Profiling the physiological impact of aberrant folded-state protein filamentation in cells

  1. Tal Levin
  2. Hector Garcia-Seisdedos
  3. Arseniy Lobov
  4. Matthias Wojtynek
  5. Alexander Alexandrov
  6. Ghil Jona
  7. Dikla Levi
  8. Ohad Medalia
  9. D. Emmanuel Levy

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Abstract

The formation of large polymeric structures such as cytoskeletal and enzyme filaments is crucial for normal cellular function. However, such filaments can also form due to mutations that create self-interactions at the surface of symmetric proteins. Often, the proteins forming these structures maintain a folded state and thereby differ from aggregates and amyloids that involve misfolding. We refer to this type of assemblies as agglomerates to mark this difference. While cells have quality control mechanisms to identify, buffer, and eliminate misfolded proteins, it is unclear whether similar mechanisms exist for agglomerates, or whether agglomerates are toxic to cells. Here, we profiled the physiological impact of mutation-induced folded-state protein filamentation in yeast cells. First, we devised a simple strategy to distinguish fluorescently labeled proteins forming agglomerates versus aggregates. We then profiled exogenous protein agglomerates in terms of their recognition by known quality control mechanisms, their effects on specific cellular processes and overall fitness on S. cerevisiae cultures. We found that agglomerates do not colocalize with the proteostasis machinery and do not result in measurable fitness defects. Proteomics profiling of cells expressing the wild type protein, agglomerating or misfolded variants revealed a consistent picture, with only minor, agglomerate-size-dependent changes observed and linked to the cell-wall and plasma-membrane proteins. Overall, our findings indicate that agglomerates form mostly benign structures in cells when compared to aggregates, and thereby offer a promising route for synthetic biology applications.

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  1. Review Commons

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  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This article investigates the phenomenon of intracellular protein agglomeration. The authors distinguish between agglomeration and aggregation, both physically characterising them and developing a simple but elegant assay to differentiate the two. Using microscopy and structural analysis, this research demonstrates that unlike aggregates, agglomerates retain their folded structures (and are not misfolded), and do not colocalise with chaperones or interact with the proteostasis machinery which targets and breaks down misfolded proteins. The inert nature of agglomerates was further confirmed in fitness assays, though they were observed to disrupt the yeast proteome. Overall, agglomerated proteins were described and characterised, and shown to be largely neutral in vivo.

    The claims and conclusions were well supported by the data. Microscopy and CD spectra (previously published) were used to confirm the nature of agglomerates and to rule out colocalistion with proteostasis machinery. This was confirmed by testing ubiquitination.

    The fitness of yeast cells carrying enzymatically-inactive agglomerates was assayed by generating growth curves over 24 hours. The growth rate and doubling time were taken from these growth curves as a proxy for relative fitness. The authors mention not wanting to mask differences in lag, log or stationary phases between mutants. This could be achieved by using the area under each growth curve, rather than growth rate or doubling time alone. No further experimentation would be needed, and area under the curve may provide a more holistic metric to measure fitness by.

    The results indicate that agglomerates confer a slight fitness advantage. The authors do not speculate on a reason for this. I would be interested to know why they thought this might be.

    Referees cross-commenting

    I have read the reports from the other reviewers and agree with their comments.

    Significance

    Protein filamentation is observed across the tree of life, and contributes greatly to cell structure and organisation (Wagstaff, J., Löwe, J. Prokaryotic cytoskeletons: protein filaments organizing small cells. Nat Rev Microbiol 16, 187-201 (2018).). Recent work in this field has shown that self-assembly is also important for enzyme function (S. Lim, G. A. Jung, D. J. Glover, D. S. Clark, Enhanced Enzyme Activity through Scaffolding on Customizable Self-Assembling Protein Filaments. Small 2019, 15, 1805558.). Previous work from several of these authors demonstrated that the ability of a protein to filament is subject to selection (Garcia-Seisdedos H, Empereur-Mot C, Elad N, Levy ED. Proteins evolve on the edge of supramolecular self-assembly. Nature. 2017 Aug 10;548(7666):244-247. doi: 10.1038/nature23320. Epub 2017 Aug 2. PMID: 28783726.). It has become increasingly clear that protein assemblies are ubiquitous, evolvable and perhaps overlooked in research.

    This research explores a specific type of filamentation, named agglomeration, unique in that the protein which assemble are not misfolded (Romero-Romero ML, Garcia-Seisdedos H. Agglomeration: when folded proteins clump together. Biophys Rev. 2023;15: 1987-2003.). This is particularly of biomedical interest due to its role in disease, such as sickle cell anaemia (J. Hofrichter, P.D. Ross, & W.A. Eaton, Kinetics and Mechanism of Deoxyhemoglobin S Gelation: A New Approach to Understanding Sickle Cell Disease*, Proc. Natl. Acad. Sci. U.S.A. 71 (12) 4864-4868, https://doi.org/10.1073/pnas.71.12.4864 (1974).) The current research adds to the field by specifically exploring agglomerates in the most detailed methodology to date.

    The novelty of this research lies especially in two areas; (1) establishing a method for distinguishing between aggregation and agglomeration, and (2) the finding that agglomerates are largely innocuous in vivo. The method established for defining agglomerates is simple, elegant and well-described in this paper's methods. The authors then probe cellular responses to agglomeration via both proteostasis machinery and cellular fitness. They noted no disruption to fitness and observed little targeting of agglomerates by chaperones. The experiments were thorough, conclusive, and resulted in interesting findings.

    The inertia of this type of protein filament is unexpected; agglomerates are large and have been associated with disease. The results of this study, however, indicate that agglomerates are non-toxic and well-tolerated in vivo. The authors speculate that agglomerates may have evolved in a non-adaptive process, which is evolutionary very interesting. They also posit that these results could lead to synthetic biology applications such as a tracking expression or as a molecular sensor. This work is of great interest and impact both in cell biology, biomedicine and in-vivo biology.

    Personal note: I come from a background of enzyme evolution and have viewed the work in this light.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This is a very interesting paper investigating the fitness and cellular effects of mutations that drive dihedral protein complex into forming filaments. The Levy group have previously shown that this can happen relatively easily in such complexes and this paper now investigates the cellular consequences of this phenomenon. The study is very rigorous biophysically and very surprisingly comes up empty in terms of an effect: apparently this kind of self-assembly can easily be tolerated in yeast, which was certainly not my expectation. This is a very interesting result, because it implies that such assemblies may evolve neutrally because they fulfill the two key requirements for such a trajectory: They are genetically easily accessible (in as little as a single mutation), and they have perhaps no detrimental effect on fitness. This immediately poses two very interesting questions: Are some natural proteins that are known to form filaments in the cell perhaps examples of such neutral trajectories? And if this trait is truly neutral (as long as it doesn't affect the base biochemical function of the protein in question), why don't we observe more proteins form these kinds of ordered assemblies.

    I have no major comments about the experiments as I find that in general very carefully carried out. I have two more general comments:

    1. The fitness effect of these assemblies, if one exists, seems very small. I think it's worth remembering that even very small fitness effects beyond even what competition experiments can reveal could in principle be enough to keep assembly-inducing alleles at very low frequencies in natural populations. Perhaps this could be acknowledged in the paper somewhere.
    2. The proteins used in this study I think were chosen such that they do not have an important function in yeast that could be disrupted by assembly This allows the effect of the large scale assemblies to be measured in isolation. If I deduced this correctly, this should probably be pointed out agin in this paper (I apologise if I missed this).
    3. The model system in which these effects were tested for is yeast. This organism has a rigid cell wall and I was wondering if this makes it more tolerant to large scale assemblages than wall-less eukaryotes. Could the authors comment on this?

    Minor points:

    In Figure 2D, what are the fits? And is there any analysis that rules out expression effects on the mutant caused by higher levels of the wild-type? The error bars in Figure 2E are not defined.

    Significance

    This is a remarkably rigours paper that investigates whether self-assembly into large structures has any fitness effect on a single celled organism. This is very relevant, because a landmark paper from the Levy group showed that many proteins are very close in genetic terms to forming such assemblies. The general expectation I think would have been that this phenomenon is pretty harmful. This would have explained why such filaments are relatively rare as far as we know. This paper now does a large number of highly rigours experiments to first prove beyond doubt that a range of model proteins really can be coaxed into forming such filaments in yeast cells through a very small number of mutations. Its perhaps most surprising result is that this does not negatively affect yeast cells.

    From an evolutionary perspective, this is a very interesting and highly surprising result. It forces us to rethink why such filaments are not more common in Nature. Two possible answers come to mind: First, it's possible that filamentation is not directly harmful to the cell, but that assembling proteins into filaments can interfere with their basic biochemical function (which was not tested for here).

    Second, perhaps assembly does cause a fitness defect, but one so small that it is hard to measure experimentally. Natural selection is very powerful, and even fitness coefficients we struggle to measure in the laboratory can have significant effects in the wild. If this is true, we might expect such filaments to be more common in organisms with small effective population sizes, in which selection is less effective.

    A third possibility is of course that the prevalence of such self-assembly is under-appreciated. Perhaps more proteins than we currently know assemble into these structures under some conditions without any benefit or detriment to the organism.

    These are all fascinating implications of this work that straddle the fields of evolutionary genetics and biochemistry and are therefore relevant to a very wide audience. My own expertise is in these two fields. I also think that this work will be exciting for synthetic biologists, because it proves that these kinds of assemblies are well tolerated inside cells.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this work, the authors used yeast cell as a model system to study the abovementioned question. They established a model protein system using fluorescently labeled proteins that can form both agglomerates and aggregates. Using imaging experiments, they arguably showed that agglomerates do not colocalize with the proteostasis machinery, echoing what was observed by proteomics results. The proteomics results after pull down assay to study the interactome revealed that agglomerate-size-dependent changes were dependent on the cell-wall and plasma-membrane proteins. On the other hand, as expected, the misfolded proteins (aggregates) showed heavy involvement of proteostasis network components.

    Although the experiments still lack some controls and failed to support some of the conclusions, I found this work is a nice complement of the field to emphasize the point that "aggregates" and "agglomerates" are two different states, which is often mistaken by lots of researchers in recent years, in particular with the membraneless organelles (LLPS). I support its publication after the authors may consider the following suggestions and make necessary improvement.

    Major concerns:

    My major concern was raised by the lack of evidence to support the model system's folding state in the cell.

    1. In Figure 1 and 2, I found the evidence to distinguish the folded state of proteins in the cells was limited. The concept of using hybrid imaging technique to prove the folding state is not a common experiment. The description of Figure 2 was very limited. I am sure the general audience can be convinced that the model proteins were actually folded and form agglomeration.
    2. In addition, for mutants formed aggregates, the authors may consider to perform fractionation or crosslinking or native page experiment to show the evidence of protein misfolding and aggregation.
    3. Have the authors considered to use FRAP assay to distinguish "aggregates" and "agglomerates" states in the cell? Does each of the state display different dynamics in the cell?

    Minor concerns:

    1. In Figure 3, it is very interesting to see such patten. I wonder why some of the chaperones were not responsive to misfolded proteins but some were very addicted to proteostasis. Could you elaborate more on this point? Are they chaperone sensitive, namely selective to 60/10, 70/40 or 90 system?
    2. In Figure 6, I suggest to add GO analysis and KEGG analysis to distinguish pathways and functional mismatch between "aggregates" and "agglomerates" interactomics.
    3. The quantity control for the proteomics studies is needed, namely the reproducibility of 3 repeats?
    4. This may beyond the scope of this work. I am interested whether the authors could point out whether similar works can be done in mammalian cells. What is the model system for mammalian cell that can form "agglomerates".

    Referees cross-commenting

    I read through the other two reviewers' comments, which I found reasonable. It seems like all reviewers agreed that this work is of enough significance for the field only with several technical concerns.

    Significance

    The submitted manuscript emphasized on a very important but often misleading concept: "aggregates" and "agglomerates" are two different states of protein structures in the cell with distinct physiological roles. However, these two states are of very similar phenotype: punctate structure in the cell. While the proteostasis network has been well-established for its central role of protein quality control and coping with misfolded and aggregated proteome, the authors attempted to profile the mechanism and physiological impact of mutation-induced folded-state protein filamentation, namely a model of "agglomerates". Such overarching goal of this work clearly pointed out the novelty of this work. Clearly, this is a new angle and aspect remained to be clarified for the field.

  5. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The response appears in a PDF document, which will be easier to read than plain text

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    This article investigates the phenomenon of intracellular protein agglomeration. The authors distinguish between agglomeration and aggregation, both physically characterising them and developing a simple but elegant assay to differentiate the two. Using microscopy and structural analysis, this research demonstrates that unlike aggregates, agglomerates retain their folded structures (and are not misfolded), and do not colocalise with chaperones or interact with the proteostasis machinery which targets and breaks down misfolded proteins. The inert nature of agglomerates was further confirmed in fitness assays, though they were observed to disrupt the yeast proteome. Overall, agglomerated proteins were described and characterised, and shown to be largely neutral in vivo.

    The claims and conclusions were well supported by the data. Microscopy and CD spectra (previously published) were used to confirm the nature of agglomerates and to rule out colocalistion with proteostasis machinery. This was confirmed by testing ubiquitination.

    The fitness of yeast cells carrying enzymatically-inactive agglomerates was assayed by generating growth curves over 24 hours. The growth rate and doubling time were taken from these growth curves as a proxy for relative fitness. The authors mention not wanting to mask differences in lag, log or stationary phases between mutants. This could be achieved by using the area under each growth curve, rather than growth rate or doubling time alone. No further experimentation would be needed, and area under the curve may provide a more holistic metric to measure fitness by.

    The results indicate that agglomerates confer a slight fitness advantage. The authors do not speculate on a reason for this. I would be interested to know why they thought this might be.

    Referees cross-commenting

    I have read the reports from the other reviewers and agree with their comments.

    Significance

    Protein filamentation is observed across the tree of life, and contributes greatly to cell structure and organisation (Wagstaff, J., Löwe, J. Prokaryotic cytoskeletons: protein filaments organizing small cells. Nat Rev Microbiol 16, 187-201 (2018).). Recent work in this field has shown that self-assembly is also important for enzyme function (S. Lim, G. A. Jung, D. J. Glover, D. S. Clark, Enhanced Enzyme Activity through Scaffolding on Customizable Self-Assembling Protein Filaments. Small 2019, 15, 1805558.). Previous work from several of these authors demonstrated that the ability of a protein to filament is subject to selection (Garcia-Seisdedos H, Empereur-Mot C, Elad N, Levy ED. Proteins evolve on the edge of supramolecular self-assembly. Nature. 2017 Aug 10;548(7666):244-247. doi: 10.1038/nature23320. Epub 2017 Aug 2. PMID: 28783726.). It has become increasingly clear that protein assemblies are ubiquitous, evolvable and perhaps overlooked in research.

    This research explores a specific type of filamentation, named agglomeration, unique in that the protein which assemble are not misfolded (Romero-Romero ML, Garcia-Seisdedos H. Agglomeration: when folded proteins clump together. Biophys Rev. 2023;15: 1987-2003.). This is particularly of biomedical interest due to its role in disease, such as sickle cell anaemia (J. Hofrichter, P.D. Ross, & W.A. Eaton, Kinetics and Mechanism of Deoxyhemoglobin S Gelation: A New Approach to Understanding Sickle Cell Disease*, Proc. Natl. Acad. Sci. U.S.A. 71 (12) 4864-4868, https://doi.org/10.1073/pnas.71.12.4864 (1974).) The current research adds to the field by specifically exploring agglomerates in the most detailed methodology to date.

    The novelty of this research lies especially in two areas; (1) establishing a method for distinguishing between aggregation and agglomeration, and (2) the finding that agglomerates are largely innocuous in vivo. The method established for defining agglomerates is simple, elegant and well-described in this paper's methods. The authors then probe cellular responses to agglomeration via both proteostasis machinery and cellular fitness. They noted no disruption to fitness and observed little targeting of agglomerates by chaperones. The experiments were thorough, conclusive, and resulted in interesting findings.

    The inertia of this type of protein filament is unexpected; agglomerates are large and have been associated with disease. The results of this study, however, indicate that agglomerates are non-toxic and well-tolerated in vivo. The authors speculate that agglomerates may have evolved in a non-adaptive process, which is evolutionary very interesting. They also posit that these results could lead to synthetic biology applications such as a tracking expression or as a molecular sensor. This work is of great interest and impact both in cell biology, biomedicine and in-vivo biology.

    Personal note: I come from a background of enzyme evolution and have viewed the work in this light.

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This is a very interesting paper investigating the fitness and cellular effects of mutations that drive dihedral protein complex into forming filaments. The Levy group have previously shown that this can happen relatively easily in such complexes and this paper now investigates the cellular consequences of this phenomenon. The study is very rigorous biophysically and very surprisingly comes up empty in terms of an effect: apparently this kind of self-assembly can easily be tolerated in yeast, which was certainly not my expectation. This is a very interesting result, because it implies that such assemblies may evolve neutrally because they fulfill the two key requirements for such a trajectory: They are genetically easily accessible (in as little as a single mutation), and they have perhaps no detrimental effect on fitness. This immediately poses two very interesting questions: Are some natural proteins that are known to form filaments in the cell perhaps examples of such neutral trajectories? And if this trait is truly neutral (as long as it doesn't affect the base biochemical function of the protein in question), why don't we observe more proteins form these kinds of ordered assemblies.

    I have no major comments about the experiments as I find that in general very carefully carried out. I have two more general comments:

    1. The fitness effect of these assemblies, if one exists, seems very small. I think it's worth remembering that even very small fitness effects beyond even what competition experiments can reveal could in principle be enough to keep assembly-inducing alleles at very low frequencies in natural populations. Perhaps this could be acknowledged in the paper somewhere.
    2. The proteins used in this study I think were chosen such that they do not have an important function in yeast that could be disrupted by assembly This allows the effect of the large scale assemblies to be measured in isolation. If I deduced this correctly, this should probably be pointed out agin in this paper (I apologise if I missed this).
    3. The model system in which these effects were tested for is yeast. This organism has a rigid cell wall and I was wondering if this makes it more tolerant to large scale assemblages than wall-less eukaryotes. Could the authors comment on this?

    Minor points:

    In Figure 2D, what are the fits? And is there any analysis that rules out expression effects on the mutant caused by higher levels of the wild-type? The error bars in Figure 2E are not defined.

    Significance

    This is a remarkably rigours paper that investigates whether self-assembly into large structures has any fitness effect on a single celled organism. This is very relevant, because a landmark paper from the Levy group showed that many proteins are very close in genetic terms to forming such assemblies. The general expectation I think would have been that this phenomenon is pretty harmful. This would have explained why such filaments are relatively rare as far as we know. This paper now does a large number of highly rigours experiments to first prove beyond doubt that a range of model proteins really can be coaxed into forming such filaments in yeast cells through a very small number of mutations. Its perhaps most surprising result is that this does not negatively affect yeast cells.

    From an evolutionary perspective, this is a very interesting and highly surprising result. It forces us to rethink why such filaments are not more common in Nature. Two possible answers come to mind: First, it's possible that filamentation is not directly harmful to the cell, but that assembling proteins into filaments can interfere with their basic biochemical function (which was not tested for here).

    Second, perhaps assembly does cause a fitness defect, but one so small that it is hard to measure experimentally. Natural selection is very powerful, and even fitness coefficients we struggle to measure in the laboratory can have significant effects in the wild. If this is true, we might expect such filaments to be more common in organisms with small effective population sizes, in which selection is less effective.

    A third possibility is of course that the prevalence of such self-assembly is under-appreciated. Perhaps more proteins than we currently know assemble into these structures under some conditions without any benefit or detriment to the organism.

    These are all fascinating implications of this work that straddle the fields of evolutionary genetics and biochemistry and are therefore relevant to a very wide audience. My own expertise is in these two fields. I also think that this work will be exciting for synthetic biologists, because it proves that these kinds of assemblies are well tolerated inside cells.

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this work, the authors used yeast cell as a model system to study the abovementioned question. They established a model protein system using fluorescently labeled proteins that can form both agglomerates and aggregates. Using imaging experiments, they arguably showed that agglomerates do not colocalize with the proteostasis machinery, echoing what was observed by proteomics results. The proteomics results after pull down assay to study the interactome revealed that agglomerate-size-dependent changes were dependent on the cell-wall and plasma-membrane proteins. On the other hand, as expected, the misfolded proteins (aggregates) showed heavy involvement of proteostasis network components.

    Although the experiments still lack some controls and failed to support some of the conclusions, I found this work is a nice complement of the field to emphasize the point that "aggregates" and "agglomerates" are two different states, which is often mistaken by lots of researchers in recent years, in particular with the membraneless organelles (LLPS). I support its publication after the authors may consider the following suggestions and make necessary improvement.

    Major concerns:

    My major concern was raised by the lack of evidence to support the model system's folding state in the cell.

    1. In Figure 1 and 2, I found the evidence to distinguish the folded state of proteins in the cells was limited. The concept of using hybrid imaging technique to prove the folding state is not a common experiment. The description of Figure 2 was very limited. I am sure the general audience can be convinced that the model proteins were actually folded and form agglomeration.
    2. In addition, for mutants formed aggregates, the authors may consider to perform fractionation or crosslinking or native page experiment to show the evidence of protein misfolding and aggregation.
    3. Have the authors considered to use FRAP assay to distinguish "aggregates" and "agglomerates" states in the cell? Does each of the state display different dynamics in the cell?

    Minor concerns:

    1. In Figure 3, it is very interesting to see such patten. I wonder why some of the chaperones were not responsive to misfolded proteins but some were very addicted to proteostasis. Could you elaborate more on this point? Are they chaperone sensitive, namely selective to 60/10, 70/40 or 90 system?
    2. In Figure 6, I suggest to add GO analysis and KEGG analysis to distinguish pathways and functional mismatch between "aggregates" and "agglomerates" interactomics.
    3. The quantity control for the proteomics studies is needed, namely the reproducibility of 3 repeats?
    4. This may beyond the scope of this work. I am interested whether the authors could point out whether similar works can be done in mammalian cells. What is the model system for mammalian cell that can form "agglomerates".

    Referees cross-commenting

    I read through the other two reviewers' comments, which I found reasonable. It seems like all reviewers agreed that this work is of enough significance for the field only with several technical concerns.

    Significance

    The submitted manuscript emphasized on a very important but often misleading concept: "aggregates" and "agglomerates" are two different states of protein structures in the cell with distinct physiological roles. However, these two states are of very similar phenotype: punctate structure in the cell. While the proteostasis network has been well-established for its central role of protein quality control and coping with misfolded and aggregated proteome, the authors attempted to profile the mechanism and physiological impact of mutation-induced folded-state protein filamentation, namely a model of "agglomerates". Such overarching goal of this work clearly pointed out the novelty of this work. Clearly, this is a new angle and aspect remained to be clarified for the field.

  9. Version published to 10.1101/2024.09.13.612878 on bioRxiv