HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation.

Tristetraprolin (TTP) is a critical negative immune regulator. It binds AU-rich elements in the untranslated-regions of many mRNAs encoding pro-inflammatory mediators, thereby accelerating their decay. A key but poorly understood mechanism of TTP regulation is its timely proteolytic removal: TTP is degraded by the proteasome through yet unidentified phosphorylation-controlled drivers. In this study, we set out to identify factors controlling TTP stability. Cellular assays showed that TTP is strongly lysine-ubiquitinated, which is required for its turnover. A genetic screen identified the ubiquitin E3 ligase HUWE1 as a strong regulator of TTP proteasomal degradation, which we found to control TTP stability indirectly by regulating its phosphorylation. Pharmacological assessment of multiple kinases revealed that HUWE1-regulated TTP phosphorylation and stability was independent of the previously characterized effects of MAPK-mediated S52/S178 phosphorylation. HUWE1 function was dependent on phosphatase and E3 ligase binding sites identified in the TTP C-terminus. Our findings indicate that while phosphorylation of S52/S178 is critical for TTP stabilization at earlier times after pro-inflammatory stimulation, phosphorylation of the TTP C-terminus controls its stability at later stages.

SPOP targets the immune transcription factor IRF1 for proteasomal degradation.

Adaptation of the functional proteome is essential to counter pathogens during infection, yet precisely timed degradation of these response proteins after pathogen clearance is likewise key to preventing autoimmunity. Interferon regulatory factor 1 (IRF1) plays an essential role as a transcription factor in driving the expression of immune response genes during infection. The striking difference in functional output with other IRFs is that IRF1 also drives the expression of various cell cycle inhibiting factors, making it an important tumor suppressor. Thus, it is critical to regulate the abundance of IRF1 to achieve a 'Goldilocks' zone in which there is sufficient IRF1 to prevent tumorigenesis, yet not too much which could drive excessive immune activation. Using genetic screening, we identified the E3 ligase receptor speckle type BTB/POZ protein (SPOP) to mediate IRF1 proteasomal turnover in human and mouse cells. We identified S/T-rich degrons in IRF1 required for its SPOP MATH domain-dependent turnover. In the absence of SPOP, elevated IRF1 protein levels functionally increased IRF1-dependent cellular responses, underpinning the biological significance of SPOP in curtailing IRF1 protein abundance.

A repetitive acidic region contributes to the extremely rapid degradation of the cell-context essential protein TRIM52.

Tripartite motif protein 52 (TRIM52) is a non-canonical TRIM family member harbouring the largest RING domain encoded in the human genome. In humans TRIM52 is conserved and has been under positive selection pressure, yet it has been lost in many non-primates. Competitive cell fitness assays demonstrated that TRIM52 ablation reduces cellular fitness in multiple different cell types. To better understand how this cell-essential factor is controlled, we investigated how expression of this non-canonical protein is regulated. Here, we show that TRIM52 mRNA is constitutively expressed from an intergenic region preceding the TRIM52 gene. Yet, TRIM52 protein is rapidly turned-over by the proteasome with a 3.5-minute half-life, one of the shortest in the human proteome. Consistent with this extremely rapid degradation rate, all three TRIM52 domains were identified to contribute to its instability. Intriguingly, a repetitive acidic loop in the RING domain was identified as one of the main destabilizing regions, which was unexpected given the prevailing notion that these sequences are poor proteasome substrates. This work indicates that the effect of such repetitive acidic regions on proteasomal degradation depends on the protein context, and it identifies TRIM52 as an attractive model protein to study what these contextual properties are.