Interferon-Induced PARP14-Mediated ADP-Ribosylation in p62 Bodies Requires an Active Ubiquitin-Proteasome System

  1. Rameez Raja
  2. Banhi Biswas
  3. Rachy Abraham
  4. Hongrui Liu
  5. Che-Yuan Chang
  6. Hien Vu
  7. Anthony K. L. Leung

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Abstract

Biomolecular condensates are cellular compartments without enveloping membranes, enabling them to dynamically adjust their composition in response to environmental changes through post-translational modifications. A recent study has revealed that interferon-induced ADP-ribosylation (ADPr), which can be reversed by a SARS-CoV-2-encoded hydrolase, is enriched within a condensate. However, the identity of the condensate and responsible host ADP-ribosyltransferase remain elusive. Here, we demonstrate that interferon induces ADPr through transcriptional activation of PARP14, requiring both its physical presence and catalytic activity for condensate formation. Interferon-induced ADPr colocalizes with PARP14, and these PARP14/ADPr condensates contain key components of p62 bodies—including the selective autophagy receptor p62 and its binding partner NBR1, along with K48-linked and K63-linked polyubiquitin chains—but lack the autophagosome marker LC3B. Knockdown of p62 disrupts the formation of these ADPr condensates. Importantly, these structures are unaffected by autophagy inhibition but depend on both ubiquitin activation and proteasome activity. Taken together, these findings demonstrate that interferon triggers PARP14-mediated ADP-ribosylation in p62 bodies, which requires an active ubiquitin-proteasome system.

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    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary In this study, Raja et al. found cytoplasmic condensates formed by the treatment of INFγ, investigated components of these condensates and identified p62, NBR1 and PARP14 as their components. INFγ treatment induced PARP14 expression, and PAPR14 inhibitor treatment inhibited condensation formation, suggesting that the amount of PARP14 and its enzymatic activity are important for the condensate formation. The ADPr-positive p62 condensates were independent of autophagic degradation, and proteasomal activity was required for their formation.

    Major comment

    1. The finding that the ubiquitin-proteasome, but not autophagy activity, is indispensable for the formation of p62 condensates is of interest. However, the molecular mechanism by which the ubiquitin-proteasome system (UPS) is involved in the regulation of the PARP14-p62 condensate is still unclear. Which step(s) is the UPS involved?

    We appreciate the reviewer's acknowledgment of the novelty of our studies on the requirement of the ubiquitin-proteasome system (UPS) in the formation of ADPr-containing PARP14/p62 condensates. We have demonstrated that condensation formation requires the first and last steps of the UPS using two distinct classes of inhibitors: (1) TAK243 inhibits the E1 enzyme by forming a covalent adduct with ubiquitin that mimics the ubiquitin-adenylate complex, thereby blocking the initial step of ubiquitin conjugation (Fig. 6F-G). (2) Three different proteasome inhibitors with varying degrees of selectivity—MG132, epoxomicin, and Bortezomib—block the final step of the UPS by inhibiting the 26S proteasome (Fig. 6B, S6D). Given that blocking the early steps of the ubiquitin conjugation pathway or the late stages of the UPS inhibits the formation of ADPr condensates, we deduce that an active UPS is required. We will explore the involvement of additional steps in the UPS.

    The p62 condensate serves as a scaffold for autophagosome formation through the assembling autophagy receptors including NBR1 and TAX1BP1, followed by recruiting ATG proteins such as FIP200. While ADPr-positive p62 condensates also contain NBR1 and polyubiquitinated proteins, they are unrelated to autophagic degradation. It is unclear what factors govern autophagy-independent function.

    As we have identified the requirement of active UPS in regulating these condensates, determining the factors that govern autophagy-independent functions, though interesting, is beyond the scope of this manuscript. Our data indicate that the formation of ADPr-containing condensates, which include p62, other autophagy receptors such as NBR1, and polyubiquitinated proteins, but lack the autophagasome membrane protein LC3B (Fig. 3B, 5E-I, and S5D). Notably. this condensate formation is not inhibited by treatment with Bafilomycin A1 and chloroquine, which target the final step of autophagy involving lysosome interaction (Fig. 6A). In response to the reviewer's comments, we will further investigate whether these condensates also include other autophagy receptors, such as TAX1BP1, as well as the downstream autophagosome protein FIP200. Additionally, we will genetically deplete the critical autophagy factor ATG5 to confirm orthogonally that the formation of these condensates is indeed independent of autophagy.

    The authors claim that the amount of PARP14 and its MAR activity are essential for the condensate formation. However, all experiments were performed only with PARP14 inhibitors, and further validation is needed. If the importance of PARP14 activity is to be directly demonstrated, experiments in which an enzyme activity mutant is introduced into PARP14 KO cells are needed.

    We would like to clarify that we have not only used PARP14 chemical inhibitors to reduce MAR activity but also employed PROTAC to reduce the amount of PARP14 (Fig. 1H). Both approaches demonstrated that the inhibition of either the amount or MAR activity of PARP14 is critical for condensate formation. Additionally, we demonstrated that condensate formation is reduced upon PARP14 knockdown using siRNA and shRNA, as well as CRISPR-mediated knockout (Fig. 1G and S1C-H).

    Furthermore, we showed that transient transfection of a PARP14 mutant deficient in ADP-ribosylhydrolase activity into U2OS cells leads to the formation of ADPr condensates that colocalize with PARP14, independent of IFNγ treatment. Notably, a subset of condensates—particularly the larger ones—that contain both PARP14 and ADPr showed strong colocalization with p62 (Fig. 4G). Treatment with PARP14 MAR activity inhibitor under these conditions resulted in the disappearance of ADPr/PARP14 condensates while p62 bodies remained (Fig. 4H), further indicating that ADPr enrichment in p62 bodies depends on the MAR activity of PARP14. To further confirm the dependence on MAR activity, we have now repeated the experiment using a PARP14 mutant deficient in MAR activity. PARP14 and ADPr condensates were not observed upon expression of this mutant, indicating that condensate formation depends on PARP14 MAR activity.

    In Figure 2a, the heatmap alone is insufficient. Neither errors nor statistical comparisons are indicated.

    We will incorporate our statistical data presented in Fig. S2A-D into Fig. 2A.

    The statistical analysis of Figure S2 is inappropriate; instead of t-tests, multiple comparisons should be used to compare three or more groups.

    We will perform multiple comparison analyses, as suggested.

    Minor comment

    1. What percentage of p62 condensates upon INFγ treatment are ADPr positive? Are all p62 bodies seen with INFγ stimulation unrelated to autophagy?

    We will perform the quantification, as suggested.

    Is ADPr condensation a PARA14-specific phenomenon? PARP9 and PARP12 were also upregulated by INFγ treatment. Are these factors also involved in condensate formation?

    Amongst all catalytically active PARPs, ADPr condensation requires only PARP14. Russo et al., J Biol Chem 2021, have shown that genetic knockout of PARP9 affects the formation of ADPr condensates; however, PARP9 is catalytically inactive as an ADP-ribosyltransferase. Ribeiro et al., EMBO 2024, have further confirmed the requirement of PARP9 by siRNA knockdown and have also shown that condensate formation does not require PARP12. Based on the reviewers' comments, we will independently confirm this observation by performing knockdown experiments.

    Figure 4D appears to be immunoprecipitation (IP) under non-denaturing conditions. If so, it is not possible to distinguish whether the MAR signal is derived from p62 or from the p62 interacting proteins (the associated ubiquitinated substrates). IP experiments should be performed under denaturing conditions.

    We will perform denaturing IP or other experiments to confirm the p62 modification upon IFNγ treatment. Additionally, we would like to note that following our submission, Kubon et al. reported in a bioRxiv preprint that p62 is ADP-ribosylated in a PARP14-dependent manner upon treatment with type I interferon, IFNβ. This finding is consistent with our study involving type II interferon, IFNγ.

    In Figure 5B, which band is HO1, the upper or lower?

    Both bands are HO1, as shown by Biswas et al., J Biol Chem 2014. One band appears at 28 kDa and the other at 32 kDa. The 32-kDa isoform is predominantly constitutive in the cytoplasm, whereas the 28-kDa HO-1 is predominant and primarily localized to the nucleus.

    There is no image for ubiquitin in S5D.

    Our original statement, “However, when inhibited with the mTOR inhibitor Torin-1, autophagy is induced, leading to increased autophagosome formation marked by LC3B on the membranes, which facilitates the recruitment of p62 and ubiquitinated proteins (Fig. S5D),” contained a misplaced figure citation. The correct statement should be: “However, when inhibited with the mTOR inhibitor Torin-1, autophagy is induced, leading to increased autophagosome formation marked by LC3B on the membranes (Fig. S5D), which facilitates the recruitment of p62 and ubiquitinated proteins.” Our intention was to show that there are conditions, such as Torin-1 treatment, where p62 and LC3B colocalize.

    Right panel in Figure 4F shows only IFγ + RBN, which should show all data sets in the same panel.

    Given the complexity of the three conditions with extensive data points and error bars on the FRAP experiments, we aim to present the data clearly. Instead of merging the panel into one figure, we initially provided a summary table in Figure 4F. However, in response to the reviewer's comments, we will provide the composite image that includes all data sets in the same panel.

    Reviewer #1 (Significance (Required)):

    Liquid droplets, which have continuously being identified in cells, are a hot topic in cell biology. Droplet formation, structure, molecular dynamics, and degradation, as well as their abnormalities and disease development due to genetic mutation and stress, are of wide-ranging interest from basic to pathological aspects. Therefore, this research has the potential to attract interest from a wide range of fields.

    General assessment Overall, the data are clear and the phenomenon is of interest. However, the molecular mechanism and biological significance of the condensate formation is unknown; It is unclear why proteasome activity is required for the formation of PARP14-mediated ADP ribosylation. It is also unclear what the consequences are for the cell if the ADPr-positive condensates are not formed. Thea authors should address these general and important issues and provide the data If not all.

    We thank the reviewer for acknowledging that our condensate investigation is timely and important in cell biology and for recognizing that “data are clear and the phenomenon is of interest”. As mentioned in the Discussion, these condensates can be reversed by the SARS-CoV-2 macrodomain in lung A549 cells, whose activity to remove ADP-ribosylation is critical for viral replication and pathogenesis, indicating the biological significance of these condensates. In addition, similar IFNγ conditions can induce PARP14 expression in melanoma, where PARP14 inhibition resensitizes these cancers to immunotherapy. Given that these ADPr condensates are also observed in A375 melanoma cells beyond lung cells (Fig. S3B), this provides additional context to investigate their biological significance in the future.

    We would like to note that we have already made significant advances by (1) revealing the identity of these condensates as related to p62 bodies (Fig. 3-5), (2) defining the responsible ADP-ribosyltransferase as PARP14 (Fig. 1-2), and (3) determining the requirements for condensation through ubiquitin-proteasome system (Fig. 6). The proposed exploration of the functional consequences and significance is beyond the scope of this manuscript. However, we will further define the mechanistic involvement of which step of ubiquitin-proteasome system.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This manuscript investigates the formation of a novel cellular structure or condensate, similar to p62 bodies, that includes PARP14 and p62. The interferon-induced PARP14-mediated ADP-ribosylation of p62 in these condensates depends on an active ubiquitin-proteasome system. These condensates are characterized by the presence of PARP14 and ADPr and include some, but not all, components of the p62 bodies. Furthermore, their formation depends on both ubiquitin activation and proteasome activity, but it is unaffected by autophagy inhibition, unlike conventional p62 bodies.

    The Introduction provides a well-delineated context of condensates, highlighting the importance of post-translational modifications in responding to environmental changes.

    Although the manuscript is well-organized with apparent logical development, there are weaknesses that diminish the impact of the reported data. A more accurate review of the structures, both from a morphological and quantitative perspective, would strengthen the conclusions and the overall impact of this work. Additionally, while the authors have analyzed the contribution of PARP14 to condensate formation, the biological significance of these structures remains unclear. For instance, performing MS (mass spectrometry) analysis on the described structures could help identify their composition and functions.

    The methodologies used in this study are standard for molecular and cellular biology research, including immunofluorescence assays, transient transfections, immunoprecipitations, and fluorescence recovery after photobleaching (FRAP) assays. These methods are described in detail and can be reproduced.

    Below, please find a list of comments and suggestions to enhance the robustness of the data:

    We thank the reviewer for acknowledging the logical progression of the manuscript and the detailed, reproducible methods. As detailed below, we will perform super-resolution microscopy experiments to examine morphology, improve data quantification, and conduct proteomics experiments to identify how the p62 interactome changes upon IFNγ treatment.

    Major Points

    1. The IF analyses are central to the conclusions reported and are employed for each of the inhibitors or other tools used to investigate the formation of these condensates. The quality of the IF images needs to be improved; the shape and contacts of the condensates should be analyzed using either super-resolution or EM microscopy, or preferably both. The lack of morphometry and quantification from cell populations needs to be addressed for all experiments. These analyses are needed to support the claim that the condensates presented in this study are indeed novel structures, rather than being transient aggregates of a different nature.

    We believe the reviewer may be referring to the size of the images rather than their quality, as they are of high resolution when zoomed in. However, we agree that larger images would enhance clarity. We will be discreet in choosing the figures to present, given that we have over 550 panels in the main figures and over 250 in the supplemental figures. We will resize our figures to ensure the images are clearly visible.

    We will perform Airyscan imaging to provide super-resolution images for a better understanding of the morphology of these condensates. We will perform the morphometry quantification (circularity and ellipticity). For quantification, we would like to point out that nearly all of our experiments were analyzed from at least 4 fields, each containing 20-50 cells (depending on the magnification of 20x or 40x). In response to the reviewer's comment, we will also provide quantification on a per-cell basis.

    Our data indicate that these are novel ADPr-containing condensates that colocalize with PARP14, p62, NBR1, and ubiquitin, but not LC3B (Fig. 1F, 3B, 5E-F, 5I). These structures are inducible by IFN treatment and can be inhibited by even 1 hour of PARP14 inhibition (Fig. 1A-B, 2D) They are dependent on PARP14 induction and its ADP-ribosyltransferase activity (Fig. 1G, 2B, 2D). These structures are not protein aggregates, as evidenced by their lack of staining with ProteoStat Dye (Fig. S6A), which stains for unfolded proteins.

    The claim that PARP14 is essential for the formation of condensates requires support by the analyses indicated above. Minor points regarding Fig. 1 are indicated below. I suggest performing KD of PARP9 and/or PARP12 (whose expression is increased upon IFN treatment) and checking ADPr condensates to validate the central role of PARP14.

    ADPr condensation requires PARP14, as demonstrated by multiple genetic depletion techniques (siRNA/shRNA/CRISPR; Fig. 1G, S1C-H) and chemical inhibitors (catalytic and PROTAC; Fig. 1H, 2B, 2D). We will provide additional image analyses to support the claim. In addition, Russo et al., J Biol Chem 2021, have shown that genetic knockout of PARP9 affects the formation of ADPr condensates; however, PARP9 is catalytically inactive as an ADP-ribosyltransferase. Ribeiro et al., EMBO 2024, have further confirmed the requirement of PARP9 by siRNA knockdown and have also shown that condensate formation does not require PARP12. Based on the reviewers' comments, we will independently confirm this observation by performing knockdown experiments.

    According to the text, "PARP14 was pulled down by ADP-ribose binding Af1521 macrodomain following IFNγ treatment (Fig. 2H)", but the legend to the figure says otherwise. A Pan-ADPr binding reagent (MABE1016) is reported in the figure. Although the conclusion is similar for the results obtained with these two tools (but they must be described and reported properly), it is still insufficient to claim that PARP14 is ADP-ribosylated. This point should be at least discussed.

    We apologize for the confusion. The Pan-ADPr binding reagent (MABE1016) is a His-tagged recombinant Af1521 macrodomain that binds to ADP-ribosylated protein (Gibson et al., Biochemistry 2017). Therefore, we used Ni-NTA resin to pull down His-tagged AF1521 for the subsequent analysis of PARP14. We will revise the text and figure legend for Fig. 2H to clarify this. We will further probe the eluted PARP14 is indeed ADP-ribosylated by western blot. Consistent with our studies, Kar et al. and Ribeiro et al. (EMBO J. 2024) also recently reported that PARP14 is ADP-ribosylated upon IFNγ treatment in A549 cells. Additionally, Higashi et al. (J. Proteome Res. 2019) reported PARP14 ADP-ribosylation in IFNγ-treated macrophage cells. We will include these references in our Discussion.

    I have difficulties analyzing the colocalization with the different organelles, even enlarging the images as much as possible. In most cases, only one condensate per image is shown. Continuities with the nuclear envelope appear in some cases: has this been investigated?

    We have provided images of at least two cells containing multiple cytoplasmic condensates in Figure S3A. We believe part of the confusion arises from the staining pattern of ADPr in the nucleus, which colocalizes with splicing speckles. However, this nuclear staining was not altered by IFNγ treatment. Therefore, we have focused on the cytoplasmic condensates in this study and will clarify this focus in the main text. However, in response to the reviewer’s question, we will also visit the possibilities of enrichment of signals at the nuclear envelope. For each colocalization study, we have analyzed at least four fields, each containing 20-30 cells. To further strengthen our claim for colocalization analyses, we will now provide quantification on a per-cell basis.

    Minor Points

    1. Fig. 1A: The DAPI images at 3 and 6 hours are reversed. Additionally, for Fig. S1a and Fig. 1A, please include quantifications.

    We apologize for the oversight and will provide quantification.

    1. Fig. 1B: Check PARP14 levels (and other IFN-PARPs) under the same experimental conditions.

    As suggested, we will assess PARP9, PARP12, and PARP14 levels.

    Fig. 1H: Explain why PARP14 IF staining is still visible upon RBN012811 treatment, while it is completely lost in WB analysis or upon PARP14 siRNA treatment (Fig. 1G). In addition, please include IF quantifications.

    To clarify, for Figure 1H, the images provided correspond to those from IFNγ-treated cells while the western blot data include both with and without IFNγ treatment. We would like to point out that RBN012811 treatment indeed shows a similar dose-dependent signal with increasing hours of treatment, comparable to the western blot results. Specifically, we observed a small amount of PARP14 remaining at the 1-hour timepoint on western blots and IF images, with the highest intensity observed compared to other timepoints. In addition, we believe part of the discrepancy is due to background staining by PARP14 antibodies. Therefore, we will examine the level of background staining in PARP14 KO cells and provide corresponding IF quantification.

    Fig. 2C: Please include quantifications.

    We will provide quantification.

    1. Fig. 2E: The RBN treatment time is not indicated. Please include this information in the figure legend.

    We apologize for the oversight and will add the treatment time (24 h) to the figure legend.

    Fig. 2G: I am not convinced about the PARP14 staining. IF images do not show an increase in PARP14 levels, while WB analysis shows a strong increase in PARP14 protein levels (see Fig. 2E). Moreover, the RBN treatment time was not indicated; please include it in the figure legend. Does RBN alone affect PARP14 localization? The reported picture shows only 2 cells, each with a different subcellular localization of PARP14. As previously suggested, quantifications are required.

    When presenting the data, our aim was to show the pattern rather than the relative intensity difference. Therefore, we used the autocontrast image function across different conditions, which resulted in an apparent change in pattern even with weak signals in control or RBN-treated samples. To address this, we will ensure the images presented across different conditions have the same exposure and are shown with consistent image contrast parameters. We will also include quantification of the condensates. Additionally, we apologize for the oversight and will add the RBN treatment time to the figure legend.

    Fig. 3B: Pearson's correlation coefficient (PCC) is reported for n=3. The images show one condensate per cell. Under these conditions, the number of cells analyzed should be at least 100 for each experiment. Additionally, the PCC between PARP14 and p62 at steady state is shown to be 60% (which is quite high). However, the IF pictures do not support this quantification. Can the authors provide higher-resolution pictures? Does PARP14 always co-localize with p62? Lines 207-208 state: "these findings suggest that PARP14 is localized to p62 bodies upon IFNγ treatment when ADP-ribosylation occurs." According to the PCC value, the two proteins co-localize even in the absence of IFN. Can the authors clarify this aspect?

    We apologize for the inaccurate description. The data should be n=4, representing different fields, each containing 20-30 cells (as indicated by the number of dots in the original graph panels in Fig. 3B). The Pearson's correlation coefficient was calculated across the cells, instead of focusing on the condensates—we will provide additional analyses on condensate colocalization analyses. We will also provide larger images and quantification to indicate the level of PARP14 colocalization with p62. For PARP14, we did not see a significant number of PARP14 condensates in control cells; PARP14 condensates were seen only after IFNγ treatment. A fraction of PARP14 condensates did not colocalize with p62. We will provide detailed quantification analyses.

    Fig. S3B: Please include quantifications.

    Quantification will be provided

    Fig. S3C: How was the condensate size quantified? It would be useful to show a quantification mask.

    We apologize for the omission in the Method Section. The condensate size quantification was performed with ImageJ. The nuclei were first identified with DAPI staining and masked out from the ADPr channel. The image was then thresholded with the “Maximum Entropy” method from ImageJ and the “Analyze Particles” function was used to identify condensates with size larger than 1 pixel. Quantification mask will be provided.

    Fig. 3D: Does p62 KD affect PARP14 localization? The reported picture shows only 2 cells, each with different staining of PARP14.

    p62 KD reduced the number of PARP14 condensates but did not change their localization. We will provide a representative image with more cells to illustrate this effect more clearly and provide quantification on the change in number of condensates.

    Fig. 4A: Please quantify the PARP14 co-IP signal with p62, normalized to PARP14 total levels. In the reported WB, it is difficult to see the interaction between PARP14 and p62 in untreated conditions. Please provide clearer WB.

    As suggested, we will quantify the PARP14 co-IP signal by normalizing it with PARP14 input levels. Additionally, we will provide a clearer WB in the revised manuscript.

    Additionally, I would expect an increased interaction between PARP14 and p62 upon IFN treatment due to PARP14 recruitment to p62 condensates, not just because of increased PARP14 levels. Since the authors show that PARP14 is not recruited to ADPr condensates upon RBN treatment (Fig. 2G), why is the interaction between p62 and PARP14 so high under RBN treatment?

    RBN treatment inhibits PARP14 catalytic activity but simultaneously increases PARP14 levels, as first described by Schenkel et al., Cell Chem Biol 2021. Western blot data indicate that the interaction between PARP14 and p62 is independent of this activity and instead depends on PARP14 protein levels. However, the formation of ADPr/PARP14-containing condensates requires catalytically active PARP14. Based on these data, we conclude that the colocalization of p62 and PARP14 depends on the catalytic activity of PARP14, which is reflected by its ADP-ribosylation.

    Fig. 4C: Please quantify WB signals of ADP-ribosylated p62 for the different conditions analyzed. ADP-ribosylation of p62 is still present in cells lacking PARP14. Are there other enzymes that can modify p62? Moreover, the authors state: "We observed an increased MARylation of p62 upon IFNγ treatment" (line 230); is this dependent on the increase in PARP14 levels or the translocation of PARP14 to ADPr condensates? Quantifications should help clarify this aspect.

    WB signals will be quantified. We agree with the reviewer's observation regarding the presence of ADP-ribosylated p62 in PARP14 KO cells. The basal levels of ADP-ribosylated p62 may be due to other PARP enzymes. However, PARP14 is critical for the increase in ADP-ribosylation under IFNγ treatment, as the increase was not observed in PARP14 KO cells. Given that PARP14 inhibitors increase PARP14 levels, we interpret that the increase in p62 MARylation requires an increase in active PARP14 levels, not just its total level. Since PARP14 activity is crucial for the localization of PARP14 to p62 condensates and its enrichment of ADPr signals, it is possible, as suggested by the reviewer, that the increase in MARylation of p62 is dependent on the translocation of PARP14 to the structure. However, the field currently lacks the tools to disrupt p62 bodies without knocking down p62 to definitively test whether colocalization is required for the MARylation increase.

    Fig. 4G: Quantificationsare required.

    Quantification will be provided

    Reviewer #2 (Significance (Required)): The role of the PARP family in cellular processes is a very active and rapidly growing field. New information about the organization of PARPs in the nucleus, cytosol, or different types of bodies/structures is certainly relevant to the field. However, the present study is too preliminary at the moment to be considered highly relevant. Both the data analysis and conclusions need to be carefully reviewed. After major revisions, the manuscript might be of general interest if well contextualized within the fields of post-translational modification and protein degradation processes. It would remain in any case interesting for the field of ADP ribosylation. We thank the reviewer for recognizing the significance of our work in the rapidly evolving field of PARP biology. We apologize for the lack of clarity that we indeed quantified over 100 cells across at least 4 fields of images for the data reported. To further address the concerns raised, we will provide additional cell-based quantification to strengthen our claims. Furthermore, we will enhance the contextualization of our findings within the broader frameworks of post-translational modification and protein degradation processes in the Introduction and Discussion sections.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, titled "Interferon-Induced PARP14-Mediated ADP-Ribosylation in p62 Bodies Requires an Active Ubiquitin-Proteasome System", Raja et al. perform fluorescence microscopy assays and molecular analyses on cultured cells ex vivo to further our understanding of ADP-ribose accumulations that form in the cytoplasm in response to IFγ stimulation. Guided by the operon-like linkage and co-expression patterns of PARP14, PARP9, and DTX3L and recent reports describing a SARS-CoV-2-dissolved cytoplasmic body induced by interferon-induced that is rich in ADP-ribose and the PARP9/DTX3L heterodimer form following IFγ stimulation, the authors provide clarity in this manuscript regarding two knowledge gaps - (i) what catalyzes mono(ADP-ribosyl)ation within these structures (as PARP9 lacks ADP-ribosyltransferase activity) and (ii) how these foci/condensates relate to similarly composed autophagy-associate "p62 bodies" that have been previously described. Using a combination of genetic depletion and inhibitor-based approaches, the authors show that these ADPr-condensates rely on the catalytic activity of PARP14. The authors also show that while these ADPr-condensates share componentry with "p62 bodies" like ubiquitin and p62 itself, these foci are distinct accumulations, as they lack both LC3B and sensitivity to autophagy inhibitors and require an active ubiquitn-mediated proteosomal degradation system.

    While this report represents a more incremental advance in our understanding of these cell signaling structures, especially considering a pair of very recently published and similar reports (Kar et al., EMBO J [2024] and Chaves Ribiero et al EMBO J [2024]), the work here is well-written and reasoned and complements these works with some novelty and distinction to that reported literature. The experiments are definitive and of high quality and the authors interpretations/conclusions are largely well-supported by the results. Thus, it is my opinion that this work is appropriate for publication with predominantly minor revisions (outlined below) and a few more substantive experimental additions.

    We thank the reviewer for recognizing the high quality of our work and for acknowledging that our interpretations are well-supported by the results. We appreciate that the reviewer deemed our work ready for publication with minor revisions. We believe the reviewer’s perception of our work as incremental arises because two related studies were published in June, after we submitted our work to Review Commons in May. According to the Scooping Protection Policy, our work should still be considered novel. The publication of these studies in EMBO J, which reported the discovery of PARP14 in ADPr-containing condensate formation, highlights the significance of our research. However, we further contribute to this discovery by demonstrating the critical role of PARP14 using multiple genetic manipulation techniques (siRNA, shRNA, and CRISPR-Cas9) and chemical inhibitors (catalytic and PROTAC), indicating the rigor of our studies. Moreover, we not only investigate PARP14 but also define the identity of these condensates related to p62 bodies and establish the requirement of an active ubiquitin-proteasome system for their formation.

    Major Comment:

    A major claim and novelty reported here is that the ADPr condensates are distinct from "p62 bodies". The evidence to support this rely largely on differences in their sensitivity to pharmacological treatments as well as somewhat subtle differences in FRAP recovery in p62 condensates after IFNgamma treatment. But, this claim would be better supported with more comprehensive mapping of differences in the componentry or functional outcomes of these condensates. The authors might consider:

    -Mass spectrometry against p62 (a common component) in standard "p62 bodies" and ADPr Condensates, followed by IF to confirm significantly different composition in, what is argued here, these distinct structures. -Fine mapping of concentration dependence of components that give rise to these distinct condensates as has been demonstrated in papers like Riback et al Nature 2020 and others. -Methodology of the author's choosing to decipher functional outcomes from these condensates followed by demonstration that components unique to ADPr condensates are dispensable for functioning "p62 bodies" and, vice versa, components unique to "p62 bodies" are dispensable for ADPr Condensate function.

    As rightly pointed out by the reviewer, our studies indicate the alteration in the composition and dynamics of p62 bodies upon IFNγ treatment. This was assessed using immunofluorescence against various known components of p62 bodies (Fig. 3B, 5E-I), quantification of the condensate size (Fig. S3C), and p62 mobility assessment by photokinetic experiments (Fig. 3C, 4F). In considering reviewer’s suggestions, we will perform p62 interactome studies with and without IFNγ treatment to identify potential changes. Additionally, we will analyze the concentration dependence of ADPr for condensate formation. However, we believe that investigating the functional outcomes is beyond the scope of this manuscript.

    Minor Comments:

    Overall, the representative microscopy images are far too small. For the benefit of future readers, please consider enlarging these images.

    More of the quantitation of microscopy images, with accompanying statistics, that are found in abundance in the supplemental material should find their way into the main figures of the manuscript. This will give room for larger and more reader-friendly representative microscopy images in the main figures/text as discussed briefly above.

    We appreciate the reviewer’s suggestions and rightly pointed out that our quantification and statistics were in supplementary materials. Given that we have provided over 800 image panels, we will restructure the manuscript so that the cell biology information is more readily available. We will move our quantification data and statistics currently in the supplementary materials to the main figures. Additionally, we will provide larger and more reader-friendly representative microscopy images in the main text as suggested.

    Can the authors test whether or not the condensates are purely driven by mono(ADP-ribosyl)ation? Or does poly(ADP-ribose) co-occupy these condensates and play a substantive role?

    We have tested for the presence of poly(ADP-ribose) in the condensates and found it is not present. We will provide the supporting data.

    The manuscript would benefit from discussing very recent and related reports (Kar et al., EMBO J [2024] and Chaves Ribiero et al EMBO J [2024]), that I suspect were not available at the time of submission.

    Yes, the reviewer is correct that our submission preceded the publication of these related reports. In light of the co-discovery, we will add a section to discuss their findings.

    IFNalpha and IFNbeta, which are used in Figure S1, do not appear as reagents in Table 1.

    We apologize for the oversight and will add the information on IFNa and IFNb to Table 1.

    On lines 113-114, it would seem more appropriate to describe the increase of PARP-14 as statistically significant and largest in magnitude. "most significant" would just mean lowest p-value, which I expect is different that the authors intend here.

    We thank the reviewer's suggestion will modify the text as follows:

    "PARP9, PARP12, and PARP14 were statistically significantly upregulated at 6 and 24 hours post-treatment, with PARP14 showing the largest increase in mRNA expression levels (Fig. 1D and S1B)."

    In Figure S1, better care should be taken to crop and align the western blots.

    We thank the reviewer for pointing this out, and we will properly align and crop the western blots in Figure S1.

    On line 154, it may be more appropriate to describe ITK as a "weaker" inhibitor of PARP14 relative to PARP11. It certainly is effective as an inhibitor (Figs. S2A and S2G) and its unclear how the authors (or anyone would) define what qualities make it "weak".

    We thank the reviewer's suggestion and will modify the text as follows:

    “…—specifically RBN012579 (hereafter RBN) and ITK7 (a potent PARP11 inhibitor with inhibitory effects on PARP14 that are weaker than RBN)—…"

    The multiple bands for PARP14 in Figure 3E should be addressed. Why does this differ from other blots from the same cells?

    We believe that the multiple bands seen can be due to insufficient blocking and using a different lot of PARP14 antibodies. We will address the issue by performing a new experiment with proper blocking conditions and using the same lot of PARP14 antibody as other blots. It should be noted that that variation is also observed in the reagent website: https://www.scbt.com/p/parp-14-antibody-c-1

    Reviewer #3 (Significance (Required)):

    I expect the advances in this work will appeal more to specialists who are interested in ADP-ribosylation as a signaling molecule and to those engaged in biotechnological efforts to drug immunological responses.

    The advances reported here are incremental. The ADPr condensates that form in response to IFNgamma, the involvement of PARP9/DTX3L, and very recently the involvement of PARP14 and its MARylation activity are all known. Less known is the notion that this condensate is distinct from other kinds of "bodies", which is a clear point of novelty, especially if buttressed by the authors as suggested in this review.

    We agree with the reviewer that our work is significant for multiple fields, including ADP-ribosylation and immunology. The perception of our work as incremental likely stems from the publication of two related recent studies in June, after our submission in May. According to the Scooping Protection Policy in Review Commons, our work should remain novel in editorial consideration. More importantly, the back-to-back EMBO J studies highlight the importance of reporting the critical role of PARP14 in ADPr-containing condensate formation. We further contribute to this discovery in three aspects: (1) we rigorously demonstrate the critical role of PARP14 through multiple genetic techniques (siRNA, shRNA, and CRISPR-Cas9) and chemical inhibitors (catalytic and PROTAC), (2) we reveal the identity of these condensates as related to p62 bodies, and (3) we define their requirement for an active ubiquitin-proteasome system.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, titled "Interferon-Induced PARP14-Mediated ADP-Ribosylation in p62 Bodies Requires an Active Ubiquitin-Proteasome System", Raja et al. perform fluorescence microscopy assays and molecular analyses on cultured cells ex vivo to further our understanding of ADP-ribose accumulations that form in the cytoplasm in response to IFγ stimulation. Guided by the operon-like linkage and co-expression patterns of PARP14, PARP9, and DTX3L and recent reports describing a SARS-CoV-2-dissolved cytoplasmic body induced by interferon-induced that is rich in ADP-ribose and the PARP9/DTX3L heterodimer form following IFγ stimulation, the authors provide clarity in this manuscript regarding two knowledge gaps - (i) what catalyzes mono(ADP-ribosyl)ation within these structures (as PARP9 lacks ADP-ribosyltransferase activity) and (ii) how these foci/condensates relate to similarly composed autophagy-associate "p62 bodies" that have been previously described. Using a combination of genetic depletion and inhibitor-based approaches, the authors show that these ADPr-condensates rely on the catalytic activity of PARP14. The authors also show that while these ADPr-condensates share componentry with "p62 bodies" like ubiquitin and p62 itself, these foci are distinct accumulations, as they lack both LC3B and sensitivity to autophagy inhibitors and require an active ubiquitn-mediated proteosomal degradation system.

    While this report represents a more incremental advance in our understanding of these cell signaling structures, especially considering a pair of very recently published and similar reports (Kar et al., EMBO J [2024] and Chaves Ribiero et al EMBO J [2024]), the work here is well-written and reasoned and complements these works with some novelty and distinction to that reported literature. The experiments are definitive and of high quality and the authors interpretations/conclusions are largely well-supported by the results. Thus, it is my opinion that this work is appropriate for publication with predominantly minor revisions (outlined below) and a few more substantive experimental additions.

    Major Comment:

    A major claim and novelty reported here is that the ADPr condensates are distinct from "p62 bodies". The evidence to support this rely largely on differences in their sensitivity to pharmacological treatments as well as somewhat subtle differences in FRAP recovery in p62 condensates after IFNgamma treatment. But, this claim would be better supported with more comprehensive mapping of differences in the componentry or functional outcomes of these condensates. The authors might consider:

    • Mass spectrometry against p62 (a common component) in standard "p62 bodies" and ADPr Condensates, followed by IF to confirm significantly different composition in, what is argued here, these distinct structures.
    • Fine mapping of concentration dependence of components that give rise to these distinct condensates as has been demonstrated in papers like Riback et al Nature 2020 and others.
    • Methodology of the author's choosing to decipher functional outcomes from these condensates followed by demonstration that components unique to ADPr condensates are dispensable for functioning "p62 bodies" and, vice versa, components unique to "p62 bodies" are dispensable for ADPr Condensate function.

    Minor Comments:

    Overall, the representative microscopy images are far too small. For the benefit of future readers, please consider enlarging these images.

    More of the quantitation of microscopy images, with accompanying statistics, that are found in abundance in the supplemental material should find their way into the main figures of the manuscript. This will give room for larger and more reader-friendly representative microscopy images in the main figures/text as discussed briefly above.

    Can the authors test whether or not the condensates are purely driven by mono(ADP-ribosyl)ation? Or does poly(ADP-ribose) co-occupy these condensates and play a substantive role?

    The manuscript would benefit from discussing very recent and related reports (Kar et al., EMBO J [2024] and Chaves Ribiero et al EMBO J [2024]), that I suspect were not available at the time of submission.

    IFNalpha and IFNbeta, which are used in Figure S1, do not appear as reagents in Table 1.

    On lines 113-114, it would seem more appropriate to describe the increase of PARP-14 as statistically significant and largest in magnitude. "most significant" would just mean lowest p-value, which I expect is different that the authors intend here.

    In Figure S1, better care should be taken to crop and align the western blots.

    On line 154, it may be more appropriate to describe ITK as a "weaker" inhibitor of PARP14 relative to PARP11. It certainly is effective as an inhibitor (Figs. S2A and S2G) and its unclear how the authors (or anyone would) define what qualities make it "weak".

    The multiple bands for PARP14 in Figure 3E should be addressed. Why does this differ from other blots from the same cells?

    Significance

    I expect the advances in this work will appeal more to specialists who are interested in ADP-ribosylation as a signaling molecule and to those engaged in biotechnological efforts to drug immunological responses.

    The advances reported here are incremental. The ADPr condensates that form in response to IFNgamma, the involvement of PARP9/DTX3L, and very recently the involvement of PARP14 and its MARylation activity are all known. Less known is the notion that this condensate is distinct from other kinds of "bodies", which is a clear point of novelty, especially if buttressed by the authors as suggested in this review.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript investigates the formation of a novel cellular structure or condensate, similar to p62 bodies, that includes PARP14 and p62. The interferon-induced PARP14-mediated ADP-ribosylation of p62 in these condensates depends on an active ubiquitin-proteasome system. These condensates are characterized by the presence of PARP14 and ADPr and include some, but not all, components of the p62 bodies. Furthermore, their formation depends on both ubiquitin activation and proteasome activity, but it is unaffected by autophagy inhibition, unlike conventional p62 bodies.

    The Introduction provides a well-delineated context of condensates, highlighting the importance of post-translational modifications in responding to environmental changes.

    Although the manuscript is well-organized with apparent logical development, there are weaknesses that diminish the impact of the reported data. A more accurate review of the structures, both from a morphological and quantitative perspective, would strengthen the conclusions and the overall impact of this work. Additionally, while the authors have analyzed the contribution of PARP14 to condensate formation, the biological significance of these structures remains unclear. For instance, performing MS (mass spectrometry) analysis on the described structures could help identify their composition and functions.

    The methodologies used in this study are standard for molecular and cellular biology research, including immunofluorescence assays, transient transfections, immunoprecipitations, and fluorescence recovery after photobleaching (FRAP) assays. These methods are described in detail and can be reproduced.

    Below, please find a list of comments and suggestions to enhance the robustness of the data:

    Major Points

    1. The IF analyses are central to the conclusions reported and are employed for each of the inhibitors or other tools used to investigate the formation of these condensates. The quality of the IF images needs to be improved; the shape and contacts of the condensates should be analyzed using either super-resolution or EM microscopy, or preferably both. The lack of morphometry and quantification from cell populations needs to be addressed for all experiments. These analyses are needed to support the claim that the condensates presented in this study are indeed novel structures, rather than being transient aggregates of a different nature.
    2. The claim that PARP14 is essential for the formation of condensates requires support by the analyses indicated above. Minor points regarding Fig. 1 are indicated below. I suggest performing KD of PARP9 and/or PARP12 (whose expression is increased upon IFN treatment) and checking ADPr condensates to validate the central role of PARP14.
    3. According to the text, "PARP14 was pulled down by ADP-ribose binding Af1521 macrodomain following IFNγ treatment (Fig. 2H)", but the legend to the figure says otherwise. A Pan-ADPr binding reagent (MABE1016) is reported in the figure. Although the conclusion is similar for the results obtained with these two tools (but they must be described and reported properly), it is still insufficient to claim that PARP14 is ADP-ribosylated. This point should be at least discussed.
    4. I have difficulties analyzing the colocalization with the different organelles, even enlarging the images as much as possible. In most cases, only one condensate per image is shown. Continuities with the nuclear envelope appear in some cases: has this been investigated?

    Minor Points

    1. Fig. 1A: The DAPI images at 3 and 6 hours are reversed. Additionally, for Fig. S1a and Fig. 1A, please include quantifications.
    2. Fig. 1B: Check PARP14 levels (and other IFN-PARPs) under the same experimental conditions.
    3. Fig. 1H: Explain why PARP14 IF staining is still visible upon RBN012811 treatment, while it is completely lost in WB analysis or upon PARP14 siRNA treatment (Fig. 1G). In addition, please include IF quantifications.
    4. Fig. 2C: Please include quantifications.
    5. Fig. 2E: The RBN treatment time is not indicated. Please include this information in the figure legend.
    6. Fig. 2G: I am not convinced about the PARP14 staining. IF images do not show an increase in PARP14 levels, while WB analysis shows a strong increase in PARP14 protein levels (see Fig. 2E). Moreover, the RBN treatment time was not indicated; please include it in the figure legend. Does RBN alone affect PARP14 localization? The reported picture shows only 2 cells, each with a different subcellular localization of PARP14. As previously suggested, quantifications are required.
    7. Fig. 3B: Pearson's correlation coefficient (PCC) is reported for n=3. The images show one condensate per cell. Under these conditions, the number of cells analyzed should be at least 100 for each experiment. Additionally, the PCC between PARP14 and p62 at steady state is shown to be 60% (which is quite high). However, the IF pictures do not support this quantification. Can the authors provide higher-resolution pictures? Does PARP14 always co-localize with p62? Lines 207-208 state: "these findings suggest that PARP14 is localized to p62 bodies upon IFNγ treatment when ADP-ribosylation occurs." According to the PCC value, the two proteins co-localize even in the absence of IFN. Can the authors clarify this aspect?
    8. Fig. S3B: Please include quantifications.
    9. Fig. S3C: How was the condensate size quantified? It would be useful to show a quantification mask.
    10. Fig. 3D: Does p62 KD affect PARP14 localization? The reported picture shows only 2 cells, each with different staining of PARP14.
    11. Fig. 4A: Please quantify the PARP14 co-IP signal with p62, normalized to PARP14 total levels. In the reported WB, it is difficult to see the interaction between PARP14 and p62 in untreated conditions. Please provide clearer WB. Additionally, I would expect an increased interaction between PARP14 and p62 upon IFN treatment due to PARP14 recruitment to p62 condensates, not just because of increased PARP14 levels. Since the authors show that PARP14 is not recruited to ADPr condensates upon RBN treatment (Fig. 2G), why is the interaction between p62 and PARP14 so high under RBN treatment?
    12. Fig. 4C: Please quantify WB signals of ADP-ribosylated p62 for the different conditions analyzed. ADP-ribosylation of p62 is still present in cells lacking PARP14. Are there other enzymes that can modify p62? Moreover, the authors state: "We observed an increased MARylation of p62 upon IFNγ treatment" (line 230); is this dependent on the increase in PARP14 levels or the translocation of PARP14 to ADPr condensates? Quantifications should help clarify this aspect.
    13. Fig. 4G: Quantifications are required.

    Significance

    The role of the PARP family in cellular processes is a very active and rapidly growing field. New information about the organization of PARPs in the nucleus, cytosol, or different types of bodies/structures is certainly relevant to the field.

    However, the present study is too preliminary at the moment to be considered highly relevant. Both the data analysis and conclusions need to be carefully reviewed. After major revisions, the manuscript might be of general interest if well contextualized within the fields of post-translational modification and protein degradation processes. It would remain in any case interesting for the field of ADP ribosylation.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In this study, Raja et al. found cytoplasmic condensates formed by the treatment of INFγ, investigated components of these condensates and identified p62, NBR1 and PARP14 as their components. INFγ treatment induced PARP14 expression, and PAPR14 inhibitor treatment inhibited condensation formation, suggesting that the amount of PARP14 and its enzymatic activity are important for the condensate formation. The ADPr-positive p62 condensates were independent of autophagic degradation, and proteasomal activity was required for their formation.

    Major comment

    1. The finding that the ubiquitin-proteasome, but not autophagy activity, is indispensable for the formation of p62 condensates is of interest. However, the molecular mechanism by which the ubiquitin-proteasome system (UPS) is involved in the regulation of the PARP14-p62 condensate is still unclear. Which step(s) is the UPS involved?
    2. The p62 condensate serves as a scaffold for autophagosome formation through the assembling autophagy receptors including NBR1 and TAX1BP1, followed by recruiting ATG proteins such as FIP200. While ADPr-positive p62 condensates also contain NBR1 and polyubiquitinated proteins, they are unrelated to autophagic degradation. It is unclear what factors govern autophagy-independent function.
    3. The authors claim that the amount of PARP14 and its MAR activity are essential for the condensate formation. However, all experiments were performed only with PARP14 inhibitors, and further validation is needed. If the importance of PARP14 activity is to be directly demonstrated, experiments in which an enzyme activity mutant is introduced into PARP14 KO cells are needed.
    4. In Figure 2a, the heatmap alone is insufficient. Neither errors nor statistical comparisons are indicated.
    5. The statistical analysis of Figure S2 is inappropriate; instead of t-tests, multiple comparisons should be used to compare three or more groups.

    Minor comment

    1. What percentage of p62 condensates upon INFγ treatment are ADPr positive? Are all p62 bodies seen with INFγ stimulation unrelated to autophagy?
    2. Is ADPr condensation a PARA14-specific phenomenon? PARP9 and PARP12 were also upregulated by INFγ treatment. Are these factors also involved in condensate formation?
    3. Figure 4D appears to be immunoprecipitation (IP) under non-denaturing conditions. If so, it is not possible to distinguish whether the MAR signal is derived from p62 or from the p62 interacting proteins (the associated ubiquitinated substrates). IP experiments should be performed under denaturing conditions.
    4. In Figure 5B, which band is HO1, the upper or lower?
    5. There is no image for ubiquitin in S5D. Right panel in Figure 4F shows only IFγ + RBN, which should show all data sets in the same panel.

    Significance

    Liquid droplets, which have continuously being identified in cells, are a hot topic in cell biology. Droplet formation, structure, molecular dynamics, and degradation, as well as their abnormalities and disease development due to genetic mutation and stress, are of wide-ranging interest from basic to pathological aspects. Therefore, this research has the potential to attract interest from a wide range of fields.

    General assessment

    Overall, the data are clear and the phenomenon is of interest. However, the molecular mechanism and biological significance of the condensate formation is unknown; It is unclear why proteasome activity is required for the formation of PARP14-mediated ADP ribosylation. It is also unclear what the consequences are for the cell if the ADPr-positive condensates are not formed. Thea authors should address these general and important issues and provide the data If not all.

  5. Version published to 10.1101/2024.05.22.595402v1 on bioRxiv