The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP

  1. Maria Körner
  2. Susanne R Meyer
  3. Gabriella Marincola
  4. Maximilian J Kern
  5. Clemens Grimm
  6. Christina Schuelein-Voelk
  7. Utz Fischer
  8. Kay Hofmann
  9. Alexander Buchberger

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Abstract

The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members V CP nuclear c ofactor f amily member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.

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  1. Version published to 10.7554/elife.92409 on eLife
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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript reports the interaction between poorly characterized FAM104 proteins to ubiquitin-dependent segregase VCP. VCP functions in protein and organelle quality control as well as in extraction of ubiquitylated proteins from chromatin to regulate DNA repair, replication and transcription. In addition, VCP mutations are causative for several human neurological disorders. The authors demonstrate that FAM104 proteins promote the nuclear localization of VCP and that their loss causes impaired growth and hypersensitivity to chemical inhibition of VCP. They show that FAM104 proteins bind to VCP directly via a non-canonical helical motif and model the interaction with AlphaFold Miner, which allowed the identification of critical amino acids that mediate the interaction, which was then validated in vivo and in vitro.

    The conclusions are supported with well-designed experiments and data of high quality, manuscript is written in a clear and precise way.

    Minor points

    • P3: The authors write that mutations in VCP are causative for cancer. This should be rephrased.
    • P3: I would suggest to add a reference to the new study that also shows that VCP is also exploited by bacteria rand not only viruses (
    • Could the authors better illustrate the difference between FAM104A and B, and provide some explanations of why A seems to interact better with VCP compared to B. Is it just matter of higher expression of FAM104A in the cells where the interaction has been tested?
    • The authors should quantify the IF results in Figure 4 and include the quantification in the main figure
    • UBXN2B interaction with FAM104A was found in HT affinity-MS (Huttlin et al) and Y2H (Luck et al) studies. Can the authors validate this interaction of UBXN2B with FAM104 proteins? This would help to understand whether FAM104 interacts mainly with nuclear adaptors.

    Significance

    The results presented in this manuscript will be of interest to the borad field of protein quality control and lay the foundation to study the functions of FAM104 proteins in chromatin-associated degradation.

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    Referee #2

    Evidence, reproducibility and clarity

    In their manuscript ‚FAM104 proteins promote the nuclear localization of p97/VCP' Maria Körner, Susanne Meyer, and coauthors describe the identification of FAM104 proteins as cofactors of p97/VCP, a central factor in ubiquitin-mediated cellular proteolytic processes. Function as p97/VCP cofactors has not previously been clearly attributed to FAM104 proteins.

    The initial observation that FAM104 proteins interact directly with p97/VCP in yeast two-hybrid assays is confirmed by in vitro pulldown experiments with recombinantly purified proteins as well as in lysates of cultured mammalian cells. Using truncated proteins and structure predictions, the interaction interface of p97/VCP and FAM104 proteins is further narrowed down to single amino acids of a characteristic C-terminal alpha helix in FAM104. Overall, these interaction studies are technically sound and include meaningful control conditions to postulate FAM104 proteins as p97/VCP binders. Subsequent functional analyses using colocalization studies and cellular fractionation suggest that FAM104 proteins determine the nuclear/chromatin-associated fraction of p97/VCP. Based on this observation, the authors speculate that FAM104 proteins are of particular importance given the established nuclear/chromatin-associated processes involving p97/VCP activity. This hypothesis is supported by the observation that FAM104A knockout cells exhibit an impaired growth phenotype that is exacerbated in the presence of a p97/VCP inhibitor and in combination with a DNA damage trigger.

    Points of concern:

    1. The authors hypothesize that FAM104 proteins enhance the nuclear/chromatin-associated function of p97/VCP by sequestering it from the cytosol into nuclear/chromatin. In the corresponding experiments, overexpression of FAM104 species (Figures 4 and 5) in otherwise unperturbed cells is used. Because recruitment of p97/VCP to client proteins is thought to depend in large part on ubiquitylation, it is unclear how overexpression of FAM104 is sufficient to enhance nuclear/chromatin localization of VCP. Is nuclear/chromatin localization accompanied by changes in ubiquitylation and/or turnover of the corresponding proteins? In other words, does enhanced localization also correlate with increased activity, or could the enhanced nuclear/chromatin association also be explained by inhibited/captured p97/VCP?
    2. The authors link the function of FAM104 proteins in nuclear targeting of p97/VCP to the absence of a unique NLS peptide. Therefore, it would be interesting to determine whether the appearance of FAM104 proteins at the evolutionary level correlates with the strength/presence of NLS peptides in p97/VCP and/or its cofactors UFD1/NPL4/FAF1/UBXN3. Do FAM104 proteins compensate for the loss of NLS peptides in p97/cofactor complexes?
    3. Re 2) It remains unclear whether FAM104 proteins are responsible for the mere sequestration of p97/VCP in the nucleus or whether FAM104 proteins also contribute to process/client specificity in other ways. In this context, the authors could investigate a possible compensation of the reduced nuclear targeting of p97/VCP in FAM104 knock-out cells by fusion with an efficient cNLS peptide. Does this compensate for both nuclear/chromatin localization and growth/drug sensitivity?
    4. Re 3) How does overexpression of FAM104 alter drug sensitivity compared to knock-out cell lines (Figure 7)?
    5. Is there experimental evidence on how FAM104 proteins can bind p97/VCP to chromatin in this context and the proposed targeting of p97/VCP to the nucleus/chromatin? Does FAM104 mRNA/protein expression increase when p97/VCP-mediated processes are disrupted (e.g., in the presence of p97/VCP inhibition or DNA damage)? Are FAM104 protein levels stabilized under these conditions? Are FAM104 proteins differentially regulated (e.g., in terms of localization) under these conditions? Figure 3A suggests that FAM104 proteins may have a different function in relation to p97/VCP protein levels: FAM104A iso1/2 have lower p97/VCP protein levels than FAM104A iso5 and B iso3. The authors suggest that this is due to the solubility of p97/VCP. It should be clarified whether lower solubility equates to increased chromatin association.
    6. It remains unclear whether a FAM104-dependent shift in nuclear/chromatin-associated p97/VCP could also be a secondary compensatory effect versus functional impairment in FAM104 overexpression/depletion. The authors might include this in their discussion.

    Significance

    In summary, the author's conclusion that FAM104 proteins represent a previously underappreciated class of p97/VCP cofactors is well supported. Given the versatile and important role of p97/VCP and cellular protein homeostasis pathways, this finding is of interest to a broad audience. However, the functional role of FAM104 proteins in p97/VCP biology remains unclear. Therefore, the authors need to further elaborate the physiological contribution of FAM104 proteins to p97/VCP function in additional experiments. The suggestions are largely based on modifications of experiments already performed in this manuscript.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The manuscript submitted by Korner et al. presents data regarding the interaction of p97 with FAM104 protein family. This part of the manuscript is performed by Y2H as well as in-vitro and in-vivo pull-down assays. After molecular mapping and characterizations, the authors continue and address the role of FAM104-p97 interaction on nuclear localization of p97 as well cell health in respect to p97 dependent activity. Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.

    Major comments:

    • Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

    Claims concerning the mapping of FAM104 and p97 (Figures 1&2) are generally well concluded. Yet, minor issues concerning Fig2D (FAM104Aiso1 cdel26) as well as Figure 2E (p97-deltaN pull down) lack of interaction-are not supported by the presented data (both show weak interactions). Claims concerning nuclear/cytosol p97 distribution impact upon FAM104 manipulations (over-expression or KO) need to be further evaluated by additional methodologies. For example, the distribution impact using the FAM104 mutants in 4B should be evaluated by cell fractionation experiments (as performed in figure 5). Cell fractionation performed for FAM104A isoforms 1&2 should be performed on isoforms 5&3, the fact that they are expressed at lower levels has no impact, as the evaluation is on p97 and they were able to show in figure 3A an impact on p97 levels. Impact on distribution performed in Figure 6 using FAM104 KO cells should also include cell fractionation experiments in order to enable clear conclusion regarding FAM104 impact on p97 nuclear distribution.

    Also, statistics presented are somewhat problematic at several points. In figure S4C the ** difference between vector and deNLS mutant make no sense (I think they should have been non-significant). Figure 7 make no sense to compare WT and KO cells (in panels b&C) if their original growth was different. One should compare the differences in respect to the drug concentration in each cell type. Also, it may be useful for statistical purposes to evaluate cell numbers rather than growth% and this may enable to obtain better statistical significances.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated time investment for substantial experiments.

    The suggested experiments are all in reasonable time frame.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Y2H is not explained at all in methods, furthermore, it would be useful to present in a table the entire list of p97 interactome obtained in this screen.

    • Are the experiments adequately replicated and statistical analysis adequate?

    See comments above

    Minor comments:

    • Specific experimental issues that are easily addressable.
    • Are prior studies referenced appropriately?

    Previous reports regarding FAM104 interaction with p97 have been reported in two papers (PMID 32296183 sup. Table9 therein and PMID 32814053 S2 therein) this has not been stated at all. Furthermore, no data concerning previous knowledge of FAM104 is referred to in the introduction.

    • Are the text and figures clear and accurate?

    The text is written well and one can easily follow

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    See major suggestion above

    Referee cross-commenting

    It seems reviewer #2 concerns are also situated close to our comments regarding nuclear function of FAM104 on p97 function. Reviewers 3 comment regarding UBXN2B possible tertiary complex with p97 and FAM104 should be attempted as it would help put p97 function in a slightly more specific context

    Significance

    Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested.

    The following aspects are important:

    • General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? The authors deal with a specific interaction of p97 with FAM104 protein family. While this interaction has been previously reported, their mapping of domains required for interaction is new. Conclusions regarding the additional binding partners of the FAM104-p97 complex would require additional double affinity and mass-spectrometry identifications (as well as possible substrates identification).
    • Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). The study advances the repertoire of p97 adaptors and interacting domains.
    • Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field? The suitable audience would be p97 basic researchers
    • Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. p97 role in protein quality control and cellular homeostasis.
  10. Version published to 10.1101/2023.07.25.550451v1 on bioRxiv