The intrinsically disordered protein TNIP1 forms puncta in cells and undergoes liquid-liquid phase separation in...

Tumor necrosis factor α-induced protein 3 (TNFAIP3)-interacting protein 1 (TNIP1) is a ubiquitin-binding protein and central repressor of cytoplasmic inflammatory signaling. We have previously shown that TNIP1 is an intrinsically disordered protein (IDP) and that endogenous TNIP1 can be found in cytoplasmic puncta. These TNIP1 protein traits led us to hypothesize that it is capable of liquid-liquid phase separation (LLPS). Here we report the ability of TNIP1 to redistribute the otherwise diffuse signal of EGFP to puncta when the TNIP1-EGFP fusion protein is transiently expressed in HaCaT keratinocyte skin cells. Formation of these puncta is independent of TNIP1 binding to polyubiquitin. The C-terminal region of TNIP1 (amino acids 410-636) contains many of the features of the full-length protein in regard to presence of intrinsic disorder, low complexity regions, and predicted droplet formation. It reversibly forms liquid droplets mainly dependent on electrostatic interactions. While this C-terminal region of TNIP1 contains the protein's ubiquitin-binding region, droplet formation was independent of interaction with polyubiquitin; this observation was mirrored in cells. This sets its in vitro performance apart from other polyubiquitin-binding proteins. These findings have helped newly identify TNIP1 as a phase separating protein. They support future studies to examine the contribution of puncta on the repressing ability of TNIP1 and will broaden understanding of biomolecular condensates in regulating cytoplasmic inflammatory signaling.