Proteostasis is differentially modulated by inhibition of translation initiation or elongation

  1. Khalyd J Clay
  2. Yongzhi Yang
  3. Christina Clark
  4. Michael Petrascheck

  • Curated by eLife

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    eLife assessment

    This manuscript is of interest for the fields of ageing, mRNA translation and C. elegans biology, as it provides new insights into the regulation of lifespan by alternate mechanisms that modulate mRNA translation in selected environmental contexts. While overall the main conclusions are supported by the data and of interest, the work would be stronger with control experiments that more fully and more consistently support all the conclusions. Furthermore, data presentation and interpretation need some attention.

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Abstract

Recent work has revealed an increasingly important role for mRNA translation in maintaining proteostasis. Here, we use chemical inhibitors targeting discrete steps of translation to compare how lowering the concentration of all or only translation initiation-dependent proteins rescues Caenorhabditis elegans from proteotoxic stress. We systematically challenge proteostasis and show that pharmacologically inhibiting translation initiation or elongation elicits a distinct protective profile. Inhibiting elongation protects from heat and proteasome dysfunction independently from HSF-1 but does not protect from age-associated protein aggregation. Conversely, inhibition of initiation protects from heat and age-associated protein aggregation and increases lifespan, dependent on hsf-1 , but does not protect from proteotoxicity caused by proteasome dysfunction. Surprisingly, we find that the ability of the translation initiation machinery to control the concentration of newly synthesized proteins depends on HSF-1. Inhibition of translation initiation in wild-type animals reduces the concentration of newly synthesized proteins but increases it in hsf-1 mutants. Our findings suggest that the HSF-1 pathway is not only a downstream target of translation but also directly cooperates with the translation initiation machinery to control the concentration of newly synthesized proteins to restore proteostasis.

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  1. Version published to 10.7554/elife.76465 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Inhibition of translation has been found as a conserved intervention to extend lifespan across a number of species. In this work, the authors systematically investigate the similarities and differences between pharmacological inhibition of protein synthesis at the initiation or elongation steps on longevity and stress resistance. They find that translation elongation inhibition is beneficial during times when proteostasis collapse is the primary phenotype such as proteasome dysfunction, hsf-1 mutants, and heat shock, but this intervention does not extend the lifespan of wt worms. While translation initiation inhibition extends the lifespan of wt worms and heat shock, but in an HSF-1 dependent manner. This work shows that a simple explanation of just inhibiting total protein synthesis and reduced folding load cannot explain all of the phenotypes seen from protein synthesis inhibition, as initiation and elongation inhibition repress overall translation similarly, but have different effects depending on the experiment tested. Using multiple interventions that target both initiation and elongation lends further support to their findings. These experiments are important for conceptualizing how translation inhibition actually extends lifespan and promotes proteostasis.

    Major Comment:

    The authors acknowledge that lifespan extension must not necessarily arise just from reducing protein synthesis, as elongation inhibition reduced protein synthesis but did not extend lifespan. Yet for the converse effects from elongation inhibition they seem to suggest that it arises from reducing protein synthesis. For example, regarding how elongation inhibition extends lifespan in an hsf-1 mutant, the authors suggest that "inhibition of elongation lowers the production of newly synthesized proteins and thus reduces the folding load on the proteostasis machinery", even though initiation inhibitors do not extend lifespan in an hsf-1 background (while presumably lowering the production of newly synthesized proteins).

    Thank you for this excellent comment. It led us to conduct a crucial experiment with a new finding that is now Figure 6. As suggested, we asked if initiation inhibitors lower the concentration of newly synthesized protein in the hsf-1(sy441) background. The surprising answer is that initiation inhibitors lower the concentration of newly synthesized proteins in N2 but dramatically increase it in hsf-1(sy441). The failure to lower the concentration of newly synthesized proteins was true for the pharmacological inhibitors as well as RNAi against ifg-1. Therefore, inhibition of initiation requires HSF1 to lower the protein concentration. These new findings enable us to make a much more precise statement now added to the discussion:

    Lines 372: “The inability of translation-initiation inhibitors to reduce the concentration of newly synthesized proteins in hsf-1(sy441) mutants and the inability to extend their lifespan shows that lowering the concentration of newly synthesized proteins is necessary for the beneficial effects. On the other hand, the finding that elongation inhibitors protect from proteotoxic stress but does not extend lifespan shows that lowering the concentration of newly synthesized proteins is sufficient to protect from proteotoxic stress but is not sufficient to extend lifespan in wild-type, which appears to require selective translation.”

    Reviewer #2 (Public Review):

    In this manuscript, Clay et al. investigate the underlying effects of reduced mRNA translation beneficial on protein aggregation and aging. They aim to test two pre-existing hypotheses: The selective translation model proposes that downregulation of overall translation increases the capacity of ribosomes to translate selected factors that in turn increase stress resistance against toxicity. The reduced folding load model suggests that during high mRNA translation rates, newly synthesized peptides and proteins can overwhelm the protein folding capacity of the cell and therefore cause protein toxicity. By generally lowering mRNA translation, lower loads of newly synthesized proteins should cause less protein folding stress and hence protein toxicity.

    To understand how reduced mRNA translation mediates its beneficial effects in the context of the proposed models, the authors use different drugs established previously in other in vitro and in vivo systems to inhibit selected steps of translation. The systemic effects of translation initiation versus elongation inhibition in C. elegans are compared during heat shock, specific protein aggregation stresses and aging. These phenotypes are further tested for dependence on hsf-1, as contradictory data on the effect of translation inhibition during thermal stress in the context of hsf-1 dependency exist.

    The data show that inhibition of translation initiation protects from heat stress and age-associated protein aggregation but on the contrary further sensitizes animals to protein toxicity induced by a misfunctioning proteasome. Further, inhibition of translation initiation increases lifespan in WT animals. The survival phenotypes observed during heat shock and regular lifespan assays are dependent of HSF-1, supporting the selective translation model. As stated in the manuscript, these findings themselves are not new, given that similar observations were made before using genetic models. Interestingly, the inhibition of translation elongation protects from heat stress, but, unlike initiation inhibition, also proteasome-misfunction-induced protein toxicity. Both phenotypes were observed to be independent of hsf-1. The authors further find that inhibiting elongation does not reduce protein aggregation in aged worms and does not prolong lifespan in wild-type animals. It does increase lifespan in short-lived hsf-1 mutants, where protein homeostasis is compromised. To a degree, these findings support the reduced folding load model. Overall, from these observations the authors summarize that the systemic consequences of lowering translation depend on the step in which translation is inhibited as well as the environmental context. The authors conclude that different ways to inhibit translation can protect from different insults by independent mechanisms.

    Impact, strengths and weaknesses:

    mRNA translation and its regulation is one of the most studied mechanisms connected to lifespan extension. However, gaps behind the protective effects of translation inhibition are so far unresolved, as stated by the authors. Therefore, testing existing hypotheses explaining the beneficial effects of translation inhibition is of great interest, not only for C. elegans researchers but a broad community working on the effects of misregulated translation during aging and disease. Overall, the conclusions made by the authors are generally supported by the data shown in this manuscript. However, some major gaps remain and need to be clarified and extended.

    Thank you for your generous comments and thorough review.

    Reviewer #3 (Public Review):

    Clay and colleagues investigate the proteostasis and longevity benefits derived from translation inhibition in C. elegans by examining the impacts of chemical translation initiation inhibitors (IIs) and translation elongation inhibitors (EIs) on thermotolerance, protein folding stress, aggregation and longevity. They observe somewhat distinct impacts by the two chemical groups. IIs increased longevity in wild-type animals in an hsf-1 dependent manner, whereas, EIs only extended hsf-1 mutants' lifespan. Only EIs protected against proteasome dysfunction. Both protected against heat stress but with differing hsf-1 dependence. The authors utilize these observations to derive conclusions regarding two dominant points of view on the mechanism by which translation inhibition improves lifespan and proteostasis.

    The study is based on interesting observations and several promising avenues of further investigation can be identified. However, the manuscript appears somewhat preliminary in nature, with many of the observations, while interesting, only explored superficially for mechanistic insights. The rationale behind some of the interpretations was also difficult to interpret. For example, the authors make conclusions about 'selective translation' being adopted upon IIs treatment without directly testing this. Protein aggregation, while possibly predictive, is not a reliable readout for selective translation of some mRNAs. Similarly, the evidence for a reduction in 'newly-synthesized protein load' by EIs is thin based on one reporter. Previous studies from the Blackwell lab have identified differential impacts of SKN-1 on select cytoprotective genes' expression and proteasomal gene expression based on inhibition of translation initiation or elongation. So there is precedence for both the differential impact of initiation vs. elongation inhibition as well as genetic background. There are several other such studies that reduce the impact of the observations presented here. With limited novelty and mechanistic insight, the impact of the study on the field is likely to be moderate.

    We thank the reviewer for the thorough analysis and candid summary. Some of the criticisms rang true, and we have made considerable efforts to address them, both increasing the thoroughness of our study by establishing that these inhibitors inhibit initiation and elongation (new Figure 1) and by providing a novel mechanism showing that the ability of the initiation machinery requires HSF1 to lower the concentration of newly synthesized proteins.

    Before we go into the specific criticisms, we would like to note that of the 30-40 eukaryotic translation inhibitors used in cell culture and yeast, very few have been validated in C. elegans. The go-to inhibitor was cycloheximide which, in our hands, is reliable in cell culture but unreliable in C. elegans, most likely due to its poor pharmacokinetics (data now added to the supplementary figures). To our knowledge, no C. elegans study investigating translation has made sure to equalize the concentration of newly synthesized proteins or could have because of a lack of validation of the chemical tools used in other organisms. Thus, the comment of reviewer #3 that we did not go far enough with the validation struck home, and in the revised version of Figure 1 we added more validation.

    We are of the opinion that it is essential to ensure that both mechanisms reduce the concentration of newly synthesized proteins to the same degree to study mechanistic differences. Otherwise, one cannot deconvolute if any phenotypic difference is caused by the mechanistic difference or the degree of translation inhibition. The importance of monitoring the level of inhibition became evident in our new Figure 6, which shows that inhibition of the translation initiation machinery no longer reduces the concentration of newly synthesized proteins but increases it in the absence of HSF1.

  3. eLife

    eLife assessment

    This manuscript is of interest for the fields of ageing, mRNA translation and C. elegans biology, as it provides new insights into the regulation of lifespan by alternate mechanisms that modulate mRNA translation in selected environmental contexts. While overall the main conclusions are supported by the data and of interest, the work would be stronger with control experiments that more fully and more consistently support all the conclusions. Furthermore, data presentation and interpretation need some attention.

  4. eLife

    Reviewer #1 (Public Review):

    Inhibition of translation has been found as a conserved intervention to extend lifespan across a number of species. In this work, the authors systematically investigate the similarities and differences between pharmacological inhibition of protein synthesis at the initiation or elongation steps on longevity and stress resistance. They find that translation elongation inhibition is beneficial during times when proteostasis collapse is the primary phenotype such as proteasome dysfunction, hsf-1 mutants, and heat shock, but this intervention does not extend the lifespan of wt worms. While translation initiation inhibition extends the lifespan of wt worms and heat shock, but in an HSF-1 dependent manner. This work shows that a simple explanation of just inhibiting total protein synthesis and reduced folding load cannot explain all of the phenotypes seen from protein synthesis inhibition, as initiation and elongation inhibition repress overall translation similarly, but have different effects depending on the experiment tested. Using multiple interventions that target both initiation and elongation lends further support to their findings. These experiments are important for conceptualizing how translation inhibition actually extends lifespan and promotes proteostasis.

    Major Comment:

    The authors acknowledge that lifespan extension must not necessarily arise just from reducing protein synthesis, as elongation inhibition reduced protein synthesis but did not extend lifespan. Yet for the converse effects from elongation inhibition they seem to suggest that it arises from reducing protein synthesis. For example, regarding how elongation inhibition extends lifespan in an hsf-1 mutant, the authors suggest that "inhibition of elongation lowers the production of newly synthesized proteins and thus reduces the folding load on the proteostasis machinery", even though initiation inhibitors do not extend lifespan in an hsf-1 background (while presumably lowering the production of newly synthesized proteins).

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Clay et al. investigate the underlying effects of reduced mRNA translation beneficial on protein aggregation and aging. They aim to test two pre-existing hypotheses: The selective translation model proposes that downregulation of overall translation increases the capacity of ribosomes to translate selected factors that in turn increase stress resistance against toxicity. The reduced folding load model suggests that during high mRNA translation rates, newly synthesized peptides and proteins can overwhelm the protein folding capacity of the cell and therefore cause protein toxicity. By generally lowering mRNA translation, lower loads of newly synthesized proteins should cause less protein folding stress and hence protein toxicity.

    To understand how reduced mRNA translation mediates its beneficial effects in the context of the proposed models, the authors use different drugs established previously in other in vitro and in vivo systems to inhibit selected steps of translation. The systemic effects of translation initiation versus elongation inhibition in C. elegans are compared during heat shock, specific protein aggregation stresses and aging. These phenotypes are further tested for dependence on hsf-1, as contradictory data on the effect of translation inhibition during thermal stress in the context of hsf-1 dependency exist.

    The data show that inhibition of translation initiation protects from heat stress and age-associated protein aggregation but on the contrary further sensitizes animals to protein toxicity induced by a misfunctioning proteasome. Further, inhibition of translation initiation increases lifespan in WT animals. The survival phenotypes observed during heat shock and regular lifespan assays are dependent of HSF-1, supporting the selective translation model. As stated in the manuscript, these findings themselves are not new, given that similar observations were made before using genetic models. Interestingly, the inhibition of translation elongation protects from heat stress, but, unlike initiation inhibition, also proteasome-misfunction-induced protein toxicity. Both phenotypes were observed to be independent of hsf-1. The authors further find that inhibiting elongation does not reduce protein aggregation in aged worms and does not prolong lifespan in wildtype animals. It does increase lifespan in short-lived hsf-1 mutants, where protein homeostasis is compromised. To a degree, these findings support the reduced folding load model. Overall, from these observations the authors summarize that the systemic consequences of lowering translation depend on the step in which translation is inhibited as well as the environmental context. The authors conclude that different ways to inhibit translation can protect from different insults by independent mechanisms.

    Impact, strengths and weaknesses:

    mRNA translation and its regulation is one of the most studied mechanisms connected to lifespan extension. However, gaps behind the protective effects of translation inhibition are so far unresolved, as stated by the authors. Therefore, testing existing hypotheses explaining the beneficial effects of translation inhibition is of great interest, not only for C. elegans researchers but a broad community working on the effects of misregulated translation during aging and disease. Overall, the conclusions made by the authors are generally supported by the data shown in this manuscript. However, some major gaps remain and need to be clarified and extended.

  6. eLife

    Reviewer #3 (Public Review):

    Clay and colleagues investigate the proteostasis and longevity benefits derived from translation inhibition in C. elegans by examining the impacts of chemical translation initiation inhibitors (IIs) and translation elongation inhibitors (EIs) on thermotolerance, protein folding stress, aggregation and longevity. They observe somewhat distinct impacts by the two chemical groups. IIs increased longevity in wild-type animals in an hsf-1 dependent manner, whereas, EIs only extended hsf-1 mutants' lifespan. Only EIs protected against proteasome dysfunction. Both protected against heat stress but with differing hsf-1 dependence. The authors utilize these observations to derive conclusions regarding two dominant points of view on the mechanism by which translation inhibition improves lifespan and proteostasis.
    The study is based on interesting observations and several promising avenues of further investigation can be identified. However, the manuscript appears somewhat preliminary in nature, with many of the observations, while interesting, only explored superficially for mechanistic insights. The rationale behind some of the interpretations was also difficult to interpret. For example, the authors make conclusions about 'selective translation' being adopted upon IIs treatment without directly testing this. Protein aggregation, while possibly predictive, is not a reliable readout for selective translation of some mRNAs. Similarly, the evidence for a reduction in 'newly-synthesized protein load' by EIs is thin based on one reporter. Previous studies from the Blackwell lab have identified differential impacts of SKN-1 on select cytoprotective genes' expression and proteasomal gene expression based on inhibition of translation initiation or elongation. So there is precedence for both the differential impact of initiation vs. elongation inhibition as well as genetic background. There are several other such studies that reduce the impact of the observations presented here. With limited novelty and mechanistic insight, the impact of the study on the field is likely to be moderate.

  7. Version published to 10.1101/2022.01.23.477418v1 on bioRxiv