DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43

  1. Andy Y.W. Lam
  2. Kotaro Tsuboyama
  3. Hisashi Tadakuma
  4. Yukihide Tomari

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Abstract

Protein misfolding and aggregation are characteristic features of neurodegenerative diseases. While molecular chaperones are well-known suppressors of these aberrant events, we recently reported that highly disordered, hydrophilic and charged heat-resistant obscure (Hero) proteins may have similar effects. Specifically, Hero proteins can maintain the activity of other proteins from denaturing conditions in vitro, while their overexpression can suppress cellular aggregation and toxicity associated with aggregation-prone proteins. However, it is unclear how these protective effects are achieved. Here, we utilized single-molecule FRET to monitor the conformations of the aggregation-prone prion-like low complexity domain (LCD) of TAR DNA-binding protein 43 (TDP-43). While we observed high conformational heterogeneity in wild-type LCD, the ALS-associated mutation A315T promoted collapsed conformations. In contrast, an Hsp40 chaperone, DNAJA2, and a Hero protein, Hero11 stabilized extended states of the LCD, consistent with their ability to suppress the aggregation of TDP-43. Our results link single-molecule effects on conformation to macro effects on bulk aggregation, where a Hero protein, like a chaperone, can maintain the conformational integrity of a client protein to prevent its aggregation.

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  1. Version published to 10.1101/2022.11.15.516569v4 on bioRxiv
  2. Version published to 10.1101/2022.11.15.516569v3 on bioRxiv
  3. Version published to 10.1101/2022.11.15.516569v2 on bioRxiv
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    Reply to the reviewers

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

    Summary:

    The submitted manuscript is comparing the effect of individual chaperones and heat-resistant obscure (Hero) proteins on the overall folding of the TDP-43 LCD-domain and its relation to aggregation propensity. Therefore, the authors apply smFRET in order to deduce eventual morphological changes of the LCD-domain from FRET efficiencies. The authors observe that the LCD domain extends its structure upon binding of chaperone/Hero proteins whereas it is collapsed in the absence of those. Furthermore, immunoblotting of filter trap assays indicate that overexpression of chaperones and Hero proteins reduce aggregation of TDP-43 in vivo. Both, the morphological effects on the LCD-domain and the aggregation propensity are significantly enhanced for the TDP-43 A315T mutant. Moreover, the authors tested a charge depleted Hero protein version with reduced "chaperone-like" behaviour. Therefore, the authors conclude that the binding or chaperone activity of the Hero protein is based on its residue specific charges. Finally, the authors conclude that Hero proteins can act similar to chaperones in order to keep protein homeostasis under stress conditions.

    We thank the Reviewer for their insightful evaluation of our study.

    Major comments:

    The similar effect of chaperones and Hero proteins on the morphology of TDP-43 found by the authors is intriguing and the applied experimental procedures seem well described and conducted.

    However, the assumption of the authors that a change in morphology of the LCD-domain by the chaperones and Hero proteins is directly connected to the reduction of TDP-43 aggregation is not entirely clear. Whether an overexpression of individual chaperones and Hero proteins has a direct effect on TDP-43 aggregation cannot be tested in vivo, only. It cannot be excluded that inside the cell the here tested chaperones and Hero proteins control intermediate processes or work as co-factors for other proteins involved in protein homeostasis rather directly influencing the aggregation of TDP-43. Therefore, I recommend in vitro aggregation experiments, using ThT signal as a readout. By adding chaperones, Hero proteins and a negative (BSA or others) control individually, a direct effect on TDP-43 aggregation could be concluded. Those experiments have been extensively used in the field and are quick and straightforward to handle.

    As the Reviewer explains, indirect effects on TDP-43 aggregation in cells may be accounted for by conducting aggregation experiments in vitro, with recombinant proteins. We are currently designing such experiments based on a previously described full-length recombinant TDP-43 with a TEV-cleavable MBP tag (Wang 2018 EMBO J). This can be incubated with Hero/DNAJA2/Control, and aggregation induced by cleavage of the tag, after which aggregation can be measured via filter trap similar to the method described in our work. We will include these results in our revised manuscript.

    We thank the Reviewer for their advice. While we note that it is controversial whether ThT binds to aggregates formed from full-length TDP-43 (used in all our assays in the current manuscript), it is reasonable to apply this assay to the LCD fragment as in the paper referenced by the Reviewer below (Lu 2022 Nat Cell Biol). Such an assay is also a reasonable method for confirming effects of Hero protein and DNAJA2 in vitro, and we can conduct this assay as a back-up if the above does not work.

    In addition, focusing on the LCD-domain as a main driver for TDP-43 aggregation is limiting this study. In particular, recent studies [1] indicate that the RRM1 and RRM2 sites of TDP-43 have a major impact on TDP-43 gelation and maturation to solid aggregates. Unfortunately, those sites have not been studied in this manuscript.

    We thank the Reviewer for their insight. While we are keen to investigate the impact of other regions on the aggregation of TDP-43 in the future, we chose to focus on the LCD in our current study because our smFRET assay is particularly suitable to monitor the dynamic conformational nature of this flexible, unfolded region.

    However, we agree with the Reviewer that it is possible the RRMs have an effect on the activities of Hero11 and DNAJA2. We will create constructs for the RRM-depleted variant, TDP43ΔRRM1&2, and RNA-binding deficient variant, TDP435FL for use in our cell-based assay. This will allow us to investigate how this domain influences the effects of Hero and DNAJA2, and we will include this in our revised manuscript.

    As an optional alternative for using Hero11KR->G could be the alteration of buffer conditions and using higher number of salts to promote charge screening. It would be of interest whether the results with the Hero11KR->G could be reproduced with wild type Hero11.

    We will perform our assays with Hero11 in high salt conditions for charge screening. While we agree that it may be a great alternative experiment, we note that changing the salt concentration may directly affect the LCD conformation, possibly complicating interpretation of results.

    [1] Lu et al. Nat Cell Biol;24(9):1378-1393 (2022)

    Minor comments:

    Overall, the text is clearly written, and the figures are appropriate.

    Whether the activity of individual chaperones or Hero proteins on TDP-43 aggregation "may result in the overall fitness of the cell" or "reinforcing the conformational health of the proteome" is disputable without knowing how the overexpression of certain chaperones or Hero proteins alter the formation of toxic TDP-43 oligomers.

    We thank the Reviewer for their balanced critique. We will remove or weaken this point regarding how Hero proteins "may result in the overall fitness of the cell" or may be "reinforcing the conformational health of the proteome" from the discussion.

    Reviewer #1 (Significance (Required)):

    Studying the mechanistic effects of chaperones on aggregating proteins is of major interest for the field in order to understand aging related disbalance of protein homeostasis and the progression of neurological decline, such as seen for amyotrophic lateral sclerosis (ALS). Furthermore, finding homolog proteins, also being able to inhibit protein aggregation, can help to understand overall mechanisms of protein aggregation and processes preventing such fatal behaviour. However, the technique used in this manuscript are not very novel and have been used numerously times before. smFRET is a common technique to look at protein folding/unfolding and is used frequently as a molecular ruler. The manuscript is of interest for the field of protein aggregation and folding, smFRET and neurodegeneration.

    My expertise lies in the field of protein aggregation and inhibition due to chaperones, measuring molecular interactions and neurodegenerative diseases.

    We greatly appreciate the Reviewer’s expert opinion on our work. As the Reviewer explains, we believe our work will contribute to the fields of protein aggregation and folding, smFRET and neurodegeneration. While the smFRET method may not be novel on its own, to our knowledge this is the first observation of the TDP-43 LCD, with the effect of a pathogenic mutation, at the single-molecule level. In fact, the production, dye-labeling and isolation of individual molecules is extremely challenging for TDP-43. This was made possible by our technical advances using genetic code expansion to site-specifically introduce an unnatural amino acid in TDP-43, purifying and labeling the TDP-43 from HEK cells, and isolation on glass slides.

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

    In this manuscript, the authors build on their findings (Tsuboyama 2020) that electrostatically charged IDPs (Heros) can protect proteins from denaturation and aggregation. In their previous work, they demonstrate that these Hero proteins could decrease the fraction of insoluble GFP-TDP43∆NLS in mammalian cell lines and that this mode of action was related to the electrostatic charge of the proteins and not sequence dependent. Although this protective mode of action appears to be similar to that of canonical chaperones, it is unclear how the Hero proteins compare. In this study, the authors compare Hero11 to a panel of canonical chaperones in their cell-based assays and show that it prevents aggregation in a comparable way to DNJA2. It appears that Hero11 decreases the GFP-TDP43∆NLS aggregates better than some other chaperones. They then utilise their expertise in smFRET analysis (Tsuboyama, 2018) to compare what effect DNJA2 and Hero11 (along with Hero11KR-->G (non-charged control)) have on the dynamic structures of the GFP-TDP43∆NLS (labelled with complementary fluorophores in the LCD domain). Based on analysis of the WT GFP-TDP43∆NLS and the A315T GFP-TDP43∆NLS, the authors suggest that the presence of Hero11 and DNJA2 maintain the LCD-domain of TDP43 in an extended conformation and that by doing so, aggregation can be prevented (as assessed in the cell-based assay).

    Despite finding the results very interesting, I feel that the study is preliminary and the conclusions drawn are not fully substantiated by the presented experimental work. Many questions need addressing to validate these findings and conclusions (please see more in the "Significance" section). I have tried to list the main concerns below.

    We thank the Reviewer for their detailed and critical assessment of our current study.

    Questions/concerns:

    Authors used double transient transfections but have not shown quantification of protein levels of the chaperones versus TDP43 - western blots to confirm proper expression (and levels) of the chaperones/Hero protein is crucial without it, we cannot assume that the differences in TDP-43 aggregation are a result of effective chaperoning or due to a lack of expression of any of the chaperone proteins, or high expression of others.

    We agree with the Reviewer that this is an important and straightforward validation experiment. We will perform the Western Blotting to confirm the proper and comparable expression of the chaperones/Hero proteins.

    Authors used quite a high BSA concentration in the smFRET work; it would be useful to see what the TDP43 smFRET trace looks like without BSA incubation (to ensure it is not causing some effect). Also, is there a concentration dependence? The Authors mention they are unable to identify a Hero/TDP43 complex; but if the amount of Hero protein is high (given that it is single molecules tethered), the change in compaction may not relate to the levels/ratios found in the cells (where changes to aggregation are occurring). have the authors considered whether positively charged polymers (poly-Lys) have any impact on the TDP-43 smFRET distribution? Given that the smFRET trace is so heterogeneous, to understand what is happening here would require the comparison of more than 2 variants.

    As the Reviewer suggests, we will include additional smFRET experiments in our revision.

    First, we will perform the smFRET experiment of the TDP-43 alone in the PBS buffer. However, we would like to clarify the reason we used BSA incubation for comparison in the current experiment is to account for the possibility of non-specific macromolecular crowding effects on the conformation of the LCD (an effect reported for IDPs in general, for example in Banks 2018 Biophys. J.); we expected that it would be fair to compare Hero11 against another protein, rather than buffer alone. As the Reviewer suggested, we can also perform the same experiments at lower concentrations of Hero11 and DNAJA2, including equimolar concentrations (as suggested below). Moreover, we can also test poly-K peptides for comparison.

    Although the A315T variant has a very distinct smFRET profile, it is clear that the effects of Hero11KR-->G (that is proposed to have no effect on aggregation or on the smFRET of WT TDP43) has a clear impact on A315T. Why is this?

    We thank the Reviewer for raising this interesting point. We envision that the observed effect is due to weak interactions between the LCD domain of TDP-43 and Hero11KR->G; even without K and R, there many other functional amino acids that are fully accessible due to the extremely disordered nature of the protein. The effect is easier to be observed with the A315T mutant, compared to the WT TDP-43, presumably because the mutant tends to take more compact conformations on its own. Nonetheless, unlike WT Hero11, Hero11KR->G fails to accumulate the very extended form of the LCD (FRET signal of ~0; please see below for the explanation of this value), which appears to be associated with suppression of aggregation. We will include these in our discussion.

    The LCD region is prone to PMTs - as the tethered protein is taken from expression in mammalian cells, how can the authors be sure that it has no PMTs? Although a clear difference is observed between WT and A315T in terms of "compactness" of the LCD domain, we cannot assume that the effect of DNAJ2 and Hero11 are the same - in fact, the Hero11 KR-->G control for the A315T is clearly different from the negative control (BSA) and the effect that was seen in WT. As the LCD domain is well-known to be the site of post-translational modifications (ie. Phosphorylation - this would have an effect on an electrostatic Hero11), could the effects be related to changes in PMTs as well?

    We thank the Reviewer for their insight. We would like to clarify that we make no assumption that our dye-labeled TDP-43 is free of post-translational modifications. Indeed, the fact that it is derived from HEK293 cells suggests it should have post-translational modifications relevant to humans and may be even considered an advantage of our method. (Most structural methods require purification of a large amount of protein, often only possible through recombinant expression in E. coli, thus lacking human-relevant PTMs.) As the Reviewer points out, the LCD is known to have many phosphorylation sites, which may help explain how the positively charged Hero11 interacts with it. Thus, we will perform mass spectrometry of TDP-43 and the A315T variant expressed in HEK cells to identify what post-translational modifications are present.

    The authors mention other studies on DNJA proteins on TDP-43; is the mechanism by which they suppress aggregation known? If the authors want to compare the unknown effects of Hero11, it would be useful to know what DNJ2A is doing, otherwise, the results are still not conclusive, only that "change is similar" in two experiments. What is known about DNJ2A interactions with TDP-43? Did the authors do any pulldown assays to detect a complex in cellulo?

    While previous studies have identified various DNAJ (specifically J-domain protein B-subfamily) proteins that suppress aggregation of overexpressed TDP-43, not much is known of this specific interaction (Udan-Johns 2014 Hum Mol Genet, Chen 2016 Brain, Park 2017 PLOS Genet). To address the Reviewer’s questions, we will include experiments characterizing the effects of DNAJA2 on TDP-43. We will perform colocalization experiments, explaining effects of DNAJA2 and Hero11 on TDP-43 in the cell. As explained below, we will also perform Pulse Shape Analysis (PulSA), a flow cytometry-based method that can be used to study protein localization patterns in cell, which will also provide insight into the effects on the distribution of TDP-43 in cells. We can also perform co-IP of TDP-43 to detect if there is a detectable, stable complex with DNAJA2 and/or Hero11. Together, these will clarify the similarities and differences between DNAJA2 and Hero11.

    It is unclear how the findings of the smFRET relate to structural understanding of the LCD-domain of TDP43 (i.e. NMR studies?); is it known whether PTMs are more prominent with the A315T variant as this may explain it's more compact nature? As well, putative helical structure in the LCD domain may lend to the changes in compaction.

    The Reviewer brings up an interesting and careful discussion. Currently, it is unknown if PTMs actually cause more compaction, or if they are more prominent in the A315T variant, but we will perform mass spectrometry to detect PTMs.

    As the Reviewer mentions, it would be very interesting to compare our smFRET results to other studies of specific LCD structures. However, it is not trivial to deduce lengths (and structure) from smFRET data as various other factors, for example, dye orientation and local chemical environment, may affect FRET efficiency. Nonetheless, we can still cautiously provide a discussion of how our FRET results compare with previous studies.

    For the dye pair used in our study, Cy3 and ATTO647N, the low/no FRET signals promoted by DNAJA2 and Hero11 correspond to a range of end-to-end distances of 6.9 nm to 10.2 nm (FRET signals of 0.1 to 0.01, respectively). Assuming that the LCD behaves like a ~140 amino acid worm-like chain (WLC) with persistence length (Lp) = 0.8 nm, we expect a mean end-to-end distance of 7.35 nm. Thus, the low FRET peak can be well explained by promotion of an extended WLC behavior of the LCD by DNAJA2 and Hero11. On the other hand, the FRET peaks of WT LCD and the A315T mutant (in the absence of Hero11 or DNAJA2) correspond to ~4 and ~3.3 nm, respectively. We will include a careful discussion of how our results relate to known structural understanding of the LCD in the revised discussion.

    It is unclear how there can be such a prominent FRET ~0 peak and in fact negative values.

    We regret that we did not clearly explain this in the manuscript. Negative values arise when applying correction factors from the alternating laser scheme (ALEX) to FRET signals. FRET efficiency, E, is the ratio of acceptor signal intensity, IA, over the total signal intensity, ID+IA, (with the application of a correction factor, γ, but this doesn’t affect the negative values and won’t be discussed further here) and is given by the equation: E=IA/(γ×ID+IA). However, due to leakage of the donor signal into the acceptor channel and direct excitation of the acceptor dye by the donor laser, raw IA values, IA,raw, are erroneously higher than in reality. For example, the ~0 FRET peaks in question appear to be around 0.1–0.2 before correction. These are accounted for by applying the respective correction factors, Dleakage and Adirect, through the equation: IA=IA,raw–Dleakage×ID–Adirect×IAA. (IAA is the acceptor signal during excitation of the acceptor dye.) These two correction factors are determined by observing the traces and choosing the mean values using iSMS software (2015 Preus Nat Methods) and applied uniformly to all traces in an experiment. When IA is especially low, such as when FRET is almost 0, the magnitude of the correction factor terms may be larger than IA,raw, resulting in negative values. This does not mean that values less than 0 are invalid, but merely that they have been overcompensated in the error application. For the dye pair in our study, FRET efficiencies less than 0.1 correspond to distances greater than 6.9 nm, meaning peaks around zero represent LCD behaviors with end-to-end distances greater than around 7 nm. Please also note that kernel density estimation often gives distributions with values beyond the (0,1) range just because of how these plots are constructed. This will be added to the methods in the revised manuscript.

    Conclusion is that Hero11 and DNJA2 maintain the TDP43 LCD-domain (soluble protein) in an extended form and that this is linked with the decrease in aggregates found in the cell; however, with the cell-based assay, no analysis to quantify the expression levels of the TDP43 and the chaperones/Hero are presented, and more importantly, no analysis on the complementary soluble fraction (to the filter assay) has been done to show that indeed, these biomolecules maintain the proteins in a soluble form. It is possible that the TDP-43 is being degraded?

    As described above, we plan to perform Western Blotting to examine the expression levels of these proteins. To address the concerns about solubility, we will perform Pulse Shape Analysis (PulSA) to quantitatively measure the expression and soluble/aggregated distribution GFP-tagged TDP43 in HEK293T cells. Measuring the soluble diffuse signals and the punctate aggregate signals will also tell us if there are differences in how GFP-TDP43 is aggregated between Hero11, DNAJA2 and controls. In addition, to support results from the FTA, we will provide sedimentation assays, where the soluble and aggregate fraction from cells is separated by centrifugation and analyzed (Krobitsch 2000 PNAS). These will provide information on TDP-43 in the soluble fraction.

    Reviewer #2 (Significance (Required)):

    Contextually, this study has novelty and potential value for basic research. Firstly, understanding the underlying mechanisms by which Hero protein prevent aggregation would be valuable towards understanding the players in protein homeostasis which can be imbalanced with respect to disease. Secondly, the use of smFRET as a tool in understanding the dynamics of TDP-43 and mutational variants can be powerful in defining structural attributes with pathological consequences in ALS. Although this work shows comparisons between the effect of a canonical chaperone (DNJA2) and Hero11 on the dynamics of monomeric protein and the effect on cellular aggregation, proposing a general mechanism on the data from two TDP-43 variants and a cell-based aggregation assay is premature and more experimental evidence is needed to define the critical link that prevents aggregation of TDP-43 within the cell. Mechanistically, the study does not give a lot of additional insight into the mode of action of Hero11 in the process of preventing aggregation (nor does it explain what DNJA2 is doing and therefore how Hero11 compares and contrasts). The proposed "extended versus collapsed" switch is simplistic and doesn't account for the complexity of TDP-43 structural dynamics. To support their proposed mechanism of action, the authors needs to examine TDP-43 mutational variants (specifically disease-related ones) using their smFRET to understand exactly what the "collapsed" and "extended" data is defining before making the leap that this effect is what is preventing aggregation. There are some structural studies about residual structure in this region (via NMR) that should be considered (https://doi.org/10.1016/j.str.2016.07.007). Although the A315T variant has a very distinct smFRET profile, it is clear that the effects of Hero11KR-->G (that is proposed to have no effect on aggregation or on the smFRET of WT TDP43) has a clear impact on A315T. Why is this? Have the authors considered that the LCD domain of TDP43 is prone to post-translational modifications? Is this variant more phosphorylated - a PMT like phosphorylation is surely to have an impact on interactions with Hero proteins as they are positively charged. Given that the protein is expressed in mammalian cells, it is likely that PMTs have occurred (but the authors should analyse for this).

    With regards to the cell-based aggregation assays, the authors again present a simplified relationship - however, a number of control experiments and additional questions arise. It appears that there is less aggregation with co-expression of some chaperones and the Hero11, but what about the soluble fraction? What is the impact of these biomolecules? Is this that it is maintaining soluble protein, enhancing degradation, propagating soluble oligomers? Equally, how do we know that the levels of the chaperones/Heros and the TDP-43 is the same in each cell - these are transient transfections, and no western blots are shown to confirm the levels of the proteins. In fact, the authors state that "co-transfection of HSP70 (HSPA8), HSP90 (HSP90AB1) or HOP all failed to suppress TDP-43 aggregation compared to GST" and mention that this is in contrast to other studies, but could this be a failure to express these in the cell models? Some western blot/lysate analysis is needed. Chaperones often form complexes with their client proteins, is there any evidence of complexes in these cell models (i.e. using immunoprecipitation)?

    We thank the Reviewer for their detailed evaluation and interest in our work. As the Reviewer describes, smFRET is a powerful tool for studying the conformational dynamics of TDP-43, and we hope that this study will contribute to our understanding of how Hero proteins and chaperones prevent aggregation.

    We are also grateful to the Reviewer for their constructive criticism of our current model, and we will revise it accordingly. We completely agree with the Reviewer that there are complex structural dynamics within the LCD that determine aggregation and phase separation behaviors. Our simple model was intended to explain how external factors that suppress aggregation, DNAJA2 and Hero11, could affect the conformation of LCD at the single-molecule level. As discussed above, we were cautious to over-interpret how our FRET observations correlate to specific conformations, leading to this simplistic model. We do not intend for our explanation of “extended versus collapsed” in the model to explain all structural dynamics of the LCD; rather, we wanted to highlight the characteristic low FRET state promoted by DNAJA2 and Hero11. We believe that the experiment plan explained above will address the Reviewer’s concerns in full, and we thank the Reviewer again for helping us to significantly improve our manuscript.

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

    - In a recent study (PLosBiol, 2020) the same authors described an interesting class of proteins they call 'Hero'. Based on their analyses in cultured cells and transgenic Drosophila models the authors concluded that 'Hero' proteins protect against protein instability and aggregation. So far, this class of proteins has not been analyzed by independent groups.

    In the current manuscript, they mainly confirm their own previous finding that Hero 11 prevents There are several concerns about the presented data:

    We thank the Reviewer for their critical comments on our current manuscript.

    - Based on the filter trap assays shown in figure 1 and 3 the authors conclude that DNAJB8 and Hero11 specifically interfere with the aggregation of TDP-43. However, they do not show that the expression levels of TDP-43 are not altered by the co-expression of the different proteins and are comparable in the different samples. In order to make a relevant statement about the anti-aggregation activity of the analyzed proteins, the ratio between soluble and aggregated TDP-43 has to be analyzed.

    We would like to clarify that the Reviewer means DNAJA2, not DNAJB8. Following the Reviewer’s advice, we will perform Western Blotting combined with sedimentation assays, where the soluble and aggregate fraction from cells is separated by centrifugation and analyzed to examine the expression levels. We will also perform colocalization experiments and Pulse Shape Analysis (PulSA), a flow cytometry-based method that can be used to study protein localization patterns in cell, which will provide insight into the anti-aggregation activities.

    - The FRET assays shown in figures 2 and 4 indicate a slightly higher FRET efficiency in the presence of Hero11 and DNAJA2 and Hero11. The authors postulate that is phenomenon is causally linked to the activity of Hero11 to prevent aggregation of TDP-43. First, it remains unclear whether the slight increase is really significant. Second, I could not find any experimental evidence to support the assumption that a more collapse conformation of the TDP-43 LCD measured in single molecule FRET assays, correlates with an increased aggregation tendency of TDP-43.

    We apologize that we are not sure what the Reviewer refers to by “a slightly higher FRET efficiency in the presence of Hero11 and DNAJA2 (and Hero11).” We would like to clarify that, in the presence of Hero11 and DNAJA2, what we observed was a very low (not slightly higher) FRET efficiency of ~0 (Figure 2g and h), suggesting an extended conformation. In contrast, the aggregation-prone A315T variant of TDP-43 shows a very high FRET efficiency of ~0.9 (Figure 4a), which indicates a collapsed conformation.

    A minor comment, if the authors would like to compare the specific activity of different proteins, they should use equal molarities of the different proteins and not equal amounts.

    As the Reviewer suggests, we will include experiments at equal molarities in the revision.

    - For a one-way ANOVA, the response variable residuals have to be normally distributed. With an n = 3 this cannot be tested. Thus, the quantifications of the results shown in figure 1 and 3 are not reliable.

    We thank the Reviewer for their critical comment on the statistical analysis. We would like to clarify that statistically significant differences in aggregation between conditions compared to a control are based on Dunnett’s test. While ANOVA is typically first performed to test for any significant difference in means before performing a post-hoc test, Dunnett’s test is independent and can be performed without ANOVA.

    Following the Reviewer’s advice, we carefully re-examined our assumption of normality for this data. It is reasonable to perform Dunnett’s test on a sample size of n = 3, and it is generally safe to assume that data from three independent experiments will be reasonably normally distributed. In support of this, performing Kolmogorov-Smirnov test on our data in Figure 1 showed none of the groups differ significantly from normal distributions with the respective mean and standard deviation (p-values greater than 0.05). Thus, we believe it is reasonable to assume the data are normally distributed, the residuals normally distributed, and our statistical analyses reliable. This analysis will be included in the revision to support the normality assumption.

    However, even if we did not assume a normal distribution of our data in Figure 1, we still would have obtained statistically significant differences; If we had relied on a Kruskal-Wallis test as a non-parametric equivalent of ANOVA, thus making no assumption of normality, we would have seen p = 0.005176, a value much lower than our significance level of α = 0.05, indicating sufficient evidence that there is a difference in aggregation among these groups.

    - The title is imprecise and overstate the presented data:

    'canonical chaperone' suggest that their results are valid for chaperones in general. However, they only tested DNAJA2 in the single -molecule FRET assay. Moreover, HAPA8, another canonical chaperone, obviously had an opposite effect on TDP-43 aggregation (Fig.1). Similarly, they only tested Hero11. Thus, 'canonical chaperone' has to be replaced by 'DNAJA2' and 'a heat-resistant obscure (Hero) protein' by 'Hero11'. Similarly, the term 'conformational modulation' is not as concise one would one expect for the title of a research paper.

    We would like to clarify that the Reviewer means HSPA8 (not HAPA8). According to the Reviewer’s suggestion, we will change the title to “DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43”.

    Reviewer #3 (Significance (Required)):

    In a recent study (PLosBiol, 2020) the same authors described an interesting class of proteins they call 'Hero'. Based on their analyses in cultured cells and transgenic Drosophila models the authors concluded that 'Hero' proteins protect against protein instability and aggregation. So far, this class of proteins has not been analyzed by independent groups.

    In the current manuscript, they mainly confirm their own previous finding that Hero 11 prevents aggregation of TDP-43 and present very few new data that would provide new insights. Specifically, only the FRET assays shown in figure 2 and 4 are really new, which, by the way, could easily be shown in one figure.

    We thank the Reviewer for their critical evaluation of our current study. As the Reviewer suggests, we believe our smFRET results provide new insights into how Hero11 and DNAJA2 function. We would like to emphasize that, rather than confirming our previous findings, our current manuscript mainly addresses a critical point that remained unknown in our previous study by investigating the mechanism of how Hero proteins prevent aggregation. Moreover, to our knowledge, this is the first observation of the TDP-43 LCD, with the effect of a pathogenic mutation, at the single-molecule level.

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    Referee #3

    Evidence, reproducibility and clarity

    • In a recent study (PLosBiol, 2020) the same authors described an interesting class of proteins they call 'Hero'. Based on their analyses in cultured cells and transgenic Drosophila models the authors concluded that 'Hero' proteins protect against protein instability and aggregation. So far, this class of proteins has not been analyzed by independent groups.

    In the current manuscript, they mainly confirm their own previous finding that Hero 11 prevents There are several concerns about the presented data:

    • Based on the filter trap assays shown in figure 1 and 3 the authors conclude that DNAJB8 and Hero11 specifically interfere with the aggregation of TDP-43. However, they do not show that the expression levels of TDP-43 are not altered by the co-expression of the different proteins and are comparable in the different samples. In order to make a relevant statement about the anti-aggregation activity of the analyzed proteins, the ratio between soluble and aggregated TDP-43 has to be analyzed.
    • The FRET assays shown in figures 2 and 4 indicate a slightly higher FRET efficiency in the presence of Hero11 and DNAJA2 and Hero11. The authors postulate that is phenomenon is causally linked to the activity of Hero11 to prevent aggregation of TDP-43. First, it remains unclear whether the slight increase is really significant. Second, I could not find any experimental evidence to support the assumption that a more collapse conformation of the TDP-43 LCD measured in single molecule FRET assays, correlates with an increased aggregation tendency of TDP-43. A minor comment, if the authors would like to compare the specific activity of different proteins, they should use equal molarities of the different proteins and not equal amounts.
    • For a one-way ANOVA, the response variable residuals have to be normally distributed. With an n = 3 this cannot be tested. Thus, the quantifications of the results shown in figure 1 and 3 are not reliable.
    • The title is imprecise and overstate the presented data: 'canonical chaperone' suggest that their results are valid for chaperones in general. However, they only tested DNAJA2 in the single -molecule FRET assay. Moreover, HAPA8, another canonical chaperone, obviously had an opposite effect on TDP-43 aggregation (Fig.1). Similarly, they only tested Hero11. Thus, 'canonical chaperone' has to be replaced by 'DNAJA2' and 'a heat-resistant obscure (Hero) protein' by 'Hero11'. Similarly, the term 'conformational modulation' is not as concise one would one expect for the title of a research paper.

    Significance

    In a recent study (PLosBiol, 2020) the same authors described an interesting class of proteins they call 'Hero'. Based on their analyses in cultured cells and transgenic Drosophila models the authors concluded that 'Hero' proteins protect against protein instability and aggregation. So far, this class of proteins has not been analyzed by independent groups.

    In the current manuscript, they mainly confirm their own previous finding that Hero 11 prevents aggregation of TDP-43 and present very few new data that would provide new insights. Specifically, only the FRET assays shown in figure 2 and 4 are really new, which, by the way, could easily be shown in one figure.

  6. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors build on their findings (Tsuboyama 2020) that electrostatically charged IDPs (Heros) can protect proteins from denaturation and aggregation. In their previous work, they demonstrate that these Hero proteins could decrease the fraction of insoluble GFP-TDP43∆NLS in mammalian cell lines and that this mode of action was related to the electrostatic charge of the proteins and not sequence dependent. Although this protective mode of action appears to be similar to that of canonical chaperones, it is unclear how the Hero proteins compare. In this study, the authors compare Hero11 to a panel of canonical chaperones in their cell-based assays and show that it prevents aggregation in a comparable way to DNJA2. It appears that Hero11 decreases the GFP-TDP43∆NLS aggregates better than some other chaperones. They then utilise their expertise in smFRET analysis (Tsuboyama, 2018) to compare what effect DNJA2 and Hero11 (along with Hero11KR-->G (non-charged control)) have on the dynamic structures of the GFP-TDP43∆NLS (labelled with complementary fluorophores in the LCD domain). Based on analysis of the WT GFP-TDP43∆NLS and the A315T GFP-TDP43∆NLS, the authors suggest that the presence of Hero11 and DNJA2 maintain the LCD-domain of TDP43 in an extended conformation and that by doing so, aggregation can be prevented (as assessed in the cell-based assay).

    Despite finding the results very interesting, I feel that the study is preliminary and the conclusions drawn are not fully substantiated by the presented experimental work. Many questions need addressing to validate these findings and conclusions (please see more in the "Significance" section). I have tried to list the main concerns below.

    Questions/concerns:

    Authors used double transient transfections but have not shown quantification of protein levels of the chaperones versus TDP43 - western blots to confirm proper expression (and levels) of the chaperones/Hero protein is crucial without it, we cannot assume that the differences in TDP-43 aggregation are a result of effective chaperoning or due to a lack of expression of any of the chaperone proteins, or high expression of others.

    Authors used quite a high BSA concentration in the smFRET work; it would be useful to see what the TDP43 smFRET trace looks like without BSA incubation (to ensure it is not causing some effect). Also, is there a concentration dependence? The Authors mention they are unable to identify a Hero/TDP43 complex; but if the amount of Hero protein is high (given that it is single molecules tethered), the change in compaction may not relate to the levels/ratios found in the cells (where changes to aggregation are occurring). have the authors considered whether positively charged polymers (poly-Lys) have any impact on the TDP-43 smFRET distribution? Given that the smFRET trace is so heterogeneous, to understand what is happening here would require the comparison of more than 2 variants.

    Although the A315T variant has a very distinct smFRET profile, it is clear that the effects of Hero11KR-->G (that is proposed to have no effect on aggregation or on the smFRET of WT TDP43) has a clear impact on A315T. Why is this?

    The LCD region is prone to PMTs - as the tethered protein is taken from expression in mammalian cells, how can the authors be sure that it has no PMTs? Although a clear difference is observed between WT and A315T in terms of "compactness" of the LCD domain, we cannot assume that the effect of DNAJ2 and Hero11 are the same - in fact, the Hero11 KR-->G control for the A315T is clearly different from the negative control (BSA) and the effect that was seen in WT. As the LCD domain is well-known to be the site of post-translational modifications (ie. Phosphorylation - this would have an effect on an electrostatic Hero11), could the effects be related to changes in PMTs as well?

    The authors mention other studies on DNJA proteins on TDP-43; is the mechanism by which they suppress aggregation known? If the authors want to compare the unknown effects of Hero11, it would be useful to know what DNJ2A is doing, otherwise, the results are still not conclusive, only that "change is similar" in two experiments. What is known about DNJ2A interactions with TDP-43? Did the authors do any pulldown assays to detect a complex in cellulo?

    It is unclear how the findings of the smFRET relate to structural understanding of the LCD-domain of TDP43 (i.e. NMR studies?); is it known whether PTMs are more prominent with the A315T variant as this may explain it's more compact nature? As well, putative helical structure in the LCD domain may lend to the changes in compaction.

    It is unclear how there can be such a prominent FRET ~0 peak and in fact negative values.

    Conclusion is that Hero11 and DNJA2 maintain the TDP43 LCD-domain (soluble protein) in an extended form and that this is linked with the decrease in aggregates found in the cell; however, with the cell-based assay, no analysis to quantify the expression levels of the TDP43 and the chaperones/Hero are presented, and more importantly, no analysis on the complementary soluble fraction (to the filter assay) has been done to show that indeed, these biomolecules maintain the proteins in a soluble form. It is possible that the TDP-43 is being degraded?

    Significance

    Contextually, this study has novelty and potential value for basic research. Firstly, understanding the underlying mechanisms by which Hero protein prevent aggregation would be valuable towards understanding the players in protein homeostasis which can be imbalanced with respect to disease. Secondly, the use of smFRET as a tool in understanding the dynamics of TDP-43 and mutational variants can be powerful in defining structural attributes with pathological consequences in ALS. Although this work shows comparisons between the effect of a canonical chaperone (DNJA2) and Hero11 on the dynamics of monomeric protein and the effect on cellular aggregation, proposing a general mechanism on the data from two TDP-43 variants and a cell-based aggregation assay is premature and more experimental evidence is needed to define the critical link that prevents aggregation of TDP-43 within the cell. Mechanistically, the study does not give a lot of additional insight into the mode of action of Hero11 in the process of preventing aggregation (nor does it explain what DNJA2 is doing and therefore how Hero11 compares and contrasts). The proposed "extended versus collapsed" switch is simplistic and doesn't account for the complexity of TDP-43 structural dynamics. To support their proposed mechanism of action, the authors needs to examine TDP-43 mutational variants (specifically disease-related ones) using their smFRET to understand exactly what the "collapsed" and "extended" data is defining before making the leap that this effect is what is preventing aggregation. There are some structural studies about residual structure in this region (via NMR) that should be considered (https://doi.org/10.1016/j.str.2016.07.007). Although the A315T variant has a very distinct smFRET profile, it is clear that the effects of Hero11KR-->G (that is proposed to have no effect on aggregation or on the smFRET of WT TDP43) has a clear impact on A315T. Why is this? Have the authors considered that the LCD domain of TDP43 is prone to post-translational modifications? Is this variant more phosphorylated - a PMT like phosphorylation is surely to have an impact on interactions with Hero proteins as they are positively charged. Given that the protein is expressed in mammalian cells, it is likely that PMTs have occurred (but the authors should analyse for this).

    With regards to the cell-based aggregation assays, the authors again present a simplified relationship - however, a number of control experiments and additional questions arise. It appears that there is less aggregation with co-expression of some chaperones and the Hero11, but what about the soluble fraction? What is the impact of these biomolecules? Is this that it is maintaining soluble protein, enhancing degradation, propagating soluble oligomers? Equally, how do we know that the levels of the chaperones/Heros and the TDP-43 is the same in each cell - these are transient transfections, and no western blots are shown to confirm the levels of the proteins. In fact, the authors state that "co-transfection of HSP70 (HSPA8), HSP90 (HSP90AB1) or HOP all failed to suppress TDP-43 aggregation compared to GST" and mention that this is in contrast to other studies, but could this be a failure to express these in the cell models? Some western blot/lysate analysis is needed. Chaperones often form complexes with their client proteins, is there any evidence of complexes in these cell models (i.e. using immunoprecipitation)?

  7. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The submitted manuscript is comparing the effect of individual chaperones and heat-resistant obscure (Hero) proteins on the overall folding of the TDP-43 LCD-domain and its relation to aggregation propensity. Therefore, the authors apply smFRET in order to deduce eventual morphological changes of the LCD-domain from FRET efficiencies. The authors observe that the LCD domain extends its structure upon binding of chaperone/Hero proteins whereas it is collapsed in the absence of those. Furthermore, immunoblotting of filter trap assays indicate that overexpression of chaperones and Hero proteins reduce aggregation of TDP-43 in vivo. Both, the morphological effects on the LCD-domain and the aggregation propensity are significantly enhanced for the TDP-43 A315T mutant. Moreover, the authors tested a charge depleted Hero protein version with reduced "chaperone-like" behaviour. Therefore, the authors conclude that the binding or chaperone activity of the Hero protein is based on its residue specific charges. Finally, the authors conclude that Hero proteins can act similar to chaperones in order to keep protein homeostasis under stress conditions.

    Major comments:

    The similar effect of chaperones and Hero proteins on the morphology of TDP-43 found by the authors is intriguing and the applied experimental procedures seem well described and conducted.

    However, the assumption of the authors that a change in morphology of the LCD-domain by the chaperones and Hero proteins is directly connected to the reduction of TDP-43 aggregation is not entirely clear. Whether an overexpression of individual chaperones and Hero proteins has a direct effect on TDP-43 aggregation cannot be tested in vivo, only. It cannot be excluded that inside the cell the here tested chaperones and Hero proteins control intermediate processes or work as co-factors for other proteins involved in protein homeostasis rather directly influencing the aggregation of TDP-43. Therefore, I recommend in vitro aggregation experiments, using ThT signal as a readout. By adding chaperones, Hero proteins and a negative (BSA or others) control individually, a direct effect on TDP-43 aggregation could be concluded. Those experiments have been extensively used in the field and are quick and straightforward to handle.

    In addition, focusing on the LCD-domain as a main driver for TDP-43 aggregation is limiting this study. In particular, recent studies [1] indicate that the RRM1 and RRM2 sites of TDP-43 have a major impact on TDP-43 gelation and maturation to solid aggregates. Unfortunately, those sites have not been studied in this manuscript.

    As an optional alternative for using Hero11KR->G could be the alteration of buffer conditions and using higher number of salts to promote charge screening. It would be of interest whether the results with the Hero11KR->G could be reproduced with wild type Hero11.

    [1] Lu et al. Nat Cell Biol;24(9):1378-1393 (2022)

    Minor comments:

    Overall, the text is clearly written, and the figures are appropriate.
    Whether the activity of individual chaperones or Hero proteins on TDP-43 aggregation "may result in the overall fitness of the cell" or "reinforcing the conformational health of the proteome" is disputable without knowing how the overexpression of certain chaperones or Hero proteins alter the formation of toxic TDP-43 oligomers.

    Significance

    Studying the mechanistic effects of chaperones on aggregating proteins is of major interest for the field in order to understand aging related disbalance of protein homeostasis and the progression of neurological decline, such as seen for amyotrophic lateral sclerosis (ALS). Furthermore, finding homolog proteins, also being able to inhibit protein aggregation, can help to understand overall mechanisms of protein aggregation and processes preventing such fatal behaviour. However, the technique used in this manuscript are not very novel and have been used numerously times before. smFRET is a common technique to look at protein folding/unfolding and is used frequently as a molecular ruler. The manuscript is of interest for the field of protein aggregation and folding, smFRET and neurodegeneration.

    My expertise lies in the field of protein aggregation and inhibition due to chaperones, measuring molecular interactions and neurodegenerative diseases.

  8. Version published to 10.1101/2022.11.15.516569v1 on bioRxiv