim Clausen, Pavel Kovarik, Johannes Zuber, Gijs A Versteeg: HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation. eLife 2023.