HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation

  1. Sara Scinicariello
  2. Adrian Soderholm
  3. Markus Schäfer
  4. Alexandra Shulkina
  5. Irene Schwartz
  6. Kathrin Hacker
  7. Rebeca Gogova
  8. Robert Kalis
  9. Kimon Froussios
  10. Valentina Budroni
  11. Annika Bestehorn
  12. Tim Clausen
  13. Pavel Kovarik
  14. Johannes Zuber
  15. Gijs A Versteeg

  • Curated by eLife

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    The RNA-binding protein Tristetraprolin (TPP) regulates the abundance of mRNAs encoding proinflammatory cytokines. The study by Scinicariello and collaborators examined mechanisms regulating the turnover of TTP in cultured cells and identified the ubiquitin E3 ligase HUWE1 as a regulator of TPP degradation. The conclusions are largely supported by the cellular and biochemical experiments. This paper thus implicates the HUWE1-TPP axis in regulating macrophage inflammatory responses at the post-transcriptional steps.

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Abstract

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.

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  1. Version published to 10.7554/elife.83159 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    1. (Schichl et al. 2011 JBC 286:38466). This publication is not cited in the current version of the manuscript. The results of Schichl et al. seem particularly relevant for the interpretation of some of the results presented here and should be considered in the final discussion and conclusions of the present work.

    This reference and related text was added in the discussion section in the revised manuscript (lines 508-517).

    1. The ubiquitination of endogenous TTP has not been demonstrated.

    New data assessing the ubiquitination of endogenous TTP was added as Figure 1 – figure supplement 1D.

    1. The type of ubiquitination detected on the overexpressed version of TTP is not characterized. This seems important in view of the results of Schichl et al. who showed non-degradative ubiquitination (K63) of TTP.

    New data with the detection of K48- or K63-linked poly-ubiquitin chain by specific antibodies was added as Figure 1 – figure supplement 1G. These data show that recombinant poly-ubiquitin chains can be readily detected with both antibodies, but that only K48-linked chains were detected on TTP IPed from cells.

    1. The half-life of the non-ubiquitinated mutant of TTP (K→R) was not precisely compared to the half-life of the wild-type TTP protein (similar to the experiment presented in 1B).

    New data from TTP-KtoR chase experiments was added as Figure 1 – figure supplement 1E. The half-life was increased substantially from 1.4 h for wtTTP to 5.7 h for the mutant.

    1. The effect of the E1 ubiquitin ligase TAk-243 on endogenous TTP levels was not tested.

    New data assessing the effect of TAK-243 on endogenous TTP was added as Figure 1 – figure supplement 1B. Consistent with our data with exogenously expressed TTP, treatment with the inhibitor increased the abundance of endogenous TTP.

    1. While they demonstrate that TTP-HA is efficiently degraded after 3 to 7h of LPS stimulation (Fig 1B) and that the stronger decrease in mCherry-TTP fusion level occurs between 4 and 6h of LPS stimulation the screen for identification of TTP modulators is performed 16h of LPS stimulation (Fig 2A). The rationale behind this experimental setting is not explicitly described.

    We found that endogenous TTP and mCherry-TTP levels were substantially lower at 16 h post-LPS stimulation compared to 6 h. (see Fig. 1D), and reasoned that this would yield the best genetic screen window in which to identify mutant cells with non-functional degradation mechanisms.

    1. The authors did not directly test the effect of HUWE1 inactivation on endogenous TTP accumulation after blocking protein synthesis. This control seems important as data presented in figure 2E could result both from an effect of Huwe1 level on LPS-induced TTP synthesis and TTP degradation.

    New data from chase experiments with endogenous TTP have been added as Fig. 2G. Consistent with the data presented in Fig. 2E, TTP levels declined during the chase period in sgROSA control cells, with an estimated half-life of 3.7 h. In contrast, TTP levels did not significantly decline during the CHX chase period in Huwe1 KO cells, resulting in an estimated TTP protein half-life of ~20 h in this genotype.

    1. In the data presented in figure 2, it is not entirely clear what exactly the authors are referring to as "endogenous TTP". In Figure 2C endogenous TTP is detected by western blot on cells transfected with an mCherry-TTP fusion. In this case, the size difference allows unambiguous identification of the endogenous form of TTP (although one could not exclude that overexpressing a TTP fusion protein might affect the level of the endogenous protein). However, TTP and mCherry-TTP cannot be distinguished by FACS (Fig2 D and E). If cells used in the experiments shown in 2C and 2D-E are distinct, this should be mentioned more explicitly in the legend of Fig. 2. Otherwise, the detection of endogenous TTP should be performed on cells that do not express mCherry-TTP.

    Results from Fig. 2D/E are indeed from cells that do not express mCherry-TTP. Endogenous TTP is detected in these cells by intracellular antibody staining. The figure legend text has been updated to reflect that panel 2C is with the RAW264.7-Dox-Cas9-mCherry-TTP cell line, and D-E is with the RAW264.7-Dox-Cas9 cell line.

    1. The third part of the manuscript aims to demonstrate that loss of Huwe1 decreases the half-life of pro-inflammatory mRNAs controlled by TTP. In my opinion, this conclusion is reliably supported by the data presented in Figure 3 and Supplementary Figure 3. As the conclusion of this paragraph refers to the effect of TTP on the stability of these mRNAs, the measurement of TNF mRNA stability (Fig. sup. 3C) should be presented in the main part of Fig. 3.

    The TNF mRNA stability figure panel was moved to the main figures as Fig. 3C.

    1. Fig 4E aims to identify kinases and phosphatases potentially involved in TTP stability (line 277, line 298). However, the approach used here (a measure of intracellular TTP level) cannot distinguish between increased production of TTP or a decrease in TTP degradation.

    One of the main points of this experiment was to assess whether the steady-state increase in TTP in HUWE1 KO cells, which stems for an important part from increased stability (Fig. 2G), was influenced by TTP phospho-status. Thus, while we do not explicitly measure TTP protein half-life in this particular assay, it is very likely to reflect changes in TTP protein stability. This idea is consistent with the fact that treatment with p38i, MK2i, and CaclycA affected TTP steady-state levels consistent with their previously reported effects on TTP protein stability.

    1. Also, the result presented in fig. 4E, are not totally consistent with the results presented in 4A. Fig4D shows a similar level of endogenous TTP accumulating after 2h of LPS stimulation in Huwe1 KO and control cells while a clear difference in TTP level is observable in the same condition in fig. 4A. Could the difference in the TTP detection method (Western vs intracellular FACS) be responsible for this discrepancy?

    We do not exactly know, but agree that this could indeed be influenced by the measurement method per se, as well as small variations in cell density, or total sample numbers in a particular experiment (as this may increase the time outside of the incubator for handling/stimulations). The much larger sample size of the experiment from panel 6E, and having multiple different stimulations, may have contributed to a slightly delayed timing of the Huwe1-dependent phenotype. It is important to note, that we have consistently demonstrated with different measurement methods, that TTP is initially stabilized post-LPS treatment (2-3 h, insensitive to Huwe1 KO), followed by TTP degradation (6-16h, sensitive to Huwe1 KO).

    1. These experiments and data presented in Fig.5D show that the level of the TTP paralog ZFP36L1 accumulates in huwe1 KO cells but do not demonstrate that HUWE1 affects ZFP36L1 protein stability.

    We agree, and changed all instances in the text that claimed ZFP36L1 ‘stabilization’ to ‘increase in abundance’.

    1. Based on data presented in fig. 6 B and sup. 6B the authors conclude that residues S52 and 178, previously identified as regulators of TTP stability, are unlikely to be involved in HUWE1-dependent TTP accumulation. The data are only based on 2 independent experiments, one of which (fig 6B) shows a difference in TTP S52/S178 mutant in Huwe1 deficient cells as compared to wt TTP. These results seem therefore too preliminary to reliably exclude the implication of S52 and 178 on the HUWE1 accumulation of TTP.

    Additional new data with the S52/178 TTP mutant of six biological replicates has been added to the manuscript as Figure 6 – figure supplement 1C. Data from these experiments are consistent with our other results, and show that protein levels similarly increase for both wtTTP and the S52/178A mutant in Huwe1 KO cells.

    1. From these data, the authors conclude (line 416) that N-terminal deletion does not affect the TTP protein level. However, TTP accumulation in Huwe1 KO cells seems mostly lost in mutant N4. As mentioned above the limited number of replicates (n=2) and the absence of a statistical test makes the interpretation of this result difficult.

    Additional new data with the Δ4 mutant of two biological replicates has been added to the manuscript as Figure 6 – figure supplement 1E. Data from these experiments are consistent with our other results, and show that protein levels similarly increase for the Δ4 mutant in Huwe1 KO cells.

    1. Several TTP C-terminal mutants show a HUWE1-independent accumulation when compared to the wt protein (Fig6. D). Is this region identical to the unstructured region identified by Ngoc (line 1255) as a potent regulator of TTP degradation? If relevant this point should be discussed.

    Ngoc showed that fusion to GFP of either the N-terminal TTP part, or the TTP Cterminal part (aa 214-436), destabilized GFP in cells. Thus, the GFP destabilization was seemingly indiscriminate, and possibly caused by the disordered nature of the fusion construct per se. Since the C-terminal TTP part fused to GFP by Ngoc included aa 214-436, we cannot rule out that part of this effect was HUWE1-dependent. However, the discrepancy with our finding that the TTP N-terminus does not contribute to HUWE1-dependent TTP regulation, may suggest that the GFP fusions by Ngoc were destabilized by more general protein principles, rather than HUWE1-specific effects. Additional text conveying this notion was added to the Discussion section (line 490-497).

  3. eLife

    eLife assessment

    The RNA-binding protein Tristetraprolin (TPP) regulates the abundance of mRNAs encoding proinflammatory cytokines. The study by Scinicariello and collaborators examined mechanisms regulating the turnover of TTP in cultured cells and identified the ubiquitin E3 ligase HUWE1 as a regulator of TPP degradation. The conclusions are largely supported by the cellular and biochemical experiments. This paper thus implicates the HUWE1-TPP axis in regulating macrophage inflammatory responses at the post-transcriptional steps.

  4. eLife

    Reviewer #1 (Public Review):

    The study by Scinicariello et al. set out to identify novel factors that controlled TTP stability and identified HUWE1 by CRISPR screening in macrophages. HUWE1 phosphorylated TTP on residues distinct from those phosphorylated by MAPKs and regulated TTP protein stability. Overall, the biochemical and cellular signaling experiments were thoughtfully designed and well executed, leading to the discovery of HUWE1 as a TTP regulator.

  5. eLife

    Reviewer #2 (Public Review):

    TPP is critical for regulating the mRNA abundance of proinflammatory cytokines. Sara Scinicariello et al., identified ubiquitin E3 ligase HUWE1 function as a key regulator of the TPP degradation, which could direct the related immune responses. However, the physiological importance and their major conclusions were not fully clarified or supported by the experimental data.

  6. eLife

    Reviewer #3 (Public Review):

    The manuscript by Scinicariello and collaborators examines the mechanisms regulating the cellular accumulation of the RNA-binding protein Tristetraprolin (TTP). This factor is a well-described regulator of mRNA stability. TTP binds to RNA AU-rich sequences localized in mRNA 3'Untranslated regions. As AU-rich elements are abundant in mRNA encoding pro-inflammatory factors, TTP has been described as a negative regulator of the inflammatory response.

    Previous reports have described that the cellular level of TTP is modulated by phosphorylation and proteasome-dependent process (see several references in the introduction of the manuscript). Non-degradative phosphorylation-dependent ubiquitination of TTP has also been reported (Schichl et al. 2011 JBC 286:38466). This publication is not cited in the current version of the manuscript. The results of Schichl et al. seem particularly relevant for the interpretation of some of the results presented here and should be considered in the final discussion and conclusions of the present work.

    In the first part of the results section, Scinicariello et al. evaluate the degradation and ubiquitination of TTP and conclude that TTP is degraded in a ubiquitin-dependent manner. By a pharmacological approach, they observed, as previously shown, that endogenous TTP is degraded by the proteasome (Fig1a). They also show that an overexpressed tagged version of TTP is degraded by the proteasome and ubiquitinated on lysine residues (Fig. 1B, C). The general conclusion of this paragraph seems premature in relation to the results presented. The ubiquitination of endogenous TTP has not been demonstrated. The type of ubiquitination detected on the overexpressed version of TTP is not characterized. This seems important in view of the results of Schichl et al. who showed non-degradative ubiquitination (K63) of TTP. The half-life of the non-ubiquitinated mutant of TTP (K→R) was not precisely compared to the half-life of the wild-type TTP protein (similar to the experiment presented in 1B). The effect of the E1 ubiquitin ligase TAk-243 on endogenous TTP levels was not tested.

    In the second part, the authors identified the E3 ligase HUWE1 as a major determinant of cellular TTP protein abundance. This demonstration is first based on the identification of HUWE1 in an unbiased CRISPR/cas9 screen to identify modulators of mCherry-TTP fusion reporter accumulation upon activation of RAW 264.7 cells by LPS. While they demonstrate that TTP-HA is efficiently degraded after 3 to 7h of LPS stimulation (Fig 1B) and that the stronger decrease in mCherry-TTP fusion level occurs between 4 and 6h of LPS stimulation the screen for identification of TTP modulators is performed 16h of LPS stimulation (Fig 2A). The rationale behind this experimental setting is not explicitly described. Nevertheless, the authors convincingly demonstrate that HUWE1 is involved in the controls of TTP cellular abundance. This demonstration mainly relies on the fact that HUWE1 inactivation induced a strong increase of both mCherry-TTP fusion and endogenous TTP (Fig. 2B and C). Ablation of HUWE1 selectively decreases the abundance of a limited number of proteins including TTP (Fig. 5A). The specificity of Huwe1 effect is confirmed by the detection of a constant level of the co-expressed BFP protein upon HUWE1 depletion (fig sup. 2E). The effect of HUWE1 depletion on TTP accumulation is observed in different cell lines and primary cells (murine, human) (Fig. sup. 2G, Fig2F).
    In this paragraph, the demonstration that Huwe1 specifically affects the stability of TTP protein appears less robust. The authors did not directly test the effect of HUWE1 inactivation on endogenous TTP accumulation after blocking protein synthesis. This control seems important as data presented in figure 2E could result both from an effect of Huwe1 level on LPS-induced TTP synthesis and TTP degradation.

    In the data presented in figure 2, it is not entirely clear what exactly the authors are referring to as "endogenous TTP". In Figure 2C endogenous TTP is detected by western blot on cells transfected with an mCherry-TTP fusion. In this case, the size difference allows unambiguous identification of the endogenous form of TTP (although one could not exclude that overexpressing a TTP fusion protein might affect the level of the endogenous protein). However, TTP and mCherry-TTP cannot be distinguished by FACS (Fig2 D and E). If cells used in the experiments shown in 2C and 2D-E are distinct, this should be mentioned more explicitly in the legend of Fig. 2. Otherwise, the detection of endogenous TTP should be performed on cells that do not express mCherry-TTP.

    The third part of the manuscript aims to demonstrate that loss of Huwe1 decreases the half-life of pro-inflammatory mRNAs controlled by TTP. In my opinion, this conclusion is reliably supported by the data presented in Figure 3 and Supplementary Figure 3. As the conclusion of this paragraph refers to the effect of TTP on the stability of these mRNAs, the measurement of TNF mRNA stability (Fig. sup. 3C) should be presented in the main part of Fig. 3.

    The authors then aim to demonstrate that HUWE1 regulates TTP phosphorylation and its increase is responsible for increased TTP stability. Taken together, data from fig. 1F, 2C, and 2F clearly show that a phosphorylated form of TTP is accumulated in Huwe1 deficient cells. The authors state that Fig 4E aims to identify kinases and phosphatases potentially involved in TTP stability (line 277, line 298). However, the approach used here (a measure of intracellular TTP level) cannot distinguish between increased production of TTP or a decrease in TTP degradation. Also, the result presented in fig. 4E, are not totally consistent with the results presented in 4A. Fig4D shows a similar level of endogenous TTP accumulating after 2h of LPS stimulation in Huwe1 KO and control cells while a clear difference in TTP level is observable in the same condition in fig. 4A. Could the difference in the TTP detection method (Western vs intracellular FACS) be responsible for this discrepancy? In addition, the absence of positive control for the various pharmacological treatments renders difficult the interpretation of these results, especially when the inhibitor shows no effect on TTP level (ex: CalyculinA). On this basis, the authors' conclusions for this paragraph seem partially over-interpreted.

    From the data presented in figure 5, the authors conclude that HUWE1 controls only a small fraction of proteasome targets and regulates the stability of TTP paralog ZFP36L1.
    A comparison of protein levels in Huwe1 and Psmb7 Ko cells reveals that Huwe1 ablation significantly changes the concentrations of only a limited number of proteins (Fig. 5A). The reliability of these data is confirmed by the identification as increased proteins in the huwe1 ko of factors previously identified as targets of HUWE1 (Fig. sup. 5C). These experiments and data presented in Fig.5D show that the level of the TTP paralog ZFP36L1 accumulates in huwe1 KO cells but do not demonstrate that HUWE1 affects ZFP36L1 protein stability.

    The next conclusion of the manuscript describes residues in the TTP234-278 region as important for their stability. Based on data presented in fig. 6 B and sup. 6B the authors conclude that residues S52 and 178, previously identified as regulators of TTP stability, are unlikely to be involved in HUWE1-dependent TTP accumulation. The data are only based on 2 independent experiments, one of which (fig 6B) shows a difference in TTP S52/S178 mutant in Huwe1 deficient cells as compared to wt TTP. These results seem therefore too preliminary to reliably exclude the implication of S52 and 178 on the HUWE1 accumulation of TTP.

    Other data from Fig. 6 further analyze the effect of deleting different regions of the TTP protein on the accumulation of this factor in HUWE1 KO and control cells. From these data, the authors conclude (line 416) that N-terminal deletion does not affect the TTP protein level. However, TTP accumulation in Huwe1 KO cells seems mostly lost in mutant N4. As mentioned above the limited number of replicates (n=2) and the absence of a statistical test makes the interpretation of this result difficult.

    Several TTP C-terminal mutants show a HUWE1-independent accumulation when compared to the wt protein (Fig6. D). Is this region identical to the unstructured region identified by Ngoc (line 1255) as a potent regulator of TTP degradation? If relevant this point should be discussed.

  7. Version published to 10.1101/2022.08.29.505645v1 on bioRxiv