Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid–liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

that it undergoes phase transition and participate in stress granules at least in Hela cells.The authors should attempt similar set of experiements (modulation of PPIA activity, assess dynamics of stress granules formation and resistance to oxidative stress) in HPSCs.In addition they should show that reintroduction of the chaperone rescues stress granule formation in aged HSPCs.Such rescue experiments should be accompanied by functional evaluation of aged HSPCs in transplantation assays to really determine how much the axis PPIA-IDR-rich proteins-effective phase separation contributes to age-associated dysfunctions of HSPCs.
Reviewer #2 (Remarks to the Author): The manuscript by Maneix et al. studied the role of Cyclophilin A (PPIA) in haematopoietic stem cell aging.The authors carried out transplantation assays in mouse models to describe premature aging phenotype upon PPIA loss.Using interaction proteomics, the authors defined PPIA target proteins and observed an enrichment of these to contain intrinsically disordered regions (IDRs).PPIA knock-down caused decreased translation and PPIA modulation affected stress granule formation.Finally, proteomics analyses revealed a correlation for PPIA and IDR in young and old HSCs.
The study addresses an interesting topic and the need to better define the role of PPIA in controlling proteostasis, IDR-containing proteins and a possible involvement in cell ageing.In the current state, the manuscript seems premature in that a number of crucial conclusions, which are critical for the logical flow of the manuscript, lack statistical rigor and necessary validation.
Major points: 1) The evidence for PPIA-mediated premature ageing is somewhat circumstantial.From the manuscript, it remains unclear how loss of PPIA shapes the cellular proteome and proteostasis.Consequently, a number of cellular pathways may be responsible for the observed effects.A more rigorous characterization of cellular changes, including classical molecular aging markers is necessary to claim the connection between PPIA and haematopoietic aging.Do PPIA levels and the specified interactions partners change when comparing old versus young HSPCs?2) Several key conclusions drawn are based on various omics data, including proteome, interactome, translatome and transcriptome changes.However, a number of these experiments lack a sufficient number of replicates to draw any conclusions with statistical significance.These design limitation in combination with a general lack of data availability (raw data, continuous quantification and statistical significance data) make it difficult to agree with the claims.Especially in that light, validation of the key claims by orthogonal methods is required.All key experiments that have not been validated orthogonally need to be carried out with at least three independent biological replicates.
3) Does PPIA loss affect translation via known cellular translation pathways regulated under stress and hence the observed reduced translation rate?4) Figure 3b should be repeated including the G104A mutant to validate the findings.5) Figure 3c, to confirm the interaction, at least one of the bait and prey should be monitored at the endogenous level without overexpression.6) The number of replicates need to be stated clearly, ideally in the experimental model or figure legend.Implying n=3 when no statement can be found is not sufficient.Drawing significance from distribution (violin) plots when there are not at least 3 biological replicates is not appropriate.Consequently, these data sets are not sufficient as single supportive evidence for major claims.7) Among the identified PPIA target proteins are a number of mitochondrial proteins (Figure 2c).Does loss of PPIA cause mitochondrial dysfunction and does that alter translation and the stem cell ageing phenotype?8) A more unbiased representation of data is prudent to assess their quality.How do interactions change across all protein in the WT versus PPIA mutant comparison?How were interactors selected?For the translatome (Fig. 2f), also heat plots per replicate should be shown.A bit more insight into which proteins go up/down would be interesting.The percentage of heavy incorporated is very low, additional validation that there is still sufficient dynamic range to monitor dynamics would be important.The transcriptome data set is quite useful and more data should be shown (e.g.quality control/correlation across replicates, some clustering or pathway analyes).9) The claim that PPIA and PABPC1 regulate phase separation needs further examination.Reduction of protein translation, as is observed upon PPIA loss, is sufficient to induce stress granules.While these are phase separated, it is not clear that PPIA and PABPC1 drive the physical aspects of phase separation.How are IDR and other target proteins changed upon rescue of PPIA?Minor points 10) PPIA Kd2 and Kd1 (293T) show major difference in expression pattern (Figure 2f).Comment on 'Cyclophilin A supports translation of intrinsically disordered proteins and mitigates haematopoietic stem cell aging' by Maneix et al.In this paper, the authors have identified Peptidyl-3 Prolyl Isomerase A (PPIA or Cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells (HSPC) as it accounts for 14% of the cytosolic proteome and its mRNA accounts for over 0.5% of all mRNAs of HSPC.Total knockout of PPIA leads to 1) myeloid skewing, an immunophenotypic but not functional increase in stem cells, and 2) impaired self-renewal with 3) accelerated exhaustion, which are three hallmarks of haematopoietic aging.Using an assay targetting differential co-immunoprecipitation between the wild-type PPIA and a G104A mutant PPIA, the authors revealed approximately 400 substrates of the wild-type enzyme.Since they performed the co-immunoprecipitation in the cytosolic cell fraction, these results suggest PPIA and substrates interact between translation and nuclear translocation.When compared to the global proteome, immunoprecipitated PPIA substrates feature higher levels of IDRs.
By surveying proline residues within PDB, the authors found that the majority of cis-prolines located in bends and turns.Their conclusion is that "PPIA and related enzymes predominantly isomerize prolines within unstructured protein regions."This conclusion is incorrect as the bends and turns are categorized as structured regions that connecting helices and beta-strands."Unstructured protein regions" are regions of proteins that they donot have stable (i.e.highly populated) conformations and are "invisible" in PDB.Further studies are required to justify this conclusion.
Using pulsed SILAC in HeLa and 293T cells with either normal or reduced levels of PPIA, the authors found that loss of PPIA significantly reduced expression of the whole proteome, PPIA clients and nonclients, in both cell types.As far as the levels of PPIA target proteins are concerned, they were lower than for other proteins and further reduced when PPIA was knockdown.In addition, the fraction of protein IDRs in the HSPC proteome of PPIA knockout animals was significantly reduced when compared with heterozygous siblings.
Largely based on these aforementioned observations, the authors claim that PPIA supports de novo translation of its target proteins.Unfortunately, this claim is not even close to be supported by the data reported.
The authors went on to confirmed a few clients of PPIA using a combination of IP and western blotting.One of the confirmed clients is PABPC1."Following PPIA knockdown, expression of PABPC1 protein was reduced by 20-30% in pulsed SILAC experiments, suggesting the chaperone engages with PABPC1 during translation."This conclusion is questionable as the pulse was 24 hours.Many other mechanisms other than, or in addition to, limited translation can account for the 20-30% reduction.
Next, the authors turned their attention to liquid-liquid phase separation (LLPS), a biophysical property of multivalent proteins including many IDR-containing ones.Essentially the authors showed that PPIA KD perturbed condensates of PABPC1-GFP (I have a hard time to figure whether PABPC1-GFP is transiently expressed or is expressed at endogenous levels) and the formation of stress granules marked by G3BP1 upon the treatment with sodium arsenite.It is unclear whether the reduced translation of PPIA's clients or the lack of PPIA activity per se was claimed to cause the perturbation to condensates of PABPC1-GFP or stress granule formation.Nevertheless LLPS is just one aspect of the global proteome Overall, the authors didn't make sufficient effort to connect pieces of observations for a coherent model explaining the role of PPIA in ageing of haematopoietic stem cells.I can't recommend its further consideration for pulication in Nature Cell Biology.
To help them improving their study for future submission, I listed some suggestions below: Major issues 1)When doing differential analysis of proteome to assess the effects of PPIA on IDR-enriched clients, the authors shall divide PPIA clients and nonclients into IDR-containing, IDR-noncontaining, four categories.
2)Genetic manipulation or pharmacological perturbation for days are rather slow to assess acute and direct functions.I suggest the authors to use an AID system or PROTAC system (if available) to chemically KD PPIA and assess whether the reported effects are direct or indirect.
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We thank the reviewers for their time and expertise in critiquing our initial manuscript.The insightful feedback has greatly assisted us in improving the robustness and clarity of our work.We are confident that addressing the highlighted limitations resulted in significant enhancements to our manuscript.To maintain transparency, responses to critiques and subsequent amendments to the manuscript are in blue font.

Reviewer #1 (Remarks to the Author):
The study from Maneix et al. aims to elucidate the role of PPIA as a co-translational chaperone in HSPCs and to identify PPIA loss as a driver of hematopoietic stem cell aging.The authors performed proteomic analysis to show that proteins with intrinsically disordered regions (IDR) are PPIA substrates and are mainly involved in liquid-liquid phase separation and the formation of membrane-less complexes with other proteins or nucleic acids.Interestingly, they found that HSPC aging (in mice) associates with decreased PPIA protein level and reduced synthesis of proteins with IDR, likely leading to reduced stress resistance and stem cell dysfunctions The main findings of the study are interesting, the topic is quite novel and the data are well-presented.
We thank reviewer #1 for his/her positive assessment, who clearly understood the novelty and importance of our study to the field.However, some of the conclusions with regard to the role of PPIA-proteins with IDR in the aging phenotype of hematopoietic stem cells are not fully supported by the data.Specifically, the molecular link between PPIA depletion and the aging phenotype through loss of liquid-liquid phase separation remains weak.My main concern is the lack of a functional interplay between loss of PPIA, loss of IDR proteins and the impact that this unbalanced cellular proteome may have on the self-renewal and transplantation fitness of murine HSPC.Similarly, whether restoration of PPIA function and increased synthesis of proteins with IDR and phase separation are sufficient to rescue age-associated dysfunctions of hematopoiesis remains to be elucidated.
We concur with reviewer #1's concerns about the molecular link between PPIA depletion, the aging phenotype, and the loss of liquid-liquid phase separation.To address these concerns, we have performed additional experiments to provide compelling evidence for the functional interplay between loss of PPIA, loss of intrinsically disordered proteins (IDPs), and the impact on self-renewal and transplantation fitness of murine HSPCs.The new data and analyses are summarized below:

Functional interplay between loss of PPIA and HSPC fitness:
To demonstrate the functional interplay, we have performed critical rescue experiments to evaluate the effect of PPIA on self-renewal and transplantation fitness in murine haematopoietic stem and progenitor cells (new Fig. 2a-d).Mouse PPIA was introduced using a lentiviral construct in aged stem and progenitor cells, which significantly improved reconstitution following competitive transplantation.We followed these animals for >6 months, which indicates that the enhanced phenotype is contributed by the long-lived stem cell pool.We further verified that the PPIA protein is increased in the blood of animals after receiving "rescued" cells and that the resulting improved haematopoiesis is not skewed towards myeloid lineages.

Functional interplay between PPIA and IDR protein expression:
PPIA is a widely expressed chaperone with a broad substrate base.While PPIA recognizes many cellular proteins with a larger fraction of disordered regions on average, it is not exclusive for disordered proteins.We, therefore, used the term "selective" instead of "specific" in our manuscript.Nonetheless, to strengthen the evidence for the interaction between PPIA and substrate proteins, which contain many well-recognized and important players in liquid-liquid phase separation, we investigated the expression and function of the stress granule protein PABPC1, the nucleolar organizer NPM1, and the P-body protein DDX6.We found that depletion of PPIA leads to reduced expression of these three essential proteins (new Fig. 4g and Extended Data Fig. 3b).Further, to substantiate that PPIA directly interacts with these three proteins, we performed coimmunoprecipitation with either wildtype PPIA or the binding-impaired G104A mutant [1].Consistent with our previous proteomic studies, we found reduced levels of PABPC1, NPM1, and DDX6 when performing immunoprecipitation with the mutant PPIA (new Fig. 4f).The dependence of substrate expression on PPIA was also noted by the group of Dr. Zweckstetter [2].Importantly, reduced levels of PPIA substrates in PPIAdeficient cells are not driven by transcription (new Extended Data Fig. 3f).
The original proteomic analysis by 2D gel electrophoresis and mass spec was performed in murine HSPCs that is notoriously a very heterogeneous cell population encompassing multipotent progenitors, committed progenitors and a rare subset of long-term HSCs.Validation of (some) of the identified targets in Fig. 1 should be performed across HSPC subsets and in a sorted subset of most primitive HSCs.
We acknowledge the potential limitations of using bulk murine cells for our proteomic analysis in the absence of single-cell mass spectrometry technology.PPIA is widely expressed across stem and progenitor cells subsets, and we focused on PPIA's overall impact on the aging haematopoietic phenotype.We are confident that haematopoietic stem cell-specific changes are contributing to this phenotype due to the long-term functional impact of PPIA deficiency and rescue in transplantation assays.However, we note that our study did not claim that PPIA is a chaperone specific for a subset of haematopoeitic stem and progenitor cells.
Recently, the laboratory of Dr. Irv Weissman published a detailed proteome catalog across different murine bone marrow stem and progenitor subsets [3].We re-examined this valuable resource and found robust expression of PPIA in mouse haematopoietic stem cells (new Extended Data Fig. 1b).Importantly, the Weissman group also observed significantly reduced PPIA protein expression in aged HSCs (new Extended Data Fig. 4a), consistent with the PPIA quantification we performed in young versus aged haematopoietic stem cells (Fig. 6a).
The data presented in the transplantation experiments with wt and PPIA KO HSPCs shown in Fig. 1c-h are clearly showing that PPIA deficiency leads to accelerated exhaustion of HSPCs but how much this dysfunction is related to PPIA-dependent regulation of the cellular proteome and deficiency of IDR proteins?
We recognize that with current technologies, it is difficult to directly quantify the exact contribution of PPIAdependent proteome regulation to the observed stem and progenitor dysfunction.To substantiate our conclusion, we have performed additional experiments to support the relevance of PPIA-dependent regulation of IDPs PABPC1, NPM1, and DDX6, and their expression and contribution to stress granules, nucleoli, and Pbodies, respectively (new Figs.4g and 5b,c).Our new data serve as a significant initial step in identifying PPIA as a crucial chaperone for IDP expression and activity, thereby influencing stem cell functionality.Importantly, we show that PPIA depletion results in a skewed proteome, disfavoring IDPs (Fig. 6c and new Extended Data Figs.4d,e and 5a-c).Identifying how loss of individual or multiple IDPs directly contributes to haematopoietic aging is beyond the scope of the current manuscript (see also response to reviewer #2, major point 1).
The experiments aimed at gaining molecular insights into the changes caused by PPIA depletion and at identifying the client proteins of PPIA are not performed in hematopoietic cells (instead in Hela or 293T).What is the reason for not performing these experiments in murine HSPCs?The authors show that in PPIA KO HSPCs the levels some interactors (G3BP1, DDX6, PABPC1..) is reduced by WB but some validation of interaction between PPIA and IDR proteins should be also performed in wt-HSPCs (by Co-IP or PLA that will require less cells).
The reviewer highlights a crucial concern regarding the interaction of PPIA with substrates and the possibility that this is cell type-specific.PPIA belongs to the extensive family of cyclophilins, which, in turn, is one of four distinct families of prolyl isomerases, including FKBPs, Parvulins, and PTPA.Consequently, compensation attenuates the effects of PPIA depletion, especially in constitutive knockout mouse models.We found more robust responses when using acute knockdown approaches.The critical proteomic and transcriptomic studies were performed in primary haematopoietic cells, and we confirmed diminished expression of PABPC1, DDX6, and NPM1 in the haematopoietic cell line, AML3, after knockdown of PPIA (new Extended Data Fig. 3b).Further, we conducted western blots in primary mouse haematopoietic stem and progenitor cells after Ppia knockout to validate our findings (Extended Fig. 3c).Unfortunately, visualization of P-bodies and stress granules by microscopy proved difficult in the haematopoietic system (our unpublished observation), prompting us to use epithelial cell lines for these assays.To specifically address the interaction between PPIA and IDPs, we performed Proximity Ligation Assays (PLA) in primary haematopoietic stem cells, which confirmed that PPIA interacts with PABPC1, DDX6, and NPM1 in mouse and human cells (new Fig. 5d).
In Fig. 3 The authors biochemically identified and confirmed that PABPC1 is a client of PPIA and show that it undergoes phase transition and participate in stress granules at least in Hela cells.The authors should attempt similar set of experiements (modulation of PPIA activity, assess dynamics of stress granules formation and resistance to oxidative stress) in HPSCs.In addition they should show that reintroduction of the chaperone rescues stress granule formation in aged HSPCs.Such rescue experiments should be accompanied by functional evaluation of aged HSPCs in transplantation assays to really determine how much the axis PPIA-IDR-rich proteins-effective phase separation contributes to age-associated dysfunctions of HSPCs.
We concur with the reviewer that performing experiments in primary haematopoietic cells would be optimal.Regrettably, quantitatively visualization of stress granules in these cells was not feasible because of the restricted size of the cytoplasm.Nonetheless, to obtain a deeper understanding of the disrupted pathways in PPIA-depleted stem and progenitor cells, we conducted RNA-sequencing analyses.We found that a majority of altered gene programs were related to initial stages of proteostasis, unlike disruptions of protein repair or clean-up pathways that lead to the accumulation of misfolded proteins.Notably, a compensatory upregulation of cytoplasmic translation emerges as one of the most prominent gene ontologies observed in Ppia knockout cells.Conversely, we did not observe molecular chaperones or the ubiquitin-proteasome system being transcriptionally activated by PPIA depletion.Our results further substantiate our hypothesis that PPIA plays a crucial role in protein biosynthesis (new Extended Data Fig. 3d).To directly test this, we carried out protein synthesis assays and found that de novo translation is markedly diminished in primary haematopoietic stem cells following treatment with a PPIA-specific inhibitor (new Extended Data Fig. 3a).
In our revised manuscript, we present unequivocal evidence that PPIA engages with substrate proteins, a majority of which possess a high level of intrinsic disorder, based on predictions with the widely-accepted IUPred2A algorithm [4].Furthermore, we report that the decrease in PPIA-an occurrence also observed during aging in haematopoietic stem cells-is associated with a significant reduction of IDPs within the proteome.Notably, numerous PPIA substrate proteins are essential for various biological processes, especially in mRNA processing.It is also noteworthy that in Ppia knockout animals, we detected a significant transcriptional upregulation of PPIA substrates (new Extended Data Fig. 3f).This phenomenon underscores the essential role of PPIA and PPIA substrates within the cellular environment.
We propose that the intimate link between prolyl isomerase and protein synthesis could be similar of the role of trigger factor, a functional homolog of human PPIA in Prokarya, in the folding of the nascent polypeptide chain as it emerges from translating ribosomes.Drawing from our observations and those documented in other studies, we postulate that PPIA may fulfill a comparable function in protein folding in Eukarya.

Reviewer #2 (Remarks to the Author):
The manuscript by Maneix et al. studied the role of Cyclophilin A (PPIA) in haematopoietic stem cell aging.The authors carried out transplantation assays in mouse models to describe premature aging phenotype upon PPIA loss.Using interaction proteomics, the authors defined PPIA target proteins and observed an enrichment of these to contain intrinsically disordered regions (IDRs).PPIA knock-down caused decreased translation and PPIA modulation affected stress granule formation.Finally, proteomics analyses revealed a correlation for PPIA and IDR in young and old HSCs.
The study addresses an interesting topic and the need to better define the role of PPIA in controlling proteostasis, IDR-containing proteins and a possible involvement in cell ageing.In the current state, the manuscript seems premature in that a number of crucial conclusions, which are critical for the logical flow of the manuscript, lack statistical rigor and necessary validation.
We appreciate the constructive criticism of reviewer #2, who considered our study an interesting topic.We respect your perspective and have thus worked diligently to strengthen our manuscript.While we understand your initial assessment, we would like to politely disagree that our revised manuscript remains in a premature state.We showed that PPIA is the first chaperone with a preference for intrinsically disordered proteins (IDPs) and provide evidence that PPIA and its substrates decline with age within the haematopoietic system, which, to the best of our knowledge, are two impactful, novel findings.In response to this reviewer's comments, we provide substantial new evidence derived from additional sets of experiments that were performed over the past year to address any perceived limitations of our study.As requested by this reviewer, we have also enhanced the statistical robustness by increasing the number of biological replicates in pivotal experiments.Importantly, we present triplicates of biologically independent analyses of isobaric proteomic studies comparing both young versus old as well as Ppia heterozygous versus knockout cells in the haematopoietic system (new Extended Data Fig. 4c-e).We believe the additions we made significantly address the concerns raised, and bring more depth, clarity, and precision to our work.

Major points:
1) The evidence for PPIA-mediated premature ageing is somewhat circumstantial.From the manuscript, it remains unclear how loss of PPIA shapes the cellular proteome and proteostasis.Consequently, a number of cellular pathways may be responsible for the observed effects.A more rigorous characterization of cellular changes, including classical molecular aging markers is necessary to claim the connection between PPIA and haematopoietic aging.Do PPIA levels and the specified interactions partners change when comparing old versus young HSPCs?
We recognize that multiple pathways can affect haematopoietic aging.To address this issue, we have now included data from RNA sequencing of three biological replicates, comparing heterozygous and knockout Ppia stem and progenitor cells (new Extended Data Fig. 3d).Notably, we find that the knockout cells exhibit a pronounced gene set enrichment that reflects data published in the recent Tabula Muris Senis study and is consistent with the transcriptome of aged haematopoietic stem cells [7] (new Extended Data Fig. 3e).Moreover, P-Selectin has been reported by several research groups [8,9] as the most consistently upregulated marker of aged stem and progenitor cells.In line with these findings, we observed a significant upregulation of this gene in Ppia knockout cells compared to heterozygous cells.Importantly, our experiments demonstrate that augmenting PPIA expression in aged stem and progenitor cells ameliorates their performance in a competitive transplantation assay (new Fig. 2a-d).This finding substantiates the proposed causal relationship between PPIA activity and the aging process within this cellular compartment.Nevertheless, while our findings underscore the significance of PPIA in aging, it is important to acknowledge that aging is a multifaceted process [10].Given that PPIA interacts with proteins involved in diverse cellular pathways, such as splicing, translation, and transcription, we assert that it would be an oversimplification and potentially misleading to concentrate solely on a single aging-driving mechanism.
In line with our data, we propose that structural, in addition to any functional features, characterize protein alterations with age.Consequently, attempts to redress PPIA deficiency by overexpressing individual substrate proteins is unlikely to further our understanding of this intricate process.We note that the substantial transcriptional upregulation of PPIA substrates observed in knockout models suggests the existence of compensatory mechanisms, thereby ensuring adequate expression of these critical RNA-processing proteins and the survival of Ppia knockout cells (new Extended Data Fig. 3f).
2) Several key conclusions drawn are based on various omics data, including proteome, interactome, translatome and transcriptome changes.However, a number of these experiments lack a sufficient number of replicates to draw any conclusions with statistical significance.These design limitation in combination with a general lack of data availability (raw data, continuous quantification and statistical significance data) make it difficult to agree with the claims.Especially in that light, validation of the key claims by orthogonal methods is required.All key experiments that have not been validated orthogonally need to be carried out with at least three independent biological replicates.
Our results were derived using a diverse range of orthogonal methods that substantially reinforce the reliability of our data.To further bolster the robustness of our findings, we have undertaken additional proteomics experiments-specifically, isobaric profiling-of young and aged cells, as well as Ppia heterozygous and knockout cells.These experiments were conducted with each condition assessed independently in biological triplicates (new Extended Data Fig. 4c-e).The raw and normalized proteome-wide quantification obtained through isobaric labeling with tandem mass tags (TMT) is now available as supplementary material.
In addition, we performed a new RNA-seq analysis on Ppia heterozygous and knockout haematopoietic stem and progenitor cells in triplicate to further enhance the significance of PPIA on transcription and transcriptional compensation (new Extended Data Fig. 3d-f).Together, this comprehensive approach adds substantial weight to our conclusions and strengthens our understanding of PPIA's role in cellular processes.
3) Does PPIA loss affect translation via known cellular translation pathways regulated under stress and hence the observed reduced translation rate?
The reviewer raises an important point as to why protein translation of proteins is impaired when PPIA is depleted or inhibited.We propose that one explanation involves compromised ribosomal biogenesis.NPM1, a master regulator of nucleolar structure and function, is a PPIA substrate.Genetic depletion or pharmacologic inhibition of PPIA reduces NPM1 levels and impairs the shape and size of nucleoli (new Fig. 5c), which may disrupt ribosomal biogenesis.In support of this, the upregulation of ribosomal genes represents one of the most significant ontological changes in Ppia knockout cells (new Extended Data Fig. 3d), while we observed no significant upregulation of genes encoding molecular chaperones or components of the ubiquitin-proteasome system.
In addition to an effect of PPIA on ribosomal biogenesis through NPM1, our data also suggest that PPIA may participate in early folding of nascent polypeptides, and thereby affect the translation efficiency of its substrates.We found several results that would corroborate such a model: First, we determined cytoplasmic association of PPIA with nuclear proteins, suggesting interactions during translation.Second, PPIA substrate translation is sensitive in the SILAC study.Third, using acute pharmacological inhibition of PPIA, we observed a robust reduction of global protein translation (new Extended Data Fig. 3a).Fourth, transcriptional upregulation of genes encoding PPIA target proteins suggests a compensatory mechanism and associates the chaperone with these substrates (new Extended Data Fig. 3f).4) and 5) Figure 3b should be repeated including the G104A mutant to validate the findings.Figure 3c, to confirm the interaction, at least one of the bait and prey should be monitored at the endogenous level without overexpression.
Previous IP-Western experiments for PPIA and its substrates were performed at endogenous protein levels (Fig. 4e).Per the reviewer's request, we have transfected HeLa cells with either 3xF-PPIA or 3xF-PPIA(G104A).Immunoprecipitation was then conducted, followed by probing with antibodies against endogenous PABPC1, DDX6, and NPM1 (new Fig. 4f).The data demonstrate a reduced ability of the mutant PPIA to interact with the three substrate proteins.Furthermore, we verified the interaction between PPIA and the aforementioned proteins at endogenous levels in haematopoietic stem cells using proximity ligation assays (new Fig. 5d).
6) The number of replicates need to be stated clearly, ideally in the experimental model or figure legend.Implying n=3 when no statement can be found is not sufficient.Drawing significance from distribution (violin) plots when there are not at least 3 biological replicates is not appropriate.Consequently, these data sets are not sufficient as single supportive evidence for major claims.
The number of replicates is now clearly stated in the experimental model or figure legend.In addition, we have performed additional as well as orthogonal experiments to substantiate the statistical validity of our findings.Specifically, the primary omics experiments, including isobaric proteomics and the new RNA-seq analysis of Ppia knockout and heterozygous haematopoietic stem and progenitor cells, are based on triplicates of independent biological replicates (n=3).The results of the cumulative and individual assays are shown in aggregated form in the main figures (Fig. 6) and individually as supplemental material (new Extended Data Fig. 4), respectively.Raw data are included as supplementary information.
7) Among the identified PPIA target proteins are a number of mitochondrial proteins (Figure 2c).Does loss of PPIA cause mitochondrial dysfunction and does that alter translation and the stem cell ageing phenotype?Like reviewer #2, we were intrigued by the abundance of mitochondrial proteins amongst PPIA target proteins.However, as a caveat, this observation may have resulted from organelle contaminations due to the use of detergents in our pulldown assay: It is possible that mitochondrial proteins have leaked and inadvertently bound to PPIA post lysis.
In the transcriptional profiling of Ppia knockout haematopoietic stem and progenitor cells, we noted an increased expression of nuclear encoded mitochondrial genes, especially those involved in oxidative phosphorylation (see Figure A below).Consistently, we also found an increase in mitochondrial proteins in Ppia knockout cells based on isobaric labeling and mass spectrometry (not shown).However, we do not have evidence linking absence of the chaperone directly to the enhanced translation of these proteins, given that Ppia knockout enhances transcription of their encoding genes.Additionally, for other PPIA substrates, we observed decreased translation in the absence of PPIA, which contrasts with the increased levels observed in the case of mitochondrial proteins.
To further examine this using a more acute cell line-based knockdown model, we performed a series of oxygen consumption assays, utilizing a Seahorse instrument on 293T and HeLa epithelial cells with and without PPIA depletion.These assays did not show a consistent alteration in mitochondrial activity (see   Taken together, we do not believe that PPIA modifies mitochondria in a distinct, directional manner, which would enable us to draw conclusions about changes in the organelle's activity.The augmentation in the number of several mitochondrial proteins, which occurred independently of respiration changes, is likely caused by transcriptional changes in nuclear-encoded mitochondrial genes within the haematopoietic compartment.While these transcriptional shifts may be associated with PPIA activity, they could also mirror an altered composition of the stem and progenitor cell pool.Specifically, a shift may have occurred in the knockout animals transitioning from quiescent stem cells towards committed progenitor cells that exhibit a higher mitochondrial abundance [11].Such a shift in cell composition would be consistent with the functional changes that we observed in transplanted animals with higher levels of CD150-positive stem cells (Fig. 1f).8) A more unbiased representation of data is prudent to assess their quality.How do interactions change across all protein in the WT versus PPIA mutant comparison?How were interactors selected?For the translatome (Fig. 2f), also heat plots per replicate should be shown.A bit more insight into which proteins go up/down would be interesting.The percentage of heavy incorporated is very low, additional validation that there is still sufficient dynamic range to monitor dynamics would be important.The transcriptome data set is quite useful and more data should be shown (e.g.quality control/correlation across replicates, some clustering or pathway analyes).
Following the reviewer's recommendation, we performed an integrative analysis of the transcriptome as well as of the proteome of haematopoietic stem and progenitor cells.The findings are summarized below: Transcriptome: The absence of PPIA does not instigate a chaperone response or enhance the expression of the ubiquitin-proteasome system.Instead, we witnessed an upregulation of cytosolic translation-related genes, notably ribosome subunits, as one of the most distinct responses (new Extended Data Fig. 3d).This observation implies that the role of PPIA is within the functional context of de novo protein synthesis as opposed to later stages of proteostasis.We hypothesize that PPIA might fulfill a role in co-translation protein folding akin to bacterial trigger factor that monitors the folding of the nascent chain.Like trigger factor, PPIA possesses prolyl isomerase activity (though employing a different catalytic domain), which is integral to protein synthesis.
The proposed involvement of PPIA in early translation is further supported by the lack of accumulation of misfolded proteins in PPIA-deficient cells (new Extended Data Fig. 3g).An increase in protein misfolding was only observed in the presence of proteasome inhibitors, suggesting that the cellular protein clearance machinery adequately compensates for PPIA deficiency.
Proteome: An unbiased analysis of protein changes based on the level of structural disorder within proteins also verifies that intrinsically disordered proteins cluster according to genotype (Ppia heterozygous versus Ppia knockout), while no such clustering is observed for proteins that lack disordered regions (new Extended Data Fig. 5a,b).In order to reciprocally validate the quality of these clusters, we conducted an unbiased hierarchical analysis of all proteins identified in the isobaric labeling experiment of Ppia expressing or knockout cells.The cluster that was upregulated the most across Ppia expressing cells is enriched for IDPs involved in cellular processes commonly found amongst its substrate proteins (i.e., mRNA binding and processing), while the top cluster upregulated in Ppia knockout cells exhibits lower-than-average levels of intrinsic disorder (new Extended Data Fig. 5c).Also of note, while genes encoding cytosolic ribosomal proteins were upregulated in Ppia deficient cells, the respective proteins were reduced in these cells, suggesting that transcriptional upregulation compensates for a translational bottleneck in cells lacking PPIA.
PPIA substrate proteins are enriched for essential RNA binding proteins.In the constitutive Ppia knockout model, overall expression of these vital proteins was only moderately reduced and likely masked by transcriptional compensation: the genes of PPIA substrates are significantly upregulated in knockout animals, potentially limiting the effect of chaperone loss at the protein level (new Extended Data Fig. 3f).Notably, when we examined the expression of PPIA substrate proteins in cell line-based PPIA knockdown systems (new Fig. 3g, Extended Data Fig. 3b), we observed a significant reduction of the substrates shortly after knockdown, which gradually diminished over time.
Translatome: By re-evaluating our SILAC data in cell lines, we confirmed that cytosolic ribosomal proteins are among the most repressed proteins in PPIA-deficient cells and that knockdown cells cluster separately from PPIA-expressing cells, even when comparing two different cell types (293T and HeLa cells).The top-scoring gene ontologies are now presented in Fig. C (below).
In summary, our results suggest the involvement of PPIA in early translation predominantly affects proteins with a high degree of structural disorder.In this context, the pronounced repression of IDPs in aged or PPIA-deficient haematopoietic cells is striking.Furthermore, observations derived from knockout models indicate potential transcriptional compensation.
9) The claim that PPIA and PABPC1 regulate phase separation needs further examination.Reduction of protein translation, as is observed upon PPIA loss, is sufficient to induce stress granules.While these are phase separated, it is not clear that PPIA and PABPC1 drive the physical aspects of phase separation.How are IDR and other target proteins changed upon rescue of PPIA?
We appreciate the reviewer's question about how PPIA influences phase separation, a topic that we find most intriguing.Prior studies support a role of PPIA in stress granule formation as PPIA co-localizes with these structures in vivo and PPIA inhibition reduces their formation [13,14].
To address the reviewer's query, it is indeed counterintuitive why depletion of a chaperone, which increases cellular stress, reduces stress granule formation.Based on our results and published findings by others, we surmise that this can be explained by PPIA enhancing the synthesis and/or function of proteins associated with stress granule assembly.Reintroducing PPIA partially rescues the stress granule deficiency in knockdown cells, suggesting that PPIA might increase the concentration of substrate proteins that are involved in phase separation of these organelles [15], or it might alter substrate protein structure, thereby modifying function and phase mixing/demixing potential [16].
Our work represents a major effort in characterizing PPIA as a chaperone modulating the levels (and likely structure) of proteins involved in phase separation.Consistent with our conclusion, the Zweckstetter lab recently reported that PPIA can modify the ability of a protein to phase separate in vitro [16].However, providing a detailed structural understanding of PPIA's effect on substrate proteins in vivo is beyond the scope of our manuscript.Future studies will be required to thoroughly address the reviewer's question.The unlabeled lanes of the total protein stain were not used for the immunoblot on the left side.We removed this subpanel for clarification.

Reviewer #3 (Remarks to the Author):
Comment on 'Cyclophilin A supports translation of intrinsically disordered proteins and mitigates haematopoietic stem cell aging' by Maneix et al.In this paper, the authors have identified Peptidyl-3 Prolyl Isomerase A (PPIA or Cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells (HSPC) as it accounts for 14% of the cytosolic proteome and its mRNA accounts for over 0.5% of all mRNAs of HSPC.Total knockout of PPIA leads to 1) myeloid skewing, an immunophenotypic but not functional increase in stem cells, and 2) impaired self-renewal with 3) accelerated exhaustion, which are three hallmarks of haematopoietic aging.Using an assay targetting differential co-immunoprecipitation between the wild-type PPIA and a G104A mutant PPIA, the authors revealed approximately 400 substrates of the wild-type enzyme.Since they performed the coimmunoprecipitation in the cytosolic cell fraction, these results suggest PPIA and substrates interact between translation and nuclear translocation.When compared to the global proteome, immunoprecipitated PPIA substrates feature higher levels of IDRs.
By surveying proline residues within PDB, the authors found that the majority of cis-prolines located in bends and turns.Their conclusion is that "PPIA and related enzymes predominantly isomerize prolines within unstructured protein regions."This conclusion is incorrect as the bends and turns are categorized as structured regions that connecting helices and beta-strands."Unstructured protein regions" are regions of proteins that they donot have stable (i.e.highly populated) conformations and are "invisible" in PDB.Further studies are required to justify this conclusion.
We agree and have eliminated these data from the revised manuscript.
Using pulsed SILAC in HeLa and 293T cells with either normal or reduced levels of PPIA, the authors found that loss of PPIA significantly reduced expression of the whole proteome, PPIA clients and non-clients, in both cell types.As far as the levels of PPIA target proteins are concerned, they were lower than for other proteins and further reduced when PPIA was knockdown.In addition, the fraction of protein IDRs in the HSPC proteome of PPIA knockout animals was significantly reduced when compared with heterozygous siblings.
Largely based on these aforementioned observations, the authors claim that PPIA supports de novo translation of its target proteins.Unfortunately, this claim is not even close to be supported by the data reported.
We disagree with reviewer #3's notion.On the contrary, a substantial body of literature supports this assertion: First, early characterizations of PPIA activity suggest the potential role of prolyl isomerization in co-translational protein folding [17,18].
Second, comprehensive research in prokaryotes revealed that trigger factor, a major prolyl isomerase in the biosphere, participates in co-translational isomerization [19].
Third, various studies demonstrated unequivocally that PPIA inhibitors obstruct protein translation [20,21].Our experiments corroborate these findings through both genetic and pharmacologic PPIA inhibition, observing significant impairment in de novo translation via SILAC, and in orthogonal puromycin-incorporation assays (Fig. 4a-c and new Extended Data Fig. 3a).
Fourth, utilizing a tRNA analog as a UV-activated chemical trap, PPIA has been located in close vicinity to functional ribosomes [22].
Fifth, the transcriptional profile of Ppia knockout animals, exhibiting increased expression of cytosolic ribosome genes, implies a compensatory mechanism responding to a translation block (new Extended Data Fig. 3f).
Sixth, in proximity ligation assays, we discerned interaction between cytosolic PPIA and NPM1, which is primarily found in the nucleus, suggesting their engagement prior to NPM1's nuclear import (new Fig. 5d).
Seventh, previous work showed that PPIA enhances the expression of alpha-synuclein [2], supporting our global analysis that PPIA enhances substrate levels.
In the revised manuscript, we have emphasized this prior research.Together with our new findings that PPIA is transcriptionally linked to the cytoplasmic translation machinery and acutely affects translation rates (new Extended Data Fig. 3a,d), our results support the overall interpretation and conclusion that PPIA interacts with nascent polypeptides.
The authors went on to confirmed a few clients of PPIA using a combination of IP and western blotting.One of the confirmed clients is PABPC1."Following PPIA knockdown, expression of PABPC1 protein was reduced by 20-30% in pulsed SILAC experiments, suggesting the chaperone engages with PABPC1 during translation."This conclusion is questionable as the pulse was 24 hours.Many other mechanisms other than, or in addition to, limited translation can account for the 20-30% reduction.
We did not find enhanced decay of PPIA substrates in PPIA-depleted cells following chase with cycloheximide for up to 24 hours (data not shown).We also did not observe increased protein aggregation or activation of the ubiquitin-proteasome system (new Extended Data Fig. 3d,g).Furthermore, puromycin incorporation studies indicated reduced translation efficiencies following acute PPIA inhibition even during short 2-hour chase experiments.However, to address the reviewer's concern, we have revised our manuscript to discuss potential alternative effects of PPIA.Furthermore, we are amenable to changing the title of the manuscript to: "Cyclophilin A Preferentially Targets Intrinsically Disordered Proteins and Mitigates Haematopoietic Aging".
Next, the authors turned their attention to liquid-liquid phase separation (LLPS), a biophysical property of multivalent proteins including many IDR-containing ones.Essentially the authors showed that PPIA KD perturbed condensates of PABPC1-GFP (I have a hard time to figure whether PABPC1-GFP is transiently expressed or is expressed at endogenous levels) and the formation of stress granules marked by G3BP1 upon the treatment with sodium arsenite.It is unclear whether the reduced translation of PPIA's clients or the lack of PPIA activity per se was claimed to cause the perturbation to condensates of PABPC1-GFP or stress granule formation.Nevertheless LLPS is just one aspect of the global proteome We concur that liquid-liquid phase separation (LLPS) represents only one facet of the global proteome.However, it is a common feature of all three PPIA substrates investigated here.To our knowledge, our preprinted manuscript describes the first identification of a chaperone that prefers to engage with intrinsically disordered proteins (IDPs) [23,24], many of which partake in LLPS.All three PPIA substrates are vital and have well-documented functions in mRNA translation, transcript turnover, ribosome biogenesis, and nuclear compartmentalization. Furthermore, the ability of these proteins to phase separate is intimately linked to their cellular function as evident from the current literature.
For the purpose of elucidation, it should be noted that the phase separation of stress granules, P-bodies, and nucleoli in Fig. 5 was analyzed at endogenous levels without overexpression of any of the proteins involved (for NPM1, endogenous tagging was utilized; DDX6, and G3BP were monitored using antibodies).According to consensus in the field, phase separation is heavily contingent on the concentration of the implicated proteins [15].It is critical to point out that both expression levels and structural conformation of PPIA substrates are likely integral to their demixing properties, which we elaborated upon in the revised discussion.Only in the supplementary videos did we employ an overexpression system, by introducing GFP-tagged PABPC1.
Overall, the authors didn't make sufficient effort to connect pieces of observations for a coherent model explaining the role of PPIA in ageing of haematopoietic stem cells.I can't recommend its further consideration for pulication in Nature Cell Biology.
We respectfully disagree with reviewer #3's assessment, which is not shared by the other two reviewers, who considered our work "novel" and "well-presented" (reviewer #1) and an "interesting topic" (reviewer #2).We wish to reiterate that our work makes a significant contribution to the field in two ways: First, we identified PPIA as a principal chaperone of disordered proteins.Second, we detected a shift in proteome composition during haematopoietic aging, reducing the ratio of disordered proteins.The latter implies that proteome alterations arise not only from functional changes in gene transcription, but also from transitions in the structural composition of polypeptides that disfavor intrinsically disordered proteins.
We have performed additional experiments to more robustly link PPIA to changes in the proteome composition: 1. Loss of intrinsically disordered proteins (IDPs) increases with age and with PPIA deficiency in the haematopoietic compartment (new Extended Data Figs.4b-e and 5a-c).2. Increased expression of PPIA in aged haematopoietic stem cells partially rescues the aging phenotype, arguing for a causal role of the PPIA chaperone in this tissue compartment (new Fig. 2).3. Loss of PPIA does not trigger a protein stress response, but instead activates the cytoplasmic translation machinery (new Extended Data Fig. 3d,g), indicating a function of this chaperone in protein synthesis.Consistently, acute inhibition of PPIA impairs translation in primary haematopoietic stem cells (new Extended Data Fig. 3a).4. Proteomic studies were validated with detailed quantitative and functional investigations of three PPIA substrates (new Figs.4f,g, 5b,c, and new Extended Data Fig. 3b).
To help them improving their study for future submission, I listed some suggestions below: Major issues 1)When doing differential analysis of proteome to assess the effects of PPIA on IDR-enriched clients, the authors shall divide PPIA clients and nonclients into IDR-containing, IDR-noncontaining, four categories.
We appreciate the reviewer's suggestion and utilized two orthogonal clustering methods when comparing the proteome in dependence of PPIA expression.We analyzed the proteome of both Ppia-expressing or knockout haematopoietic cells and considered both their genotypes as well as the expression levels of intrinsically disordered proteins.Both methodologies yielded congruent results, indicating that IDR-rich proteins are particularly sensitive to PPIA loss and that PPIA disproportionally affects IDR-rich proteins (new Extended Data Fig. 5a-c).In contrast to the SILAC experiments that employ acute PPIA knockdown (Fig. 4c), we did not observe a comparable loss of PPIA target proteins in the constitutive knockout model.This is presumably due to transcriptional compensation (new Extended Data Fig. 3f) and the upregulation of genes encoding PPIA substrates (see our response to Reviewer 2's query #8).
2)Genetic manipulation or pharmacological perturbation for days are rather slow to assess acute and direct functions.I suggest the authors to use an AID system or PROTAC system (if available) to chemically KD PPIA and assess whether the reported effects are direct or indirect.
Beyond genetic perturbation, we utilized pharmacological intervention with the PPIA-specific inhibitor TMN355 [25].While degron-based systems are an option, they can be leaky and require high doses of auxin [26].Prior studies have demonstrated that PPIA inhibitors obstruct stress granule formation [13].Using TMN355, we find that acute PPIA inhibition (24-48 h) disrupts nucleoli formation and protein translation (new Extended Data Fig. 3a and data not shown).However, we did not observe a substantial alteration in the number and morphology of nucleoli or stress granules during shorter treatment.This is consistent with previously published work [13] and may be explained by the slow synthesis rates of PPIA substrates [12].These findings suggest that PPIA influences the protein function of newly synthesized polypeptides, which drive phase separation, rather than affecting the functionality of pre-existing membraneless organelles.
Minor issues 1) There are no legends for SI-Videos.
Legends for SI-Videos are now included in the revised manuscript and can be found with the Extended Data.

Decision Letter, first revision:
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Dear Dr Catic, I apologize once again for this very long delay.Furthermore, we had also gone back to Reviewer #1 for further clarification on their report.Your manuscript, "Cyclophilin A supports translation of intrinsically disordered proteins and mitigates haematopoietic stem cell aging", has now been seen by our original referees, who are experts in stem cell senescence (referee 1); proteostasis and proteomics (referee 2); and biomolecular condensation (referee 3).As you will see from their comments (attached below) they find this work of interest, but have raised some important points.
Although we are also very interested in this study, we believe that their concerns should be addressed before we can consider publication in Nature Cell Biology.
Nature Cell Biology editors discuss the referee reports in detail within the editorial team, including the chief editor, to identify key referee points that should be addressed with priority, and requests that are overruled as being beyond the scope of the current study.To guide the scope of the revisions, I have listed these points below.We are committed to providing a fair and constructive peer-review process, so please feel free to contact me if you would like to discuss any of the referee comments further.
In particular, it would be essential to: A) If the data can be obtained within this revision's timeframe (1 month), then we'd ask for providing this data regarding potential effects of PPI2 overexpression on stem cell compartments with immunostaining of cell surface markers of HSCs/MPPs (Reviewer #1).We hope this data can be obtained within a reasonable timeframe and please get in touch with us if you would like to discuss.
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We strongly recommend the presentation of source data for graphical and statistical analyses as a separate Supplementary ---------Please don't hesitate to contact NCB@nature.com should you have queries about any of the above requirements ---------Author Rebuttal, first revision: 1/4 We would like to express our sincere gratitude to the reviewers for their continued dedication in evaluating our manuscript.We value their insightful feedback and are eager to address the outstanding concerns in the revised manuscript and in our point-by-point response to their queries.For clarity and to facilitate a seamless review process, we have highlighted our responses below and the relevant modifications to the manuscript in blue font.

Reviewer #1
Remarks to the Author: This Reviewer recognizes that the authors have done substantial work to address the raised concerns and discussed potential limitations of their study, which is good.This includes the challenges of performing more mechanistic studies and applying mass spec analysis in the relevant cell type (e.g.HSC) under study.Some questions remain on new data included, for example, the data presented in Figure 2 are important, but it would be great if the authors could better characterize the impact of PPI2 overexpression on the stem cell compartment as well, together with the lineage output presented.As a general consideration, statistical analysis with n=3 should not be presented and instead, descriptive statistics should be applied.
We thank this reviewer for the thorough assessment of our work.We concur that several questions warrant further exploration, such as the changes in stem cells (alterations in proliferation versus apoptosis), variations in progenitor cell pools, and whether blood cell functions are altered beyond their output numbers.Our experimental design aimed to ensure the most rigorous phenotypic outcomes.In our study, modified haematopoietic stem cells were transplanted into lethally irradiated recipient mice, and the outcomes were monitored in the peripheral blood over a six-month period.This duration is commonly recognized in the field to capture phenotypic differences attributable to the long-term stem cell pool, thus enabling us to attribute the observed results to this cell type.
Regrettably, due to natural attrition of the transplanted animals following submission of the revised manuscript in July, we are unable to further explore how PPIA overexpression impacted the progenitor pool within the context of this specific experiment.We appreciate the reviewer's comment and plan to bridge this knowledge gap in future work.However, experiments to comprehensively address these queries would require a substantial amount of time and effort, which are beyond the scope of the current manuscript.Importantly, we note that these results, although desirable, would not fundamentally alter our conclusions derived from the experiment presented in Fig. 2. The updated discussion addresses our study's limitations with regard to the progenitor and immune cell pool.
Following conversations with the Nature Cell Biology editorial team, we have taken additional steps to clarify the distinction between "sample size" and "independent biological replicate" as stated in our figure legends.In our previous manuscript, the term 'n' denoted the number of times the experiment was conducted independently, rather than representing sample size or technical replicates.The revised manuscript does not feature experiments with sample sizes of less than three, and we agree with the reviewer that such a sample size would preclude the use of statistical significance testing.
In response to earlier critiques, we have omitted results related to the potential effects of primary or secondary structures on Cyclophilin A substrate affinity, and as such, this data does not appear in the final manuscript.
2) Supplemental videos are not available to reviewers.
We apologize and have informed the editorial team to release the supplementary videos.

References:
Theillet FX, Kalmar L, Tompa P, Han Thank you for submitting your revised manuscript "Cyclophilin A supports translation of intrinsically disordered proteins and mitigates haematopoietic stem cell aging" (NCB-A48335B).It has now been seen by the original referees and their comments are below.The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Cell Biology, pending minor revisions to satisfy the referees' final requests and to comply with our editorial and formatting guidelines.
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Figure
Figure A: A subset of nuclear-encoded mitochondrial genes is upregulated in Ppia knockout cells.Haematopoietic stem and progenitor cells (lin-/cKit+) show significant transcriptional upregulation of gene sets involved in oxidative phosphorylation.Analysis performed with GSEA software package ver.4.3.2.

Figure B :
Figure B: No change in mitochondrial function following knockdown of PPIA.PPIA-deficiency in 293T cells (shown) and HeLa cells (not shown) shows no consistent alterations in mitochondrial respiration or ATP production, as measured by Seahorse assay (n>10).P-values were not significant (ns) as calculated by the two-sided Wilcoxon rank-sum test.

Figure C :
Figure C: Gene ontology analysis of proteins most affected in de novo translation following PPIA depletion.Pulsed SILAC analysis of 293T and HeLa cells following PPIA knockdown shows that proteins most reduced in PPIA-depleted conditions are involved in mRNA splicing and translation, consistent with the ontologies of PPIA substrates.Listed are the top unique ontologies and p-values.
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