PEBP1 amplifies mitochondrial dysfunction induced integrated stress response

Reviewer #1 (Public review):

Summary:

In this study, the authors use thermal proteome profiling to capture changes in protein stability following a brief (30 min) treatment of cells with various mitochondrial stressors. This approach identified PEBP1 as a potentiator of Integrated Stress Response (ISR) induction by various mitochondrial stressors, although the specific dynamics vary by stressor. PEBP1 deletion attenuates DELE1-HRI-mediated activation of the ISR, independent of its known role in the RAF/MEK/ERK pathway. These effects can be bypassed by HRI overexpression and do not affect DELE1 processing. Interestingly, in cells, PEBP1 physically interacts with eIF2alpha, but not its phosphorylated form (eIF2alpha-P), leading the authors to suggest that PEBP1 functions as a scaffold to promote eIF2alpha phosphorylation by HRI.

Strengths:

The authors present a clear and well-structured study, beginning with an original and unbiased approach that effectively addresses a novel question. The investigation of PEBP1 as a specific regulator of the DELE1-HRI signaling axis is particularly compelling, supported by extensive data from both genetic and pharmacological manipulations. Including careful titrations, time-course experiments, and orthogonal approaches strengthens the robustness of their findings and bolsters their central claims.

Moreover, the authors skillfully integrate publicly available datasets with their original experiments, reinforcing their conclusions' generality and broader relevance. This comprehensive combination of methodologies underscores the reliability and significance of the study's contributions to our understanding of stress signaling.

Weaknesses:

While the study presents exciting findings, there are a few areas that could benefit from further exploration. The HRI-DELE1 pathway was only recently discovered, leaving many unanswered questions. The observation that PEBP1 interacts with eIF2alpha, but not with its phosphorylated form, suggests a novel mechanism for regulating the Integrated Stress Response (ISR). However, as they note themselves, the authors do not delve into the biochemical or molecular mechanisms through which PEBP1 promotes HRI signaling. Given the availability of antibodies against phosphorylated HRI, it would have been interesting to explore whether PEBP1 influences HRI phosphorylation. Furthermore, since the authors already have recombinant PEBP1 protein (as shown in Figure 1D), additional in vitro experiments such as in vitro immunoprecipitation, FRET, or surface plasmon resonance (SPR) could have confirmed the interaction with eIF2alpha. Future studies might investigate whether PEBP1 directly interacts with HRI, stimulates its auto-phosphorylation or kinase activity, or serves as a template for oligomerization, potentially supported by structural characterization of the complex and mutational validation.

Another point of weakness is the unclear significance of the 1.5-2x enhanced interaction with eIF2alpha upon PEBP1 phosphorylation, as there is little evidence to show that this increase has any downstream effects. The ATF4-luciferase reporter experiments, comparing WT and S153D overexpression, may have reached saturation with WT, making it difficult to detect further stimulation by S153D. Additionally, expression levels for WT and mutant forms are not provided, making it challenging to interpret the results. It would also be interesting to explore whether combined mitochondrial stress and PMA treatment further enhance the ISR.

Lastly, while the authors claim that oligomycin does not significantly alter the melting temperature of recombinant PEBP1 in vitro, the data in Figure S1D suggest a small shift. Without variance measures across replicates or background subtraction, this claim is less convincing. The inclusion of statistical analyses would strengthen the interpretation of these results.

Impact on the field:

The study's relevance is underscored by the fact that overactive ISR is linked to a broad range of neurodegenerative diseases and cognitive disorders, a field actively being explored for therapeutic interventions, with several drugs currently in clinical trials. Similarly, mitochondrial dysfunction plays a well-established role in brain health and other diseases. Identifying new targets within these pathways, like PEBP1, could provide alternative therapeutic strategies for treating such conditions. Therefore, gaining a deeper understanding of the mechanisms through which PEBP1 influences ISR regulation is highly pertinent and could have far-reaching implications for the development of future therapies.

Reviewer #2 (Public review):

Summary:

In this work, Cheng et al use the TPP/MS-CETSA strategy to discover new components for the mitochondria arm of the Integrated Stress Response. By using short exposures of several drugs that potentially induce mitochondrial stress, they find significant CETSA shifts for the scaffold protein PEBP1 both for antimycinA and oligomycin, making PEBP1 a candidate for mitochondrial-induced ISR signaling. After extensive follow-up work, they provide good support that PEBP1 is likely involved in ISR, and possibly act through an interaction with the key ISR effector node EIF2a.

Strengths:

The work adds an important understanding of ISR signaling where PEBP1 might also constitute a druggable node to attenuate cellular stress. Although CETSA has great potential for dissecting cellular pathways, there are few studies where this has been explored, particularly with such an extensive follow-up, also giving the work methodological implications. Together I therefore think this study could have a significant impact.

Weaknesses:

The TPP/MS-CETSA experiment is quite briefly described and might have a too relaxed cut-off. The assays confirming interactions between PEBP1 and EIF2a might not be fully conclusive.

Reviewer #3 (Public review):

Summary:

In this paper, Chang and Meliala et al. demonstrate that PEBP1 is a modulator of the ISR, specifically through the induction of mitochondrial stress. The authors utilize thermal proteome profiling (TPP) by which they identify PEPB1 as a thermally stabilized protein upon oligomycin treatment, indicating its role in mitochondrial stress. Moreover, RNA-sequencing analysis indicated that PEBP1 may be specifically modulating the mitochondrial stress-induced ISR, as PEBP1 knock-out reduces phosphorylation of eIF2α. They also show that PEBP1 function is independent of ER stress specifically tunicamycin treatment and loss of PEBP1 does affect mitochondrial ISR but in an OMA1, DELE1 independent manner. Thus, the authors hypothesized that PEBP1 interacts directly with eIF2α, functioning as a scaffolding protein. However, direct co-immunoprecipitation failed to demonstrate PEBP1 and eIF2α potential interaction. The authors then used a NanoBiT luminescence complementation assay to show the PEBP1-eIF2a interaction and its disruption by S51 phosphorylation.

Strengths:

Taken together, this work is novel, and the data presented suggests PEBP1 has a role as a modulator of the mitochondrial ISR, enhancing the signal to elicit the necessary response.

Weaknesses:

The one major issue of this work is the lack of a mechanism showing precisely how PEBP1 amplifies the mitochondrial integrated stress response. The work, as it is described, presents data suggesting PEBP1's role in the ISR but fails to present a more conclusive mechanism.

Author response:

We thank all the reviewers for their insightful comments to help further improving this work.

Response to Reviewer #1:

We greatly appreciate your comments on the general reliability and significance of our work. We fully agree that it would have been ideal to have additional evidence related to the role of PEBP1 in HRI activation. Unfortunately, we have not been able to find phospho-HRI antibodies that work reliably. The literature seems to agree with this as a band shift using total-HRI antibodies is usually used to study HRI activation. However, with the cell lines showing the most robust effect with PEBP1 knockout or knockdown, we are yet to convince ourselves with the band shifts we see. We may attempt to address this by optimizing phos-tag gels although these gels can be a bit tricky with complex samples such as cell lysates which contain many phosphoproteins.

To address the interaction between PEBP1 and eIF2alpha more rigorously we were inspired by the insights you and reviewer #2 provided. We now think we might be able to do this with either using the purified proteins and/or CETSA WB. These experiments could also provide further evidence for the role of PEBP1 phosphorylation. Although phosphorylation of PEBP1 at S153 has been implicated as being important for other functions of PEBP1, we are not sure about its role here. It may indeed have little relevance for ISR signalling. The CETSA WB assay could also provide further insight into the in vitro stability changes of PEBP1 in response to oligomycin.

For the currently shown in vitro thermal shift assay, we have performed replicate experiments and can provide the requested statistical analysis. The ultimate conclusion of this experiment remains incomplete as there could be alternative explanations despite the apparent simplicity of the assay. As for the signal saturation in ATF4-luciferase reporter assay, this is a valid point and we aim to address this via careful titration of the plasmids. We can also provide evidence for the expression levels of the mutants.

Response to Reviewer #2:

We strongly agree that CETSA has a lot of potential to inform us about cellular state changes and this was indeed the starting point for this project. We apologize for being (too) brief with the explanations of the TPP/MS-CETSA approach and we will certainly add more detail. With regard to the cut-offs used for the mass spectrometry analysis, you are absolutely right that we did not establish a stringent cut-off that would show the specificity of each drug treatment. Our take on the data was that using the p values (and ignoring the fold-changes) of individual protein changes as in Fig 1D, we can see that mitochondrial perturbations display a coordinated response. We now realize that the downside of this representation is that it obscures the largest and specific drug effects. We will therefore be incorporating the log fold-changes as suggested in order to enhance the clarity of the data.

As mentioned in the response to Reviewer #1, we now think that we can more conclusively address the potential interaction between PEBP1 and eIF2alpha using CETSA-based assays.

Response to Reviewer #3:

Thank you for your assessment, we agree that this manuscript would have been made much stronger by having clearer mechanistic insights. As mentioned in the responses to other reviewers above, we aim to address this limitation in part by looking at the putative interaction between PEBP1 and eIF2alpha with orthogonal approaches. However, we do realize that analysis of protein-protein interactions can be notoriously challenging due to false negative and false positive findings. As with any scientific endeavor, we will keep in mind alternative explanations to the observations, which could eventually provide that cohesive model explaining how precisely PEBP1, directly or indirectly, influences ISR signalling.