ClpC1-targeting peptide natural products differentially dysregulate the proteome of Mycobacterium tuberculosis

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: minor_revision

Abstract
Targeting the protein quality control system in Mycobacterium tuberculosis represents a promising and underexplored opportunity for antibiotic development. The ClpC1:ClpP1P2 protease is an essential component of the system that mediates both regulatory and stress-related protein degradation. Several non-ribosomal peptide natural products, including ecumicin, ilamycins (rufomycins) and cyclomarins, have been discovered that bind to the ClpC1 chaperone of the complex and exhibit potent antimycobacterial activity, leading to significant interest in the ClpC1:ClpP1P2 system as a bona fide target for the new tuberculosis drugs. In this study, we combine quantitative proteomics, bioinformatics, transcriptomics, CRISPRi knockdown, and targeted biochemical and biophysical assays to dissect the mechanisms of ecumicin, ilamycin and cyclomarin in clinically relevant Mycobacterium tuberculosis. Strikingly, despite exhibiting similar binding modes to ClpC1, each compound induces distinct effects on protein degradation. Notably, ilamycin and ecumicin do not trigger the ClpC2 rescue mechanism that mitigates cyclomarin-induced mycobacterial toxicity. In addition, we identify a novel interaction between ecumicin and stress-response chaperone Hsp20. The differential disruption of ClpC1 substrates, stress-response chaperones, and distinct reshaping of the Mycobacterium tuberculosis proteome by the three natural products, unveils new opportunities for the development of protein quality control-targeted antimycobacterials.

Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death worldwide1. The bacteria are transmitted via airborne droplets that typically infect the lungs and cause structural damage to the organ2,3. It is estimated that a quarter of the global population is infected with latent Mtb and are therefore at risk of developing active disease. In 2023, the World Health Organisation reported that TB was responsible for an estimated 1.25 million deaths, with an additional 10.8 million morbidities1. Current TB antibiotics generally target a small number of common biological pathways; cell wall synthesis, ATP synthesis, transcription, and translation4,5. The overuse and misuse of frontline antibiotics has contributed to the steady incidence of multi-drug resistant TB and extensively-drug resistant TB, which poses a significant threat to the future control of the disease and therefore to global health1,6. It is widely accepted that there is a crucial need for the development of new antimycobacterials that target novel pathways, are fast acting, and non-toxic to reduce the TB disease burden globally1.

Proteostasis is guided by environmental signals and executed through the balance of synthesis, folding and degradation7,8. The capacity of a pathogen to detect environmental cues and adapt accordingly is a key determinant of its virulence9. Several first- and second-line TB drugs used clinically work by disrupting biosynthetic machinery at various stages of protein production, including rifampicin, streptomycin, capreomycin, amikacin, and linezolid10,11. Beyond protein synthesis, perturbation of protein quality control (PQC) is a comparatively unexplored approach for the generation of potent anti-TB agents. Chaperones and proteases are the key components of the PQC system, responsible for the maintenance of native protein conformations and clearing terminally damaged proteins8,12. Through binding to misfolded proteins, chaperones minimise the consequences of denaturation and prevent the formation of intracellular aggregates. In the event a protein becomes irreparably damaged, it is degraded by proteases enabling amino acid building blocks to be recycled.

Mtb contains several proteases, including Clp, FtsH, and eukaryotic-like proteases9. A functional Clp proteolytic complex is essential for growth and survival of Mtb, coordinating post-translational regulation and proteome maintenance13–15. The Mtb Clp protease (ClpP1P2) is comprised of two heptameric serine proteases, ClpP1 and ClpP2, that associate to form an active cylindrical chamber containing 14 active sites for hydrolysis of peptide bonds within proteins16–18. Regulated proteolysis requires the association of ATPase chaperones, such as ClpC1, to form the ClpC1:ClpP1P2 (ClpC1P1P2) complex (Fig. 1a). Active ClpC1 exists as a hexamer that constitutes a central axial pore for substrate interactions. Each ClpC1 monomer contains an N-terminal domain (NTD) connected via an inherently disordered 26 amino acid linker to the ATPase domains, D1 and D219,20. The D1 and D2 domains hydrolyse ATP to actively unfold and translocate client proteins to the proteolytic core of ClpP1P2 for degradation18,19. Substrate recognition by Mtb ClpC1 is complex and not well defined. However, recent advances have demonstrated that recognition is influenced by features such as unstructured N- or C-terminal regions, which facilitate engagement with ClpC1-NTDs14,21.

The relevance of the Clp system as a potential antimycobacterial target was reinforced by the discovery of several ClpC1-binding peptide natural product antibiotics, including non-ribosomal peptides (NRPs) ecumicin, cyclomarins, ilamycins (also called rufomycins) and the ribosomally-synthesised lassomycin22–25. In this study, we focus on the three main NRP classes. Ecumicin is a non-ribosomal tridecadepsipeptide isolated from the actinomycete Nonomuraea sp MJM5123. The molecule contains extensive backbone N-methylation and two non-canonical amino acids: N-methyl-4-methoxy-L-tryptophan and threo-β-hydroxy-L-phenylalanine22,26. The ilamycins/rufomycins and cyclomarins are both cyclic heptapeptides produced by Streptomyces sp23,24. Ilamycin E (Ila E), the most potent congener in its family, contains several non-canonical amino acids, including: L-2-amino-4-hexenoic acid, N-tert-prenylated L-tryptophan, L-3-nitrotyrosine, and an interesting hydroxypiperidinone motif23. Cyclomarin C contains N-tert-prenylated β-hydroxytryptophan, 2-amino-3,5-dimethyl-4-hexenoic acid, 5-hydroxyleucine, and β-methoxyphenylalanine27. Each NRP has been shown to bind to the NTD of ClpC1 and proposed to mimic exposed hydrophobic regions of unfolded proteins that would normally be cleared through ClpC1:ClpP1P2 proteolysis19,28–30. There is considerable overlap in the ClpC1-binding interface between the three compounds and mutagenesis studies have revealed mutations at F80 and V13 confer resistance to more than one natural product and a shared contact, K85, influences degradation capacity in the presence of cyclomarin (Fig. 1b and Supplementary Fig. 1a)28–31. Distinct effects on the ATPase activity and degradation of model substrates have also been reported30,32. Taken together, this suggests an elaborate and nuanced interplay between substrate recognition and NRP activity, yet the broader effects on the Mtb proteome and native substrates are yet to be elucidated. Leveraging their capacity to bind to the ClpC1-NTD, one of these NRPs, cyclomarin, recently inspired the design of bacterial proteolysis targeting chimeras (BacPROTACS), enabling targeted protein degradation in mycobacteria33–35. Given the emerging interest in targeting proteolysis in Mtb, furthering our understanding of the underlying mechanisms of the each of these NRPs is crucial for informing development of novel and innovative Clp-targeting antibacterials in the future.

In this study, we combined systematic proteomics, bioinformatics, transcriptomics, CRISPRi knockdown, and in vitro biochemical assays to investigate the effects of three NRPs, an ecumicin analogue (Ecu*)36, ilamycin E (also termed rufomycin 22)37, and desoxycyclomarin (dCym)34 on clinically relevant Mtb H37Rv. Each compound caused distinct, substrate specific dysregulation of ClpC1. Small stress response chaperones were differentially affected. All three NRPs bound ClpC2, but only dCym triggered its upregulation and exhibited enhanced activity in ClpC2 knockdown strains. Hsp20 was strongly upregulated by Ecu* and, to a lesser extent IlaE, with binding assays confirming a direct Ecu*:Hsp20 interaction. Collectively, these results demonstrate that the NRPs perturb PQC through distinct mechanisms, including differential dysregulation of ClpC1 substrate recognition and engagement of stress-response chaperones.

Results
NRPs cause widespread changes to the Mtb proteome
In this study, we have examined the activity of a representative congener (or related synthetic analogue) from the three families of ClpC1-binding NRPs, including: a synthetic ecumicin analogue (Ecu* or 1) with a simplified amino acid composition, and superior antimycobacterial potency, to the parent natural product (previously described by Hawkins et al.)36, ilamycin E (IlaE or 2)38, and a simplified synthetic analogue of cyclomarin A, desoxycyclomarin (dCym or 3)39 (Fig. 1c and Supplementary Fig. 1b). Each compound exhibited potent antimycobacterial activity against virulent Mtb strain H37Rv with these data used to direct downstream experiments (Fig. 1c and Supplementary Fig. 1c).

The NRPs target the chaperone component of the ClpC1P1P2 proteolysis system28–30,34,40. To assess their impact on the Mtb proteome, we employed label-free quantification (LFQ) proteomics to monitor changes in protein abundance. Sublethal doses and treatment durations were established for each NRP derivative (1–3) to minimise cell death, which could confound proteomic analysis. Initial assessment of (1) at 16 h, below one doubling time, revealed minimal changes (Supplementary Fig. 2a). Pronounced, reproducible effects emerged after 48 h, which was selected for all subsequent analysis maintaining 70–80% mycobacterial survival (Supplementary Fig. 2b, c). Stability of each NRP in culture media for 48 h was confirmed by LC-MS (Supplementary Fig. 2d). Following NRP exposure, cells were lysed using SDS with an overnight freezing step and ultra sonication to maximise extraction efficiency. Proteins were then trypsin-digested, peptides desalted and analysed by quantitative LC-MS/MS (Fig. 1d). We detected 3175 proteins across all samples, achieving ~80% coverage of the known Mtb proteome (Supplementary Data 1). Hundreds of proteins were significantly altered after NRP treatment (Benjamini–Hochberg-adjusted p ≤ 0.05, log2 FC > 0.5 or < −0.5, Fig. 1e–g and Supplementary Data 1). Ecu* (1) caused the most widespread effects, with nearly 17% of the proteome altered and 36 proteins showing changes between ten and 96-fold. IlaE (2) and dCym (3) affected 12% and 7% of the proteome, respectively with fewer large changes (Fig. 1e, f). These data provide evidence that NRPs alter a substantial portion of the 4173 protein Mtb proteome41 through ClpC1 dysregulation.

Although all three NRPs bind the ClpC1 NTD, their proteomic consequences were distinct. The changes induced by IlaE (2) showed greater overlap with those caused by Ecu* (1) and dCym (3) than Ecu* (1) and dCym (3) did with each other, suggesting that Ecu* (1) and dCym (3) elicit the most divergent responses. Only 72 proteins were consistently affected across all treatments and most of these were depleted (68 of 72, Fig. 1h), suggesting each NRP alters ClpC1 substrate recognition in different ways.

To probe impacted pathways, gene ontology enrichment revealed broadly similar clusters across compounds, despite differences in individual proteins (Supplementary Fig. 3 and Supplementary Data 2). Depleted processes included stress responses and diverse metabolic pathways (lipid, amino acid, small molecule and macromolecule metabolism). Ecu* (1) additionally depleted transcription-related proteins while enriching those linked to fatty acid synthesis and stress response processes. IlaE (2) and dCym (3) primarily depleted fatty-acid biosynthesis proteins, with few enriched pathways. These data are consistent with ClpC1 interaction studies14,42, reinforcing the widespread role of the ClpC1P1P2 system in essential mycobacterial processes.

BTZ broadly inhibits protein degradation in Mtb
Bortezomib (BTZ) is a well-characterised eukaryotic proteosome inhibitor that also dysregulates the ClpP1P2 protease complex (Fig. 2a, b)43–45. BTZ inhibited Mtb growth with a MIC of 5.6 µM (Supplementary Fig. 4a), consistent with previous reports and both 16 h and 48 h incubations resulted in sublethal conditions (Supplementary Fig. 4b). Using LFQ proteomics, we observed 325 and 881 differentially abundant proteins at 16 and 48h-post treatment, respectively. More proteins were enriched than depleted at both timepoints, reflecting impaired protein turnover owing to ClpP1P2 and Mtb proteasomal inhibition (Fig. 2c and Supplementary Data 4). Notably, the number of depleted proteins increased at 48 h, consistent with prolonged inhibition of proteolysis suppressing protein synthesis and transcription46. While recent in vitro studies show BTZ can activate ClpC1P1P2 model substrate proteolysis45, our data demonstrate net inhibition of protein degradation within Mtb cells.

Comparisons of BTZ-induced changes with those elicited by Ecu*(1), IlaE (2), and dCym (3) revealed limited overall overlap, with IlaE (2) and Ecu* (1) showing greater similarity to BTZ than dCym (3) (Fig. 2d, e). ClpC1P1P2 substrates comprise regulatory targets and terminally damaged proteins that are generated through stress and translational errors. Validated regulatory ClpC1P1P2 substrates include PanD, Hsp20, ClgR, CarD, and Vap/Rel antitoxins14,15,42,47,48. In addition to ClpP1P2, BTZ also inhibits the 20S eukaryotic-like protease in Mtb49. The 20S proteosome recognises substrates tagged with the prokaryotic ubiquitin-like protein, Pup, which serves as a degradation signal. Known 20S proteosome substrates include Mpa, HspR, FabD, and Pup itself50–52. The NRPs did not trigger global enrichment of all ClpC1P1P2 substrates, instead they exhibited substrate-dependent modulation. Ecu* (1) significantly enriched PanD and Hsp20 while depleting CarD, and IlaE (2) enriched ClgR and Hsp20 (Supplementary Fig. 5a). As anticipated, the NRPs did not lead to the enrichment of 20S proteosome substrates (Supplementary Fig. 5b). Several Vap/Rel antitoxins were differentially affected, being either enriched or depleted by Ecu* (1) and IlaE (2) (Supplementary Fig. 5c). Interestingly, ClgR and Hsp20 were the two most enriched proteins in response to BTZ (4) (Fig. 2c). Both were also strongly enriched by IlaE (2), whereas only Hsp20 was enriched by Ecu* (1) and neither were enriched by dCym (3) (Fig. 1e–g and Supplementary Fig. 5a). BTZ broadly enriched both ClpC1P1P2 and 20S proteosome substrates, confirming a general inhibitory impact on protein degradation within Mtb cells (Fig. 2f, g and Supplementary Fig. 5d). Together, these data indicate that, unlike BTZ, the NRPs modulate ClpC1 chaperone activity rather than global inhibition of mycobacterial protease activity.

Intrinsic protein disorder and substrate-specific effects of NRPs
ClpC1P1P2 also clears unfolded proteins to prevent aggregation18,53–55 (Fig. 3a). We examined the relationship between NRPs and intrinsically disordered proteins that contain regions of disorder, resembling unfolded polypeptides, yet are functional and stable rather than aggregation-prone56. Using the mobiDB server57,58 AlphaFold2-derived disorder predictions, which cover 68% of the Mtb proteome, Mtb proteins were assigned disorder scores. Statistical analysis revealed that intrinsically disordered proteins were 5.68 times more likely to be depleted with Ecu* (1), yet 4.76 and 6.25 times more likely to be enriched with IlaE (2) and dCym (3), respectively (Fig. 3b and Supplementary Table 1). In contrast, proteins with disordered termini did not show statistically significant global enrichment or depletion in the context of the NRPs (Fig. 3c, d, Supplementary Table 2, and Supplementary Data 3). These results indicate that overall intrinsic disorder, rather than terminal disorder, is more predictive of susceptibility to NRP-induced changes. However, as this conclusion is derived from a global proteomic analysis, it likely encompasses both direct, ClpC1 dysregulation-dependent effects, and broader protein responses that would result from the dramatic changes to the Mtb proteome that occurs following NRP treatment. Because these effects cannot be readily disentangled, genuine associations with terminal disorder may be underrepresented in the aggregated data.

Perturbation of ClpC1P1P2 substrate degradation by NRPs
Proteomic analysis captures protein abundance at a specific point in time but does not directly measure turnover. To complement our observations, we performed in vitro degradation assays using both a model disordered protein and physiologically relevant ClpC1P1P2 substrates. Degradation of β-casein, the archetypal disordered protein model34, was strongly inhibited by Ecu* (1), whereas IlaE (2) and dCym (3) had no observable effect (Fig. 3d and Supplementary Fig. 6a). It is worth noting that BTZ (4) has been described as an activator of the Mtb ClpC1P1P2 protease in vitro, particularly at low concentrations45. We did not observe a discernible effect on β-casein degradation in the presence of BTZ, which likely reflects the relatively high concentration utilised in the assay and the absence of a fluorescence-based reporter, which limits assay sensitivity (Supplementary Fig. 6a).

We next examined PanD and Hsp20, two native Mtb substrates with disordered C-terminal regions and demonstrate in vitro evidence of their degradation by ClpC1P1P2 and the influence of the NRPs on this degradation. In contrast to β-casein, each of the NRPs inhibited PanD degradation in vitro, despite only Ecu* (1) triggering an enrichment of PanD in Mtb cells in our proteomics experiments (Fig. 3e and Supplementary Fig. 6b). Hsp20 degradation was inhibited only by dCym (3). Despite strong enrichment of Hsp20 by Ecu* (1), IlaE (2) and BTZ (4) in

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an interesting article, that explores a very important question concerning the mode of action of MtbClpC1antibiotics. Very little is know on how ecumicin, cyclomarin and rufomycin lead to cell death, particularly the death of persisters. While the approach is interesting, demonstrating the power of modern proteomics, the results obtained are mostly descriptive and fail to explain the mechanistic differences between these 3 antibiotics. Following this article we will know which substrates are up or down regulated by each antibiotic but very little on how this happens. The statement that the differences between the different antibiotics are the result of specific binding of ecumicin or rufomycin to particular chaperones is not fully supported by the data presented, at least in the current version of the article. Indeed, to really understand the differences between antibiotics it would require extensive mechanistic studies that are totally absent from the work.\n\nMinor Point\nThere are multiple previous articles using the therminology rufomycin instead of ilamycin. It is true that the authors explain that ilamycins are the same as rufomycins, but perhaps this should be done earlier in the text.\n\nMajor Points\nMajor Point 1:\nThe authors state:\n“Ecu*(1) bound to MtbClpC1NTD with a KD of 8.58μM, IlaE(2) a KD of 0.11μM and dCym(3)aKD of 0.013 μM. These values are consistent with those reported in previous studies 41.”\nWe believe that the reported values for ecumicin and rufomycin are significantly lower as for example in the following article (that is not cited here), “ High-Resolution Structure of ClpC1-Rufomycin and Ligand Binding Studies Provide a Framework to Design and Optimize Anti-Tuberculosis Leads” by Wolf et al, the values described for ecumicin and rufomycin (also using SPR) are very different. For ecumicin binding to ClpC1-NTD the KD value was described as 50 nM and for rufomycin the value was 63 nM. The KD for ecumicin in that previous publication is therefore circa 200x lower than the one you describe here and that is even more puzzling when you state here the following: “a synthetic ecumicin analogue (Ecu*, 1) with a simplified amino acid composition, and superior potency, to the parent natural product”.\nSo either your ecumicin analog is in fact less potent than you propose or the KD value you present here is not correct. This paradox is even more puzzling when you report a MIC90 of 0.15 µM for ecumicin (supplementary Fig1) which was used in the proteomics experiments.\nOf note, if the ecumicin analog is not as potent as you propose, it would be perhaps important to do this kind of studies with the more potent compound.\n\nMajor Point 2:\n“Increased temperature has been shown to increase the bactericidal effect of cyclomarin; this is thought to be due to occupation of ClpC2 by unfolded proteins, thus allowing cyclomarin to act on ClpC1 41.”\nAnother reason for this high temperature effect, which is more likely in our view, is that high temperature increases the burden of unfolded proteins, challenging the protein quality system and therefore making the cell more sensitive to its dysregulation by antibiotics. To test the author´s hypothesis, it would be important to inhibit just ClpP1P2 (for example with bortezomib), this way bypassing ClpC1, and check if you would obtain similar results with temperature increase. If not, your hypothesis would be supported.\n\nMajor Point 3:\nThe authors cite and refer multiple times to the article “The unfoldase ClpC1 of Mycobacterium tuberculosis regulates the expression of a distinct subset of proteins having intrinsically disordered termini” by Lunge et al. In this specific publication the authors show that ClpC1 is responsible for the degradation of Hsp20, it is clearly stated:\n“Further, we observed that Hsp20 is poorly expressed in WT Mtb and that its expression is greatly induced upon depletion of clpC1 or clpP2. Remarkably, high Hsp20 protein levels were detected in the clpC1(2) or clpP2(2) knock- down strains but not in the parental bacteria, despite significant induction of hsp20 transcripts. In summary, the cellular levels of oligomeric proteins such as Hsp20 are maintained post-translationally through their recognition, disassembly, and degradation by ClpC1, which requires disordered ends in its protein substrates.”\nConsidering the results previously reported, it is not at all surprising that in the presence of ecumicin, an inhibitor of ClpC1P1P2 protein degradation, the levels of Hsp20 should increase. Moreover, it also explains the result with ilamycin, that is also an inhibitor of ClpC1P1P2 protein degradation. Cyclomarin, in contrary, has been proposed to enhance protein degradation in vivo: see “Toxic Activation of an AAA+ Protease by the Antibacterial Drug Cyclomarin A” by Maurer et al. However, and despite this previous evidence the authors advance the thesis that since ecumicin also binds to Hsp20, this somehow would protect the Hsp20 chaperone from degradation. Several issues arise here:\n1) The authors report a MIC90 of 0.15 µM for ecumicin, a concentration that was used in the experiments. With this concentration of ecumicin, and the reported Hsp20 KD (circa 20 µM) , the occupancy of Hsp20 with ecumicin would be certainly very low – incompatible with your thesis.\n2) Ecumicin was recently proposed by the Clausen group to mimick unfoldeded protein substrates. It is therefore not surprising that it binds to a chaperone that binds unfolded proteins like Hsp20. Did you test other random chaperones? Like Hsp70, HtpG or even ClpB. Since the KD is so high it would not be surprising that some binding can also be observed with other chaperones.\n3) The authors make a possible analogy between the roles of ClpC2 and Hsp20 but several key differences exist – the most important being the fact that ClpC2 is a very good binder of cyclomarin (KD 2 nM) as described in “ClpC2 protects mycobacteria against a natural antibiotic targeting ClpC1-dependent protein degradation” by Taylor et al.\n\nMajor Point 4:\nOne major problem of using proteomics with the ClpC1P1P2 system is the fact that some of the targets of the protease are themselves cellular regulators. For example ClgR, that is degraded by ClpC1P1P2, is a transcriptional activator that activates the expression of multiple other proteins. It has been shown that ClgR positively regulates several other genes, including its own gene, clgR, as well as acr2, encoding a chaperone. This to say, inhibiting ClpC1P1P2, will lead to an increase of ClgR and several other proteins as consequence. So it is not surprising that some protein levels are increased while others are decreased making the very analysis complex. This is not discussed at any point of the article.\nIn our view, to exclude effects like the above, it would be important to test in vitro for degradation with ClpC1P1P2 the substrates with different results between antibiotics, that is - the ones that are specifically increased or decreased with one specific antibiotic.\nThat way you could test if indeed there is a direct impact of ecumicin, cyclomarin and rufomycin on ClpC1 that makes it more prone for the degradation of some specific substrates? This is a very important question that cannot be answered only using systematic proteomics.\nOverall this is an interesting article, but likely more fitting to publications dedicated to proteomics. A broad journal of excellence such as Nature Communications appears perhaps not appropriate unless a detailed mechanistical analyis is performed on the ClpC1P1P2 different substrates.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The protein quality control system of mycobacteria is a promising and developing target for mycobacterial drug discovery, which can also be utilized for targeted protein degradation. This manuscript explores the ClpC1:ClpP1P2 protease system in virulent M. tuberculosis, focusing on the response to ClpC1 NPA targeted antibiotics - ecumicin, ilamycin, and cyclomarin using a proteomic approach. These studies show key differences in the effects of the antibiotics studied despite their common targeting of ClpC1. The roles of ClpC2 and Hsp20, which help provide compensatory and potentially protective effects to the differing agents, as highlighted in Figure 5, represent the most interesting aspect of this study. Other significant results include studies of the effects of NPA treatment on the abundance of proteins with unstructured domains and termini that are believed to be ClpC1 native substrates, and in the potentiation of the heat shock response.\nOverall, this is an excellent proteomics study, in an important topic area on the drug effects of ClpC1 targeted antibiotics. The manuscript is well written and the supporting information adds to the scientific rigor of the reported study.\nPrimary concerns:\n1. Methodological: the proteomic sampling is performed at a single time point after 48h of treatment at the predetermined low OD MIC90 level of the respective drugs. This is too long to study mechanistic effects as compensatory effects will likely dominate the response. Proteomic sampling should be performed in less than one doubling time (24h), and 3-6h is recommended. The authors should also establish that there is not an inoculum effect at the higher cell concentration used in the proteomics experiments that negates the drug action.\n2. The second concern relates to the limited scope of the study, which only uses one method of proteomic profiling. Adding complementary unbiased RNAseq or thermal proteome (CETSA) profiling experiments would strengthen the conclusions.\n3. Due to its limited scope, this study does not adequately prove the author's conclusion that these antimycobacterial drugs disrupt protein quality control beyond a single protein target.\nOther comments and suggestions:\n1. The authors use flattering terms like “cutting-edge” and “state of the art” to describe their mass-spectrometry proteomics, which is overstated as these methods are widely employed elsewhere.\n2. NPA drug stability after 48h should be confirmed under the incubation conditions.\n3. Incorporating a protease ClpP1P2 inhibitor control would be a useful comparator in this study to help decipher the mechanistic effects that are ClpC1-specific.\n4. Conclusions summary: given that the three inhibitors lack cross-resistance, is it surprising they have differential impacts?\n5. The final stated conclusion “we expect that the findings of this study, combined with more detailed structural data in the future, will greatly facilitate the rational design of next generation potent ClpC1 and PQC-targeting TB drug leads.” How? It is unclear to this reviewer how this study would aid further drug development; for instance, have any new vulnerabilities been revealed?\n6. Materials and methods – Mtb growth conditions: please report oxygen and carbon dioxide levels of culture.\n7. Materials and methods: cell viability assay; please report the time, drug concentration, and cellular density at which the Mtb-treated samples were prepared.", "reviewer_4": "Tuberculosis (TB) remains one of the leading causes of death from infection worldwide. The high virulence of Mycobacterium tuberculosis (Mtb) is partly due to its ability to survive and replicate within the alveolar macrophages, establishing reservoirs of live bacteria that promote the persistence and recurrence of the disease. An estimated one-quarter of the global population harbours a latent TB infection (LTBI)2, which, although asymptomatic, represents a reservoir for the potential reactivation and transmission of TB. Additionally, there is an increasing concern regarding the number of the multidrug resistant cases. Bater et al., have tested several antimycobacterial cyclic peptide natural products that bind the ClpC1 chaperone component of the system and claimed that the have employed cutting edge mass-spectrometry-based proteomics to determine the effects of NPAs on virulent Mtb H37Rv. The study reports 3, 175 protein IDS of hundreds of proteins were differentially abundant with different NPA treatment. The authors claimed that “this study provides a deeper understanding of the distinct mechanisms of antimycobacterial activity of different NPAs through dysregulation of the ClpC1:ClP1P2 proteolysis system. However as is stands the manuscript is merely descriptive and the results lacks orthogonal validation.\nMajor points:\nAccording to the authors, NPAs are known to target a major chaperoning element in the ClpC1-P1P2 protein degradation mechanism. However, there is no evidence in this work sowing that NPAs interact with the ClpC1-P1P2 protein system; rather, the authors refer/rely on changes in protein abundance. The authors must offer supporting evidence that the changes observed in the Mtb proteome are the result of NPA interactions with the ClpC1-P1P2 protein system, rather than a general reaction to a foreign peptide. For this, I recommend that the authors use current techniques like cross-linking mass spectrometry to demonstrate that NPAs bind to the complex. The amount of peptide-protein interaction is then assessed using molecular docking simulation.\nThe study lacks supportive functional assays that aid in understanding not only the mechanism of action but also the extent of NPA in Mtb viability. For example, it has been demonstrated that CLpC2 protects against CymA-induced toxicity; it would be interesting to know how NPA treatment interferes with such a protective model system or simply compare the presence of NPA to the clpC2 knockout strain."}, "author_response": "Responses to Referees:\nReferee #1:\nMinor Point\nThere are multiple previous articles using the terminology rufomycin instead of ilamycin. It is\ntrue that the authors explain that ilamycins are the same as rufomycins, but perhaps this should\nbe done earlier in the text.\nWe thank the reviewer for this comment. We have moved this clarification to the abstract (page\n2, line 8) and also reiterated in the main text (page 4, line 76).\nPoint 1:\nThe authors state:“Ecu*(1) bound to MtbClpC1NTD with a KD of 8.58μM, IlaE(2) a KD of 0.11μM and dCym(3) a KD of 0.013 μM. These values are consistent with those reported in previous studies 41.”\nWe believe that the reported values for ecumicin and rufomycin are significantly lower as for\nexample in the following article (that is not cited here), “ High-Resolution Structure of ClpC1-\nRufomycin and Ligand Binding Studies Provide a Framework to Design and Optimize Anti-\nTuberculosis Leads” by Wolf et al, the values described for ecumicin and rufomycin (also using\nSPR) are very different. For ecumicin binding to ClpC1-NTD the KD value was described as\n50 nM and for rufomycin the value was 63 nM. The KD for ecumicin in that previous\npublication is therefore circa 200x lower than the one you describe here and that is even more\npuzzling when you state here the following: “a synthetic ecumicin analogue (Ecu*, 1) with a\nsimplified amino acid composition, and superior potency, to the parent natural product”.\nSo either your ecumicin analog is in fact less potent than you propose or the KD value you\npresent here is not correct. This paradox is even more puzzling when you report a MIC90 of\n0.15 µM for ecumicin (supplementary Fig1) which was used in the proteomics experiments.\nOf note, if the ecumicin analog is not as potent as you propose, it would be perhaps important\nto do this kind of studies with the more potent compound.\nWe thank the reviewer for their comment. We do agree that our reported K for Ecu* was low\nD\nand, after further consulting the published literature, have remeasured SPR data with a revised\nprotocol. Specifically, we have repeated the SPR experiments at a more suitable concentration\nrange and fit the sensorgram to multi-site parameters, in alignment with the reported 2:1\nbinding mode of the Ecu natural product to ClpC1 (Acta Crystallogr D Struct Biol, 2020, 458).\nWe have recalculated the affinity of Ecu* to ClpC1 as K = 0.39 µM and K = 1.7 M. These\nD1 D2\nchanges have been updated in Figure 4c, Supplementary Figure 9a and associated text.\nbinding affinity to ClpC1-NTD measured by SPR and the MIC against Mtb. For example,\nohmyungsamycin A (OMSA) is a related congener of ecumicin and exhibits weaker binding to\nClpC1 by SPR (K = 135 nM vs 22.5 nM (Acta Crystallogr D Struct Biol, 2020, 458) but\nD1\nexhibits more potent activity against Mtb (MIC = 110 nM vs 360 nM for the parent ecumicin\n90\nnatural product, Chem Eur J, 2020, 15200). It would therefore be reasonable to observe a\ndiscrepancy between the binding affinity of the Ecu natural product and analogue Ecu* to the\nimmobilised monomeric fragment of ClpC1 and the activity against the target microorganism,\nwhere permeability, intracellular stability, and the interaction with a hexameric ClpC1 in the\nClpC1P1P2 complex are all important considerations. With respect to ilamycin E, the K\nD\nreported by Wolf et al. is for rufomycin 4 (ilamycin C) which is a different compound to the\none we have synthesized and analysed (ilamycin E). Nonetheless, the binding affinity measured\nfor Ruf4 against ClpC1 (K = 63 nM) is similar to that measured for ilamycin E in our study\nD\n(K = 107 nM).\nD\nNew sensorgrams and affinity data are included in the revised Figure 4c and Supplementary\nFig 7 and are also provided below:\nFigure 4c. Surface plasmon\nresonance (SPR) binding affinities for\ninteraction between NRPs and ClpC1-\nNTD and ClpC2-CTD. Raw data for\nthree replicates is presented in\nSupplementary Figure 7.\nSupplementary Figure. 9| Raw data for surface plasmon\nresonance (SPR) binding assays shows each NRP bound another\nchaperone in addition to ClpC1. a SPR plots for all replicates of\nNRPs 1-3 ligand binding with recombinant Mtb ClpC1-NTD\nreplicates. b SPR plots for all replicates of NRPs 1-3 interaction with\nrecombinant Mtb ClpC2-CTD. c SPR plots for NRP ligand binding to\nrecombinant Mtb Hsp20.\nPoint 2:\n“Increased temperature has been shown to increase the bactericidal effect of cyclomarin; this\nis thought to be due to occupation of ClpC2 by unfolded proteins, thus allowing cyclomarin\nto act on ClpC1 41.”\nAnother reason for this high temperature effect, which is more likely in our view, is that high\n2\ntemperature increases the burden of unfolded proteins, challenging the protein quality system\nand therefore making the cell more sensitive to its dysregulation by antibiotics. To test the\nauthor´s hypothesis, it would be important to inhibit just ClpP1P2 (for example with\nbortezomib), this way bypassing ClpC1, and check if you would obtain similar results with\ntemperature increase. If not, your hypothesis would be supported.\nWe thank the reviewer for this comment and suggested experiment. The effect of cyclomarin\nunder heat stress described in the manuscript is based on conclusions drawn from prior work\nby Hoi et al. (Cell 2023, 2176). Nonetheless, in new experiments, we inhibited Mtb proteases\nwith bortezomib (BTZ) to bypass ClpC1 and assessed Mtb survival under mild heat stress. BTZ\ntreatment in combination with elevated temperatures caused a significant drop in viability\n(Figure 5h), consistent with heat increasing the burden of unfolded proteins and sensitizing\ncells to protease inhibition. The new data can be found in Figure 5h of the revised manuscript,\nand these new results are described on page 11, from line 322.\nFig. 5 | h Colony forming unit (CFU) viability assay for heat stressed and non-heat stressed\nMtb H37Rv treated with NRPs 1-3 and BTZ (4). Data presented as mean  SD, n = 3 for each\ncondition. Statistical significance was calculated with a 2-way ANOVA, * = p-value < 0.05,\n** = p-value < 0.01. Source data are provided in Source data file.\nPoint 3:\nThe authors cite and refer multiple times to the article “The unfoldase ClpC1 of Mycobacterium\ntuberculosis regulates the expression of a distinct subset of proteins having intrinsically\ndisordered termini” by Lunge et al. In this specific publication the authors show that ClpC1 is\nresponsible for the degradation of Hsp20, it is clearly stated:\n“Further, we observed that Hsp20 is poorly expressed in WT Mtb and that its expression is greatly induced upon depletion of\nclpC1 or clpP2. Remarkably, high Hsp20 protein levels were detected in the clpC1(2) or clpP2(2) knock- down strains but not in the parental bacteria, despite\nsignificant induction of hsp20 transcripts. In summary, the cellular levels of oligomeric proteins such as Hsp20 are maintained post-translationally through their recognition,\ndisassembly, and degradation by ClpC1, which requires disordered ends in its protein substrates.”\nConsidering the results previously reported, it is not at all surprising that in the presence of\necumicin, an inhibitor of ClpC1P1P2 protein degradation, the levels of Hsp20 should increase.\nMoreover, it also explains the result with ilamycin, that is also an inhibitor of ClpC1P1P2\nprotein degradation. Cyclomarin, in contrary, has been proposed to enhance protein\ndegradation in vivo: see “Toxic Activation of an AAA+ Protease by the Antibacterial Drug\nCyclomarin A” by Maurer et al. However, and despite this previous evidence, the authors\nadvance the thesis that since ecumicin also binds to Hsp20, this somehow would protect the\nHsp20 chaperone from degradation. Several issues arise here:\n1) The authors report a MIC90 of 0.15 µM for ecumicin, a concentration that was used in the\nexperiments. With this concentration of ecumicin, and the reported Hsp20 KD (circa 20 µM),\nthe occupancy of Hsp20 with ecumicin would be certainly very low – incompatible with your\nthesis.\nWe thank the reviewer for these comments. Upon revision of the manuscript (including support\nfrom new experiments suggested by all reviewers) we agree that our earlier thesis that Hsp20\nmay sequester Ecu* was likely incorrect. As part of our revision we repeated SPR experiments\nacross a more suitable concentration range and recalculated the affinity of Ecu* for Hsp20 to\nbe 3.5 µM (Figure 5c, Supplementary Figure 9c, line 299 page 10). While this represents an\nimprovement in affinity, the interaction remains markedly weaker than that of Ecu* and ClpC1,\nmaking an Hsp20 sequestration mechanism unlikely. We have revised the manuscript text to\nreflect these data in the results and explicitly in the discussion (line 425, page 14).\n2) Ecumicin was recently proposed by the Clausen group to mimick unfolded protein\nsubstrates. It is therefore not surprising that it binds to a chaperone that binds unfolded\nproteins like Hsp20. Did you test other random chaperones? Like Hsp70, HtpG or even ClpB.\nSince the KD is so high it would not be surprising that some binding can also be observed\nwith other chaperones.\nWe agree that as the non-ribosomal peptides (NRPs) are proposed to mimic unfolded proteins,\ninteractions with other chaperones or protein quality control proteins are possible. However,\nour proteomic analysis shows that each of the NRPs lead to strong dysregulation of Hsp20 and\nClpC2, whereas levels of other major chaperones including Hsp70, HtpG and ClpB remained\nlargely unaffected (see Supplementary Figure 8 and Figure 1e-g). This striking response guided\nour focus on Hsp20 and ClpC2, supporting their functional relevance in our system. We believe\nthat investigation into other chaperones is out of the scope of our already comprehensive study.\n3) The authors make a possible analogy between the roles of ClpC2 and Hsp20 but several\nkey differences exist – the most important being the fact that ClpC2 is a very good binder of\ncyclomarin (KD 2 nM) as described in “ClpC2 protects mycobacteria against a natural\nantibiotic targeting ClpC1-dependent protein degradation” by Taylor et al.\nWe agree with the reviewer that Hsp20 and ClpC2 are not directly comparable; Mtb ClpC2\nbinds dCym with high affinity (K 13 nM), providing potential for a clear protective\nD\nmechanism, whereas the weaker interaction between Ecu* and Hsp20 makes efficient\nsequestration less likely. As described for the comment from the same reviewer above, we have\nupdated the text in the discussion of the revised manuscript to clarify this point.\nPoint 4:\nOne major problem of using proteomics with the ClpC1P1P2 system is the fact that some of\nthe targets of the protease are themselves cellular regulators. For example ClgR, that is\ndegraded by ClpC1P1P2, is a transcriptional activator that activates the expression of multiple\nother proteins. It has been shown that ClgR positively regulates several other genes, including\n4\nits own gene, clgR, as well as acr2, encoding a chaperone. This to say, inhibiting ClpC1P1P2,\nwill lead to an increase of ClgR and several other proteins as consequence. So it is not\nsurprising that some protein levels are increased while others are decreased making the very\nanalysis complex. This is not discussed at any point of the article.\nIn our view, to exclude effects like the above, it would be important to test in vitro for\ndegradation with ClpC1P1P2 the substrates with different results between antibiotics, that is -\nthe ones that are specifically increased or decreased with one specific antibiotic. That way you\ncould test if indeed there is a direct impact of ecumicin, cyclomarin and rufomycin on ClpC1\nthat makes it more prone for the degradation of some specific substrates? This is a very\nimportant question that cannot be answered only using systematic proteomics.\nWe thank the reviewer for highlighting the complexities of interpreting proteomic changes in\nresponse to dysregulation of the ClpC1P1P2 system. To address this, we have now assessed\nthe activity of each of the NRPs on in vitro the degradation of model substrate β-casein and\nnative Mtb ClpC1P1P2 substrates, PanD and Hsp20, by reconstituted ClpC1P1P2. The new\ndata can be found in Figure 3, Supplementary Figure 6 in the revised submission and below for\nconvenience. It is worth noting that all previous studies on Mtb ClpC1P1P2 have assessed\nactivity on exclusively model substrates and this data represents the first direct evidence that\nthe NRPs may affect ClpC1P1P2 activity on specific substrates in a NRP-dependent manner.\nIn our hands, ClgR could not be generated with the purity necessary to use in a degradation\nassay. The text associated with this new data can be found under Perturbation of ClpC1P1P2\nsubstrate degradation by NRPs, (page 8, line 227).\nIn addition to these experiments, on the suggestion of reviewer 3, we carried out transcriptomic\nanalysis on Ecu* treated Mtb to distinguish between changes due to dysregulation at the protein\nand transcript level. We particularly focused on ClgR and the proteins it regulates including\nHsp20, ClpC1, ClpP1 and ClpP2. The new data and discussion for these experiments can be\nfound in Figure 5 of the revised manuscript and described from line 307, page 11.\npresence of 100 µM NRP. Timepoints taken at 0, 10, 20 and 60 min for -casein and PanD, 0, 60, 120\nand 180 min for Hsp20. e Quantification of in-vitro degradation assays. Data is presented as mean \nSD, n ≥ 3. Statistical significance between endpoints was calculated using two-tailed unpaired t-tests, *\n= p-value < 0.05, ** = p-value < 0.01 *** = p-value <0.001, **** p-value = < 0.0001. f Analytical size\nexclusion chromatography depicting oligomeric state of Hsp20 under degradation assay conditions.\nSource data are provided in Source data file.\nOverall this is an interesting article, but likely more fitting to publications dedicated to\nproteomics. A broad journal of excellence such as Nature Communications appears perhaps\nnot appropriate unless a detailed mechanistical analysis is performed on the ClpC1P1P2\ndifferent substrates.\nWe would like to express our great appreciation for the reviewer’s careful assessment and\nfeedback on our manuscript. We believe the additional experiments we have performed on the\nsuggestion of the reviewer have greatly improved the impact of our work, particularly\nsurrounding the influence of NRPs on the degradation on specific substrates; our paper now\ndescribes the first report of the effect of NRPs on bona fide Mtb ClpC1 substrates. We hope\nthat with these experiments and those suggested by reviewers 2-4, the reviewer is satisfied with\nthe importance of our work and its appropriateness for publication in Nature Communications.\nReviewer #2 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author)\nThe protein quality control system of mycobacteria is a promising and developing target for\nmycobacterial drug discovery, which can also be utilized for targeted protein degradation.\nThis manuscript explores the ClpC1:ClpP1P2 protease system in virulent M. tuberculosis,\nfocusing on the response to ClpC1 NPA targeted antibiotics - ecumicin, ilamycin, and\ncyclomarin using a proteomic approach. These studies show key differences in the effects of\nthe antibiotics studied despite their common targeting of ClpC1. The roles of ClpC2 and\nHsp20, which help provide compensatory and potentially protective effects to the differing\nagents, as highlighted in Figure 5, represent the most interesting aspect of this study. Other\nsignificant results include studies of the effects of NPA treatment on the abundance of\nproteins with unstructured domains and termini that are believed to be ClpC1 native\nsubstrates, and in the potentiation of the heat shock response.\nOverall, this is an excellent proteomics study, in an important topic area on the drug effects of\nClpC1 targeted antibiotics. The manuscript is well written and the supporting information\nadds to the scientific rigor of the reported study.\nWe thank the reviewers for their thoughtful and encouraging comments. We are grateful for\ntheir recognition of the importance our study, particularly regarding the differential effects of\nCpC1-targeted NPAs, the roles of ClpC2 and Hsp20, and the insights provided by our\nproteomic analyses.\nPrimary concerns:\n1. Methodological: the proteomic sampling is performed at a single time point after 48h of\ntreatment at the predetermined low OD MIC90 level of the respective drugs. This is too long\nto study mechanistic effects as compensatory effects will likely dominate the response.\nProteomic sampling should be performed in less than one doubling time (24h), and 3-6h is\nrecommended. The authors should also establish that there is not an inoculum effect at the\nhigher cell concentration used in the proteomics experiments that negates the drug action.\nWe thank the reviewer(s) for this thoughtful comment. We selected 48 hours to capture robust,\nproteome-wide changes and maintain consistency with the literature that had used the same\ntime point for cyclomarin studies (Cell, 2023, 2176). We initially assessed the impact of Ecu*\non the Mtb proteome at 16 hours (less than one doubling time) and observed minimal changes\nto the proteome. We have now included these data in Supplementary Figure 2 of the revised\nsubmission and inserted below. This aligns with previous reports indicating that effects become\npronounced only after one or more doubling times (J Med Chem, 2022, 4893; Tuberculosis,\n2025, 102594; Cell, 2023, 2176).\nRegarding the inoculum effect, which refers to the reduction in apparent antibiotic efficacy at\nhigher bacterial densities, we observed no increase in optical density between 16 and 48 hours\n(data now presented in Supplementary Figure 2 of the revised submission and inserted below),\nindicating that the effective drug to cell ratio is maintained and that classical inoculum effects\nare unlikely to influence the observed proteomic changes.\nH37Rv treated with MIC of 1 following a 16 h or a 48 h incubation, n ≥ 3. b change in optical\n90\ndensity at 600 nm following treatment at 16 h and 48 h. Data is presented as mean  SEM, n  3. c\nColony forming units (CFU) Mtb H37Rv cell viability assay was performed to determine lethality of a\ntreatment with the MIC of each of the NRPs 1-3 for 48 h. Data is presented as mean  SEM, n = 3.\n90\n2. The second concern relates to the limited scope of the study, which only uses one method\nof proteomic profiling. Adding complementary unbiased RNAseq or thermal proteome\n(CETSA) profiling experiments would strengthen the conclusions.\nWe thank the reviewer for this suggestion and agree that a complementary technique would\nprovide orthogonal validation of our proteomics data. We have now performed transcriptomic\nprofiling following Ecu* treatment. Substantial remodeling of the transcriptome was observed,\nwith enriched proteins showing greater overlap with upregulated transcripts, indicating that\nprotein depletion is largely post-translational, while enrichment reflects both transcriptional\nactivation and reduced turnover. Notably PQC related genes, including Hsp20, ClgR, ClpC1,\nClpP1 and ClpP2 were significantly upregulated at the transcript level, and this may explain\nthe mild enrichment of ClpC1, ClpP1 and ClpP2 in the proteomic dataset. The data for these\nexperiments can be found in Figure 5 (inserted below) and are described from line 307, page\n11. These results reinforce the added value of integrating RNA-seq with proteomics to dissect\nthe complex response of ClpC1 dysregulating NRPs.\ndots represent downregulated transcripts (< -1 log fold change, p-value < 0.05). Blue dots represent\n2\nupregulated transcripts (> 1 log fold change, p-value < 0.05). Clp proteins and highly upregulated\ntranscriptomic and proteomic datasets for Ecu* (1) treated Mtb, upregulated (top) or downregulated\n(bottom). Enriched or depleted transcripts/ proteins were those significantly dysregulated greater than\n± 1-fold. g Extracted normalised counts for the transcripts for specific PQC proteins. Data presented\nas mean  SD, n = 4 for each condition and data was analysed by two-tailed Wald test, **** = p-value\n< 0.0001. Source data are provided in Supplementary Data file 5.\n3. Due to its limited scope, this study does not adequately prove the author's conclusion that\nthese antimycobacterial drugs disrupt protein quality control beyond a single protein target.\nWe thank the reviewer for this comment. Based on the additional experiments suggested by all\nreviewers, including degradation assays with Mtb client proteins, RNA sequencing and Mtb\nknockdown strains, we have refined the key conclusions of the manuscript and have adjusted\nthe discussion and title of the manuscript accordingly. While the NRPs clearly modulate\nClpC1P1P2 activity, we now place less emphasis on broad, generalized disruption of protein\nquality control and instead highlight substrate and compound specific effects supported by both\nin vitro and in cell data.\nOther comments and suggestions:\n1. The authors use flattering terms like “cutting-edge” and “state of the art” to describe their\nmass-spectrometry proteomics, which is overstated as these methods are widely employed\nelsewhere.\nWe have adjusted the language accordingly throughout the manuscript.\n2. NPA drug stability after 48h should be confirmed under the incubation conditions.\nWe have now confirmed the stability of each of the NRPs under incubation conditions, and the\nexperimental data can be found in Supplementary Figure 2d in the revised submission and\nincluded below:\nµM) in Middlebrook 7H9 media containing 0.2% glycerol at 37 °C. Samples were analysed\ndirectly by UPLC-MS (0-100% MeCN in H O over 5 min with 0.1 vol.% FA). a and b denote\nconditions.\n3. Incorporating a protease ClpP1P2 inhibitor control would be a useful comparator in this\nstudy to help decipher the mechanistic effects that are ClpC1-specific.\nThis is a great suggestion. We have now performed LC MS/MS based proteomics on Mtb\nH37Rv treated with well documented protease inhibitor bortezomib (BTZ). As this experiment\nhad not been performed previously in the literature, we chose to analyze the effects BTZ on the\nproteome of Mtb at two timepoints, 16 h and 48 h and compared both to the NRP datasets.\nInterestingly, BTZ treatment led to dramatic effects on the Mtb proteome, but these were quite\ndifferent to the changes induced by the ClpC1-targeting NRPs. The data for this experiment\ncan be found in Figure 2 (provided below), Supplementary Figures 4, 5 and 9) in the revised\nsubmission. The description of the results can be found under BTZ inhibits protein degradation\nin Mtb, line 167, page 6 and within the discussion section.\n4). Minimum inhibitory concentration (MIC) to inhibit 50% of bacteria (MIC ) indicated below\n50\nstructure. Raw data from the resazurin MIC assay found in Supplementary Fig. 1d and source data\nprovided in Source data file. b Schematic of ClpC1P1P2 complex with compound target site indicated,\nNRPs bind to the NTD of ClpC1 and BTZ (4) interacts with the protease. c Volcano plots representing\nthe effects of BTZ (4) on the proteome of Mtb H37Rv (top = 16 h incubation, bottom = 48 h incubation).\nStatistical significance was calculated with a two-way ANOVA (* = p-value < 0.05). Red dots represent\ndepleted proteins (< -0.5 log fold change, p-value < 0.05). Blue dots represent enriched proteins (> 0.5\n2\nlog fold change, p-value < 0.05). Clp proteins and highly upregulated Hsp20 and ClgR are labelled. 16\n2\nh dataset includes n = 4 biological replicates and 48 h includes n= 3 biological replicates. d, e Venn\ndiagrams showing number of similarly or uniquely affected proteins, depleted (left) or enriched (right)\n11\nbetween NRPs (1-3) and BTZ (4) 16 h (d) and BTZ (4) 48 h (e). f, g Raw abundance of ClpC1 substrates\n(f) and 20S proteosome substrates (g) following BTZ (4) treatment. Data is presented as mean  SEM,\n16 h n = 4, 48 h n= 3, * = p-value < 0.05, ** = p-value < 0.01 *** = p-value <0.001, **** p-value = <\n0.0001. Source Data are provided in Supplementary Data 4.\n4. Conclusions summary: given that the three inhibitors lack cross-resistance, is it surprising\nthey have differential impacts?\nWe thank the reviewer for this comment. We believed it was important to comment that subtle\ndifferences in the precise positioning and contacts can markedly influence how ClpC1 engages\nits substrates, highlighting that even closely related compounds can exert distinct effects\nthrough the same binding interface.\nThe statement in our originally submitted manuscript was incorrect; mutations at F80 and V13\nconfer resistance to more than one natural product (see Supplementary Figure 1a). We have\namended this in the revised manuscript to:\n“There is considerable ClpC1-interface overlap between the three compounds and\nmutagenesis studies have revealed mutations at F80 and V13 confer resistance to more than\none natural product. (Figure 1b, Supplementary Figure 1a).”\n5. The final stated conclusion “we expect that the findings of this study, combined with more\ndetailed structural data in the future, will greatly facilitate the rational design of next generation\npotent ClpC1 and PQC-targeting TB drug leads.” How? It is unclear to this reviewer how this\nstudy would aid further drug development; for instance, have any new vulnerabilities been\nrevealed?\nWe would like to thank the reviewer for raising this. As a result of the new data that we have\nincluded based on the suggestions from the four reviewers we have added additional\nclarification to our concluding paragraph. Specifically, based on our data it is unlikely that\nEcu* and IlaE are sequestered by ClpC2 (unlike dCym). This reveals that not all NRPs are\nsusceptible to inbuilt Mtb protective mechanisms and positions ecumicin and ilamycins as\npromising molecular scaffolds for rational design of ClpC1-targeting drug candidates,\nincluding BacPROTAC molecules. The new sentence in the concluding paragraph is\nprovided below:\n“Indeed, we show that Ecu* (1) and IlaE (2) cause extensive proteome dysregulation at\nsublethal doses but do not engage the ClpC2 rescue mechanism that reduces dCym (3) toxicity.\nThe ability of ecumicin and the ilamycins to overcome ClpC2 rescue, that would serve to reduce\ntheir antimycobacterial activity, makes these molecules promising molecular scaffolds for\nrational drug design of ClpC1-targeting ligands and as ClpC1 recruiters for next-generation\nBacPROTACs. Taken together, the distinct interactions, combined with the differential\ndysregulation of ClpC1P1P2 proteolysis, highlight the multifaceted ways NRPs perturb\nproteostasis. Given the essentiality of ClpC1 and protein quality control in Mtb, these findings\nreinforce the potential to exploit stress-adaptive pathways to enhance antimycobacterial\nactivity and provide the foundation for the rational design of next-generation ClpC1- and PQC-\ntargeting TB drug candidates.”\n6. Materials and methods – Mtb growth conditions: please report oxygen and carbon dioxide\nlevels of culture.\nincluded and highlighted in the Materials and Methods section of the manuscript (line 461,\nPage 15)\n7. Materials and methods: cell viability assay; please report the time, drug concentration, and\ncellular density at which the Mtb-treated samples were prepared.\nWe have now included these important details in the Methods of the revised submission (line\n521, page 17).\nReviewer #4 (Remarks to the Author):\nTuberculosis (TB) remains one of the leading causes of death from infection worldwide. The\nhigh virulence of Mycobacterium tuberculosis (Mtb) is partly due to its ability to survive and\nreplicate within the alveolar macrophages, establishing reservoirs of live bacteria that\npromote the persistence and recurrence of the disease. An estimated one-quarter of the global\npopulation harbours a latent TB infection (LTBI)2, which, although asymptomatic, represents\na reservoir for the potential reactivation and transmission of TB. Additionally, there is an\nincreasing concern regarding the number of the multidrug resistant cases. Bater et al., have\ntested several antimycobacterial cyclic peptide natural products that bind the ClpC1\nchaperone component of the system and claimed that the have employed cutting edge mass-\nspectrometry-based proteomics to determine the effects of NPAs on virulent Mtb H37Rv.\nThe study reports 3, 175 protein IDS of hundreds of proteins were differentially abundant\nwith different NPA treatment. The authors claimed that “this study provides a deeper\nunderstanding of the distinct mechanisms of antimycobacterial activity of different NPAs\nthrough dysregulation of the ClpC1:ClP1P2 proteolysis system. However as is stands the\nmanuscript is merely descriptive and the results lacks orthogonal validation.\nWhile our original manuscript relied primarily on proteomic profiling, we have now\nincorporated multiple orthogonal approaches to strengthen mechanistic insights, including\nClpC1 degradation assays with client proteins, RNAseq analysis and comparisons of the effects\nof the NRPs on the Mtb proteome with the protease inhibitor BTZ. Collectively these\nexperiments provide evidence that the observed effects of NRPs on ClpC1 are substrate- and\ncompound-specific. We have amended the text in the revised manuscript to include these\nvalidation experiments meaning that our discussion is now less ‘descriptive’ than the original\nsubmission.\nPoint 1:\nAccording to the authors, NPAs are known to target a major chaperoning element in the\nClpC1-P1P2 protein degradation mechanism. However, there is no evidence in this work\nshowing that NPAs interact with the ClpC1-P1P2 protein system; rather, the authors\nrefer/rely on changes in protein abundance. The authors must offer supporting evidence that\nthe changes observed in the Mtb proteome are the result of NPA interactions with the ClpC1-\nP1P2 protein system, rather than a general reaction to a foreign peptide. For this, I\nrecommend that the authors use current techniques like cross-linking mass spectrometry to\ndemonstrate that NPAs bind to the complex. The amount of peptide-protein interaction is\nthen assessed using molecular docking simulation.\ndegradation assays using model substrate β-casein and genuine Mtb ClpC1P1P2 substrates\nPanD and Hsp20. These assays demonstrate that NRPs differentially modulate ClpC1P1P2\ndependent proteolysis in a substrate-specific manner (line 227, page 8). In addition, we used\nbortezomib as a comparator to distinguish ClpC1-specific effects, further supporting that the\nproteomic changes observed in cells arise from targeted modulation of ClpC1P1P2 system\nrather than inhibition of other cellular pathways (line 167, page 6).\nThe interaction of the NRPs with ClpC1 is also well established in the literature through\nbiochemical, biophysical and structural studies (Acta Crystallogr D Struct Biol, 2020, 458;\nACS Infect Dis, 2019, 829; Cell, 2023, 2176; Antimicrob Agents Chemother, 2019, e02317;\nCell Chem Biol, 2019, 1169; Commun Biol, 2023, 301), reinforcing that ClpC1 targeting is the\nprimary mechanism of action.\nThe study lacks supportive functional assays that aid in understanding not only the mechanism\nof action but also the extent of NPA in Mtb viability. For example, it has been demonstrated\nthat CLpC2 protects against CymA-induced toxicity; it would be interesting to know how NPA\ntreatment interferes with such a protective model system or simply compare the presence of\nNPA to the clpC2 knockout strain.\nWe thank the reviewer for this great suggestion. As part of this revision we generated a ClpC2\nknockdown strain of Mtb H37Rv and assessed susceptibility to each NPA. Consistent with\nprevious studies, susceptibility to dCym was significantly increased in the ClpC2 knockdown.\nNotably, this is the first report testing Ecu* and IlaE in the context of ClpC2 depletion, and we\nfound their activity was unaffected, consistent with the lack of ClpC2 enrichment observed in\nour proteomic analysis. These data are now presented in Figure 4 of the revised manuscript\n(see below) and associated text can be found under compound specific modulation of ClpC2\nand impacts on NRP toxicity, line 259, page 9.\nFigure 4| e Relative mRNA levels of ClpC2 knockdown in Mtb mc26026 determined by qPCR and\nprotein abundance determined by LC-MS/MS based proteomics. The control strain contained non-\ntargeting sgRNA. Data is presented as mean  SD and each dot represents an individual biological\nreplicate, n = 3. Statistical significance was calculated with two-tailed unpaired t-tests, ** = p-value <\n14\n0.01. f Growth inhibition curves form resazurin cell viability assay comparing NRP activity in control\nand ClpC2 knockdown Mtb mc26026 strains. Data presented as mean  SD from one biological\nreplicate with three technical replicates. g IC quantification from resazurin cell viability assays. Data\n50\nis presented as mean  SEM, dots represent data from three individual biological replicates, n  3 and\nanalysed by two-tailed unpaired t-test on log-transformed data. Source data for all replicates are\nprovided in Source data file.\n15"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have clearly made a significant effort to improve the manuscript, and there is no question about their commitment to addressing the previous concerns. However, we still find that the overall message of the paper is not sufficiently strong for publication in Nature Communications, although we believe this is an interesting topic of research. That said, we also believe it is not the reviewer’s role to impose unrealistic demands on a complex topic, particularly when doing so could unnecessarily impact the careers of postdoctoral researchers or PhD students involved in the work – and we are aware it was a massive amount of work. In our view, the study appears to have been carefully executed, but the data are inherently complex, making it difficult to draw clear and definitive conclusions.\nOverall, we acknowledge the authors’ efforts to improve the article — to do better science — which, in our view, should be the main goal of any review process. Below, we outline several points that we believe will improve the manuscript if addressed before publication:\nThe Kd of the Ecu*, corrected by the authors here, has been also reported by the Clausen group to be 8 µM (one-to-one stoichiometry) using also SPR (see Hoi Cell 2023) - Dr. Richard Payne, the senior author, is also an author in that publication. Should we assume the data there was incorrect - is that true? Maybe you should refer to this fact somewhere in the manuscript as other researchers will be misguided by the conflicting results between publications.\nPage 3\nThe authors describe the ClpC1P2P1 system but rely on general references that are not specific to this complex. In addition, several recent structural studies on the ClpC1P2P1 system are omitted – including structures of ClpP1P2, ClpC1, and two recent publications on the ClpC1P2P1 complex. These should be properly cited and discussed.\nPlease verify whether reference 15 is correct and appropriate in the context where ClpP1 and ClpP2 association for protein hydrolysis is discussed. It would be more relevant to cite the ClpP1P2 structural studies from the Sauer and Goldberg groups.\nPage 4, line 76\nThe authors should mention lassomycin, as it is another well-characterized ClpC1-NTD binding natural product.\nPage 5 line 124\ndCym was not first described by Hoy et al. – but by Barbie, P. and Kazmaier in 2016.\nMechanistic discussion\nThere are fundamental differences in the in vitro mechanisms of ecumicin, cyclomarin, and rufomycin when tested with purified proteins. Notably, cyclomarin has not been described as a strong ClpC1P2P1 inhibitor; in fact, prior studies suggest that it activates the ClpC1P1P2 complex (see Mogk Heidelberg group on cyclomarin publications). This distinction should be discussed.\nPage 8, line 209\nThe statement that IDPs are structurally similar to unfolded substrates is inaccurate. IDPs are typically stable and functional, whereas unfolded substrates are often prone to aggregation. This section should be clarified to avoid conceptual confusion.\nPage 8, line 232\nThe manuscript states that bortezomib has no effect on in vitro degradation of casein by the ClpC1P2P1 complex, using a concentration of 100 µM. This seems inconsistent and potentially confusing. 100 µM is an extremely high concentration; please justify this choice and, by the way, the reference cited shows that bortezomib activates protein degradation at lower concentrations than 100 µM. Of note, the effect of the active site activators is also well described in the literature as a bell-shaped curve – that is, activation at low concentrations but inhibition at higher concentrations – please use the citations in the proper context.\nIn addition, why was 100 µM used in vitro while 5 µM? MIC50 or MIC90 was used in vitro? Normally, in vitro assays require lower concentrations to achieve target engagement. Related to this, please clarify what were the concentrations used in the proteomics experiments? Why put the bortezomib MIC50 in the figure when you used the MIC90 in the experiment (was 100 µM the MIC90?)\nAdditionally, how were the in vitro degradation experiments performed without a ClpP1P2 activator? You observed activity in the absence of any ClpP1P2 activator? It has been reported by several groups that the ClpP1P2 is not active in the absence of active site activators. Can you explain this?\nPage 10, line 281\nThe authors state that Hsp20 is specifically enriched by NRP action, yet a similar enrichment is observed with bortezomib – a compound that does not bind to ClpC1 and preferentially targets the M. tuberculosis proteasome rather than ClpP1P2. This raises concerns about the specificity of the reported effect. The data appear to contradict the claim of selective enrichment – likely any stress in protein quality control will lead to an Hsp20 increase.\nOf note, the authors choose not to test the binding of the compounds to other chaperones as we suggested before, we do understand that – but we do believe you would likely get binding as well – but we agree this is not a key issue for review.\nHeat stress section\nThe discussion of heat stress is unclear. Are the authors suggesting that heat stress potentiates the effects of drugs acting on protein quality control? If so, this concept is already well established in the literature and does not constitute a novel finding. The claims in this section should be moderated accordingly, as the data is largely confirmatory.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have done an excellent job responding to the prior critique. No further changes are suggested.", "reviewer_4": "The authors have addressed my previous comments and have now submitted an improved version of the manuscript."}, "author_response": "We thank the reviewer for recognizing the effort we have invested in addressing the previous comments in our revised the manuscript and that the work has been carefully executed.\nWhile we agree that the area of mycobacterial protein degradation is complex, our work uncovers several pivotal discoveries that open new research pathways in mycobacterial biology and provides a foundation for new TB drug discovery efforts. Below we have summarised four key findings as exemplars to emphasise the importance and impact of our work:\n1. ClpC1-binding natural products exert distinct effects on the proteome of virulent Mtb, despite overlapping binding interfaces.\n2. We demonstrate that, unlike dCym, Ecu* and IlaE do not trigger the ClpC2-mediated ClpC1-rescue mechanism. This was confirmed through binding studies, proteomic profiling, and inhibitory activity evaluation in a Mtb ClpC2 knockdown strain.\n3. We show that Ecu* and IlaE each trigger an increase in small heat shock protein Hsp20 in Mtb cells (32-95-fold increase over controls). While bortezomib also triggers Hsp20 production, this molecule broadly inhibits protein degradation in Mtb cells and triggers a complete, coordinated protein quality control response.\n4. We demonstrate that ecumicin binds to Hsp20, a non-Clp chaperone, in addition to ClpC1.\nThe K of the Ecu*, corrected by the authors here, has been also reported by the Clausen group\nD\nto be 8 µM (one-to-one stoichiometry) using also SPR (see Hoi Cell 2023) - Dr. Richard Payne,\nthe senior author, is also an author in that publication. Should we assume the data there was\nincorrect - is that true? Maybe you should refer to this fact somewhere in the manuscript as\nother researchers will be misguided by the conflicting results between publications.\nSince publication of the Hoi et al. Cell paper we have optimised the protein loading and buffers\nused in SPR experiments with the NTD of ClpC1 (Hawkins et al. ACS Infect. Dis. 2025, 3298).\nIt is known that the protein loading (NTA chip with a His-tagged protein in the Hoi paper vs\nimmobilisation on a CM5 chip in our work) can influence binding SPR (Capelli et al. Trends\nAnal. Chem. 2023, 117079). We believe our optimised SPR conditions are the most appropriate\nfor measuring affinities of the NRP natural products and their analogues. As suggested by the\nreviewer, we have added additional information into the revised manuscript to clarify this for\nthe reader (page 10, line 272). We also note that our decision to model 2:1 binding\nstoichiometry for the SPR analysis was based on crystallographic data showing 2:1 binding for\necumicin (Wolf et al. Acta Crystallogr. D Struct. Biol. 2020, 76:458–471). That being said, the\nK for 2:1 binding (K = 0.39 µM) and for 1:1 binding (K = 0.39 µM) were identical. We\nD D1 D\nhave added this information into the revised manuscript and have also added the sensorgram\nfor the 1:1 binding into the revised Supplementary Information file for completeness.\nPage 3\nThe authors describe the ClpC1P2P1 system but rely on general references that are not specific\nto this complex. In addition, several recent structural studies on the ClpC1P2P1 system are\nomitted – including structures of ClpP1P2, ClpC1, and two recent publications on the\nClpC1P2P1 complex. These should be properly cited and discussed.\nPlease verify whether reference 15 is correct and appropriate in the context where ClpP1 and\nClpP2 association for protein hydrolysis is discussed. It would be more relevant to cite the\nClpP1P2 structural studies from the Sauer and Goldberg groups.\nWe thank the reviewers for pointing this out. We have now cited recent structural studies on\nMtb ClpP1P2, ClpC1, and the ClpC1P2P1 complex (Weinhäupl, Fraga and coworkers (2025)\nbioRxiv and J. Biol. Chem. 2022, 102553). At the reviewer’s request we have included\npublications from the Sauer and Goldberg groups (Schmitz et al. PNAS 2014, 111, E4587, Li\net al. J. Biol. Chem. 2016, 7465) when discussing ClpP1 and ClpP2 association and hydrolysis.\nPage 4, line 76\nThe authors should mention lassomycin, as it is another well-characterized ClpC1-NTD\nbinding natural product.\nDuring our initial revision we opted to remove lassomycin to focus on the non-ribosomal\npeptide (NRP) molecules that were the focus of the study. Nonetheless, we agree that it is an\nimportant member of the ClpC1-targeting natural product family and so have reinstated\nlassomycin back into the manuscript on page 4 line 76.\nPage 5 line 124\ndCym was not first described by Hoy et al. – but by Barbie, P. and Kazmaier in 2016.\nWe have corrected the reference accordingly on page 5 line 125\nMechanistic discussion\nThere are fundamental differences in the in vitro mechanisms of ecumicin, cyclomarin, and\nrufomycin when tested with purified proteins. Notably, cyclomarin has not been described as\na strong ClpC1P2P1 inhibitor; in fact, prior studies suggest that it activates the ClpC1P1P2\ncomplex (see Mogk Heidelberg group on cyclomarin publications). This distinction should be\ndiscussed.\nThank you for raising this important point. The literature on cyclomarin is indeed complex,\nwith some studies showing activation of the ClpC1P1P2 complex in vitro rather than inhibition.\nHowever, direct comparisons between studies are challenging due to differences in assay\ndesign, substrates, ClpC constructs, compound, and compound concentrations, which vary\nbetween our study and those by the Mogk group (Maurer et al. Cell Chem. Biol. 2019, 26:\n1169-1179, Taylor et al. J. Biol. Chem. 2022, 102202). We have included this in our discussion\nin the revised manuscript on page 13, line 396.\nPage 8, line 209\nThe statement that IDPs are structurally similar to unfolded substrates is inaccurate. IDPs are\ntypically stable and functional, whereas unfolded substrates are often prone to aggregation.\nThis section should be clarified to avoid conceptual confusion.\nThank you for highlighting this distinction. We have revised the sentence to accurately reflect\nthe differences between intrinsically disordered proteins (IDPs) and unfolded substrates (page\n8, line 210).\nPage 8, line 232\nThe manuscript states that bortezomib has no effect on in vitro degradation of casein by the\nClpC1P2P1 complex, using a concentration of 100 µM. This seems inconsistent and potentially\nconfusing. 100 µM is an extremely high concentration; please justify this choice and, by the\nway, the reference cited shows that bortezomib activates protein degradation at lower\nconcentrations than 100 µM. Of note, the effect of the active site activators is also well\ndescribed in the literature as a bell-shaped curve – that is, activation at low concentrations but\ninhibition at higher concentrations – please use the citations in the proper context.\nIn addition, why was 100 µM used in vitro while 5 µM? MIC50 or MIC90 was used in vitro?\nNormally, in vitro assays require lower concentrations to achieve target engagement. Related\nto this, please clarify what were the concentrations used in the proteomics experiments? Why\nput the bortezomib MIC50 in the figure when you used the MIC90 in the experiment (was 100\nµM the MIC90?)\nAdditionally, how were the in vitro degradation experiments performed without a ClpP1P2\nactivator? You observed activity in the absence of any ClpP1P2 activator? It has been reported\nby several groups that the ClpP1P2 is not active in the absence of active site activators. Can\nyou explain this?\nWe acknowledge that 100 μM BTZ is a high concentration and have noted this in the revised\nmanuscript (page 8, line 234). This choice was based on our standard screening approach and\nthe expectation that higher concentrations might reveal inhibitory effects, as noted regarding\nbell-shaped activator profiles. The Zhou et al. (Nat. Commun. 2025, 16:3466) study was\npublished after our experimental design. However, their data show only mild activation (10–\n20%) of FITC-casein degradation at 80–160 μM. Given our endpoint-based assay lacks\ncontinuous monitoring and fluorescent reporters, modest activation (expected 10– 20%) would\nlikely remain undetected. Therefore, we consider that our data are consistent with the cited\nstudy.\nConcentrations of all compounds used in proteomic experiments have now been reported more\nexplicitly in the revised manuscript (page 21, line 673). MIC and MIC values for all\n50 90\ncompounds were reported in the Supplementary Information file.\nRegarding the requirement for ClpP1P2 activators, while early studies suggested that synthetic\nN-blocked dipeptide activators (e.g., Z-Leu-Leu) were necessary for ClpP1P2 activity,\nsubsequent work has demonstrated that this is not the case when AAA⁺ partners and substrates\nare present (Leodolter et al. PloS One 2025, 10:e0125345, Schmitz and Sauer Mol. Microbiol.\n2014, 93:617-628). Consistent with this, studies from our laboratories have routinely\nperformed degradation assays without activators and observed robust activity (Hoi et al. Cell\n2023, 186: 2176–2192; Morreale et al. Cell 2022, 185: 2338–2353).\nPage 10, line 281\nThe authors state that Hsp20 is specifically enriched by NRP action, yet a similar enrichment\nis observed with bortezomib – a compound that does not bind to ClpC1 and preferentially\ntargets the M. tuberculosis proteasome rather than ClpP1P2. This raises concerns about the\nspecificity of the reported effect. The data appear to contradict the claim of selective enrichment\n– likely any stress in protein quality control will lead to an Hsp20 increase.\nOf note, the authors choose not to test the binding of the compounds to other chaperones as we\nsuggested before, we do understand that – but we do believe you would likely get binding as\nwell – but we agree this is not a key issue for review.\nWe have clarified this in the discussion (page 10, line 288) and avoided referring to this effect\nas selective. While BTZ also induces Hsp20, it does so as part of a broad PQC response\ninvolving all major chaperones and proteases. In contrast, NRPs trigger what we had previously\ncoined selective changes, with convergence only at Hsp20 for Ecu* and IlaE; while dCym does\nnot induce Hsp20. Thus, Hsp20 upregulation is not universal and its strong induction by\nEcu*—comparable to BTZ despite the absence of a broader PQC response—remains\nnoteworthy.\nRegarding the reviewer’s comment on the potential interaction of NRPs with other chaperones,\nwe agree that it represents an exciting avenue for future investigation, and we have mentioned\nthis in the discussion (page 15, Line 454).\nHeat stress section\nThe discussion of heat stress is unclear. Are the authors suggesting that heat stress potentiates\nthe effects of drugs acting on protein quality control? If so, this concept is already well\nestablished in the literature and does not constitute a novel finding. The claims in this section\nshould be moderated accordingly, as the data is largely confirmatory.\nWe have moderated the language in this section to clarify that our findings are confirmatory\nand consistent with prior literature. (Page 12, Line 348, Page 12, Line 363)."}]

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