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.

The MFS efflux pump EmrKY contributes to the survival of Shigella within macrophages.

Efflux pumps are membrane protein complexes conserved in all living organisms. Beyond being involved in antibiotic extrusion in several bacteria, efflux pumps are emerging as relevant players in pathogen-host interactions. We have investigated on the possible role of the efflux pump network in Shigella flexneri, the etiological agent of bacillary dysentery. We have found that S. flexneri has retained 14 of the 20 pumps characterized in Escherichia coli and that their expression is differentially modulated during the intracellular life of Shigella. In particular, the emrKY operon, encoding an efflux pump of the Major Facilitator Superfamily, is specifically and highly induced in Shigella-infected U937 macrophage-like cells and is activated in response to a combination of high K+ and acidic pH, which are sensed by the EvgS/EvgA two-component system. Notably, we show that following S. flexneri infection, macrophage cytosol undergoes a mild reduction of intracellular pH, permitting EvgA to trigger the emrKY activation. Finally, we present data suggesting that EmrKY is required for the survival of Shigella in the harsh macrophage environment, highlighting for the first time the key role of an efflux pump during the Shigella invasive process.

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.

Author Correction: The MFS efflux pump EmrKY contributes to the survival of Shigella within macrophages.

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Human tripartite motif protein 52 is required for cell context-dependent proliferation.

Tripartite motif (TRIM) proteins have been shown to play important roles in cancer development and progression by modulating cell proliferation or resistance from cell death during non-homeostatic stress conditions found in tumor micro-environments. In this study, we set out to investigate the importance for cellular fitness of the virtually uncharacterized family member TRIM52. The human TRIM52 gene has arisen recently in evolution, making it unlikely that TRIM52 is required for basic cellular functions in normal cells. However, a recent genome-wide ablation screening study has suggested that TRIM52 may be essential for optimal proliferation or survival in certain genetic cancer backgrounds. Identifying genes which fit this concept of genetic context-dependent fitness in cancer cells is of interest as they are promising targets for tumor-specific therapy. We report here that TRIM52 ablation significantly diminished the proliferation of specific glioblastoma cell lines in cell culture and mouse xenografts by compromising their cell cycle progression in a p53-dependent manner. Together, our findings point to a non-redundant TRIM52 function that is required for optimal proliferation.