Water content influences the vitrified properties of CAHS proteins
2021; Elsevier BV; Volume: 81; Issue: 3 Linguagem: Inglês
10.1016/j.molcel.2020.12.009
ISSN1097-4164
Autores Tópico(s)Spaceflight effects on biology
ResumoIn their Letter, "Reconsidering the "glass transition" hypothesis that intrinsically unstructured CAHS proteins contribute to desiccation tolerance of tardigrades" in this issue of Molecular Cell, Arakawa and Numata, 2021Arakawa K. Numata K. Reconsidering the "glass transition" hypothesis that intrinsically unstructured CAHS proteins contribute to desiccation tolerance of tardigrades.Mol. Cell. 2021; 81 (this issue): 409-410Abstract Full Text Full Text PDF Scopus (3) Google Scholar use thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) to examine residual water in the CAHS protein RvCAHS1 and two species of tardigrades. Please note that due to the need for a timely response and a difficulty in acquiring the food source for R. varieornatus from Arakawa due to an export restriction, the editor at Molecular Cell requested that animal studies not be included in this response, which is confined to in vitro experiments and reexamination of Arakawa and Numata's data. The first assertion Arakawa and Numata make is that other materials not traditionally thought of as protective during desiccation can also vitrify, and therefore observing vitrification of a material cannot be inferred as protection. We agree with this statement, but since we made this point in Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar we fail to see how restating it adds or detracts for our original findings. Furthermore, in our original paper we did not infer protection from vitrification, but rather observed vitrification and then went on to perform experiments aimed at empirically correlating vitrification with survival in keeping with norms in the field (Sakurai et al., 2008Sakurai M. Furuki T. Akao K. Tanaka D. Nakahara Y. Kikawada T. Watanabe M. Okuda T. Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki.Proc. Natl. Acad. Sci. USA. 2008; 105: 5093-5098Crossref PubMed Scopus (143) Google Scholar). We would also point out that some of the molecules noted by Arakawa and Numata as vitrifying that "are obviously not contributing to desiccation tolerance," such as BSA, have in fact been shown to be protective during drying (Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Piszkiewicz et al., 2019Piszkiewicz S. Gunn K.H. Warmuth O. Propst A. Mehta A. Nguyen K.H. Kuhlman E. Guseman A.J. Stadmiller S.S. Boothby T.C. et al.Protecting activity of desiccated enzymes.Protein Sci. 2019; 28: 941-951Crossref PubMed Scopus (15) Google Scholar). Second, Arakawa and Numata assert that while CAHS_77580 was observed to vitrify, RNAi and heterologous expression studies did not show this protein to be necessary or sufficient for desiccation tolerance. However, because RNAi is not fully penetrant, it is possible that CAHS_77580 RNAi was simply less efficient than RNAi targeting other CAHS genes that did show loss of tolerance. Additionally, a conservative approach to assessing significance for functional assays was taken and any results with p > 0.01 were considered not significant (Figure 4 of Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The p value for RNAi knockdown of CAHS_77580 was p > 0.01, but p < 0.05, which is in line with typical assessments of significance and would be interpreted by most to indicate that this protein is in fact necessary for robust desiccation tolerance in tardigrades. The fact that heterologous expression of CAHS_77580 in yeast resulted in a novel glass transition, but not a statistical increase in desiccation tolerance, is not evidence that vitrification is unimportant. Other parameters, such as expression level, could affect the ability of a vitrifying molecule to confer protection. For example, vitrification of trehalose is important for its protective capabilities, but the intracellular level of trehalose also dictates levels of protection (Tapia et al., 2015Tapia H. Young L. Fox D. Bertozzi C.R. Koshland D. Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. USA. 2015; 112: 6122-6127Crossref PubMed Scopus (75) Google Scholar). Similarly, in vitro CAHS protection shows a clear concentration dependence (Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Piszkiewicz et al., 2019Piszkiewicz S. Gunn K.H. Warmuth O. Propst A. Mehta A. Nguyen K.H. Kuhlman E. Guseman A.J. Stadmiller S.S. Boothby T.C. et al.Protecting activity of desiccated enzymes.Protein Sci. 2019; 28: 941-951Crossref PubMed Scopus (15) Google Scholar), which likely extends in vivo. In conclusion, results from functional studies in Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar should not be used to infer that CAHS proteins do not work via vitrification. Third, Arakawa and Numata assert that the reported glass transitions of CAHS proteins at ~60°C are due exclusively to residual water. Residual water remains in anhydrobiotic organisms even after drying (Clegg, 1978Clegg J. Crowe J.H. Clegg J.S. Dry Biological Systems. Academic Press, New York, USA1978: 117-153Crossref Google Scholar, Clegg, 1986Clegg J. Leopold A.C. Membranes, metabolism and dry organisms. Cornell University Press, New York, USA1986: 169-187Google Scholar; Potts, 1994Potts M. Desiccation tolerance of prokaryotes.Microbiol. Rev. 1994; 58: 755-805Crossref PubMed Google Scholar) and it is known that vitrified materials can contain water (Blasi et al., 2005Blasi P. D'Souza S.S. Selmin F. DeLuca P.P. Plasticizing effect of water on poly(lactide-co-glycolide).J. Control. Release. 2005; 108: 1-9Crossref PubMed Scopus (208) Google Scholar; Farahnaky et al., 2005Farahnaky A. Badii F. Farhat I.A. Mitchell J.R. Hill S.E. Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state.Biopolymers. 2005; 78: 69-77Crossref PubMed Scopus (36) Google Scholar). Thus, Arakawa and Numata observing water in their samples is (1) expected and (2) does not suggest that these samples do not vitrify. In fact, a reexamination of Arakawa and Numata's data suggests that the thermal features observed by DSC are not exclusively due to water evaporation, and based on literature cited by Arakawa and Numata in their Letter it suggests vitrification (below). Arakawa and Numata observed two thermal features in their RvCAHS1 samples stored at ambient conditions, a "lower" (~80°C) and "higher" (~160°C) feature. In RvCAHS1 samples stored in the presence of silica drying agent, the protein's lower thermal feature becomes shallower after drying and the higher feature disappears. The disappearance of this higher feature is, according to their Letter, "because the transition temperature moved above the measured range. An increase in the transition temperature Tg with decreased initial water content is in accord with previous observations as also shown in Farahnaky et al., 2005Farahnaky A. Badii F. Farhat I.A. Mitchell J.R. Hill S.E. Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state.Biopolymers. 2005; 78: 69-77Crossref PubMed Scopus (36) Google Scholar." In other words, this higher feature disappeared in the silica dried sample due to a known phenomenon where removing water from a vitrified material results in an increase in the Tg of that material (Farahnaky et al., 2005Farahnaky A. Badii F. Farhat I.A. Mitchell J.R. Hill S.E. Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state.Biopolymers. 2005; 78: 69-77Crossref PubMed Scopus (36) Google Scholar), which here was outside of the temperature range measured. Somewhat surprisingly, the authors did not comment on the fact that the lower feature not only becomes shallower, but also up-shifts from ~80°C to ~95°C, which according to their own logic and literature cited (Farahnaky et al., 2005Farahnaky A. Badii F. Farhat I.A. Mitchell J.R. Hill S.E. Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state.Biopolymers. 2005; 78: 69-77Crossref PubMed Scopus (36) Google Scholar) is indicative of the strengthening of a vitrified material due to reduced hydration prior to DSC/TGA measurements. The authors may have missed this up-shift in their lower feature since it is partially obscured due to the original y axis scaling of their thermogram, but it is clearly visible when the y axis is rescaled (Figure S1B). This is consistent with the claim that CAHS proteins vitrify (Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). While these up-shifts in Tg with drying are themselves indicative of vitrification, we opted to further examine the vitrifying properties of CAHS proteins by adding humidity back to dried proteins. Adding humidity should have the opposite effect, resulting in a plasticization or weakening of vitrified materials and a down-shift in Tg (Farahnaky et al., 2005Farahnaky A. Badii F. Farhat I.A. Mitchell J.R. Hill S.E. Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state.Biopolymers. 2005; 78: 69-77Crossref PubMed Scopus (36) Google Scholar). We dried purified CAHS-D from Hypsibius exemplaris in a vacuum concentrator overnight and then incubated these samples in triplicate at 60%, 70%, and 95% relative humidity (RH) before measuring Tg. For these partially hydrated samples, an obvious decreasing trend in the Tg that correlates with the increasing humidity was observed (Figure S1C), which one would expect if the CAHS proteins were vitrified (Blasi et al., 2005Blasi P. D'Souza S.S. Selmin F. DeLuca P.P. Plasticizing effect of water on poly(lactide-co-glycolide).J. Control. Release. 2005; 108: 1-9Crossref PubMed Scopus (208) Google Scholar; Farahnaky et al., 2005Farahnaky A. Badii F. Farhat I.A. Mitchell J.R. Hill S.E. Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state.Biopolymers. 2005; 78: 69-77Crossref PubMed Scopus (36) Google Scholar), but not if water were exclusively responsible for the thermographic features. Additionally, one 95% RH replicate had two distinct thermal features, likely a result of uneven rehydration of the CAHS protein in that sample (Figure S1C). The appearance of two distinct thermal features is antagonistic to the idea that these features are due to water alone as one would expect that evaporation of water would take place over a single range, not two discrete temperatures. Based on these new observations, one would conclude that CAHS DSC features are not due exclusively to water and that CAHS proteins vitrify in vitro. Fourth, Arakawa and Numata assert that the fact that the Tg of dried tardigrades and CAHS proteins differ indicates that CAHS proteins are not contributing to vitrification in tardigrades. This may seem logical; however, it has been shown that the glass transition temperature of a material is affected by environmental parameters, such as co-solutes and humidity (Blasi et al., 2005Blasi P. D'Souza S.S. Selmin F. DeLuca P.P. Plasticizing effect of water on poly(lactide-co-glycolide).J. Control. Release. 2005; 108: 1-9Crossref PubMed Scopus (208) Google Scholar; Farahnaky et al., 2005Farahnaky A. Badii F. Farhat I.A. Mitchell J.R. Hill S.E. Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state.Biopolymers. 2005; 78: 69-77Crossref PubMed Scopus (36) Google Scholar; Kasapis et al., 2003Kasapis S. Al-Marhoobi I.M. Mitchell J.R. Molecular weight effects on the glass transition of gelatin/cosolute mixtures.Biopolymers. 2003; 70: 169-185Crossref PubMed Scopus (35) Google Scholar). Therefore, one would not expect a purified protein in a simple buffer to have the same Tg when vitrified in a complex cellular environment, and this difference does not indicate that CAHS proteins are dispensable with regard to tardigrade vitrification contrary to Arakawa and Numata's fourth assertion. Fifth, Arakawa and Numata assert that the ~98°C feature in the thermogram of preconditioned tardigrades presented in Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar is due to noise. While it is true that these data were not replicated due to the large input of animals needed for such studies, it should be noted that Arakawa and Numata's data were also not replicated, nor do they address why their data is missing other high-temperature features present in Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar that are clearly not the result of noise. Potential sources of discrepancies might be Arakawa and Numata's use of different drying methods or their not taking into account survival of the animals. Additionally, Arakawa and Numata do not explain why their tardigrade DSC data did not replicate an independent study on tardigrade vitrification, which identified vitrification in 6 different tardigrade samples with Tgs closely mirroring the 98°C Tg Boothby et al. found (e.g., 92.9°C ± 5.3°C and 91.8°C ± 9.4°C). It is unclear why the reader should accept Arakawa and Numata's conclusion that tardigrades are not vitrifying when anhydrobiotic given that (1) their data is not replicated, (2) a survival control was not performed to ensure that animals without an observable Tg are desiccation tolerant, (3) their data did not replicate higher temperature DSC features that are clearly not due to noise, and (4) their experiment failed to replicate data from an independent study that found similar Tgs to the one reported in Boothby et al., 2017Boothby T.C. Tapia H. Brozena A.H. Piszkiewicz S. Smith A.E. Giovannini I. Rebecchi L. Pielak G.J. Koshland D. Goldstein B. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.Mol. Cell. 2017; 65: 975-984.e5Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar in multiple tardigrade species. In summary, Arakawa and Numata's tardigrade DSC data do not offer a compelling rebuttal to multiple observations that tardigrades vitrify. Finally, Arakawa and Numata suggest that "high affinity of CAHS proteins to water" may be linked to tardigrade desiccation tolerance. That CAHS proteins help tardigrades retain water and that this is protective seems unlikely given the fact that their TGA data show that desiccation-tolerant tardigrades have less water by mass than desiccation-sensitive tardigrades. If anything, their data suggest that tardigrades expressing CAHS proteins retain less water than those lacking these proteins. In conclusion, the five assertions made in Arakawa and Numata's Letter fail to demonstrate that tardigrades or CAHS proteins cannot vitrify upon desiccation, and their TGA data suggests their proposed mechanism of water retention by CAHS proteins is unlikely. While there is evidence that CAHS proteins vitrify, the vitrification hypothesis is not mutually exclusive with other mechanisms of desiccation tolerance. Work by Sakurai et al., 2008Sakurai M. Furuki T. Akao K. Tanaka D. Nakahara Y. Kikawada T. Watanabe M. Okuda T. Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki.Proc. Natl. Acad. Sci. USA. 2008; 105: 5093-5098Crossref PubMed Scopus (143) Google Scholar demonstrates that in the larva of Polypedilum vanderplanki trehalose mediates desiccation tolerance via vitrification and water replacement. It is possible that the vitrified matrices formed by CAHS proteins may work in concert with what water is left, similar to the anchorage model, which posits that a highly viscous or vitrifying material could help to coordinate residual water molecules to allow desiccation-sensitive proteins to remain solvated and properly folded (Bellavia et al., 2011Bellavia G. Giuffrida S. Cottone G. Cupane A. Cordone L. Protein Thermal Denaturation and Matrix Glass Transition in Different Protein−Trehalose−Water Systems.J. Phys. Chem. 2011; 115: 6340-6346Crossref PubMed Scopus (51) Google Scholar). It will be exciting to see what new experiments shed light on other potential mechanisms by which CAHS proteins mediate protection. Kazuharu Arakawa and Keiji Numata are thanked for their work and for providing their original DSC data for reanalysis. This publication was made possible by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 2P20GM103432 as well as NASA ( 80NSSC20K0283 ) and DARPA ( W911NF2020137 ) awards to TCB. Download .pdf (.26 MB) Help with pdf files Document S1. Figure S1 Tardigrades Use Intrinsically Disordered Proteins to Survive DesiccationBoothby et al.Molecular CellMarch 16, 2017In BriefTardigrades (water bears) survive a number of extreme stresses, including desiccation. Boothby et al. show that tardigrade disordered proteins are required for desiccation tolerance and exogenously protect cells and purified enzymes from drying. When dry, these proteins form glasses, the integrity of which correlates with their protective capabilities. Full-Text PDF Open ArchiveReconsidering the "glass transition" hypothesis of intrinsically unstructured CAHS proteins in desiccation tolerance of tardigradesArakawa et al.Molecular CellFebruary 04, 2021In BriefWater is essential for all living systems, yet a number of meiofaunal organisms are able to withstand almost complete desiccation through a state of suspended animation called anhydrobiosis. Tardigrades are especially unique in that many of them can enter anhydrobiosis in a very short period of time compared to anhydrobiotic arthropods such as sleeping chronomid and artemia that accumulate a large amount of disaccharide trehalose during transition (Wełnicz et al., 2011). However, anhydrobiosis in tardigrades remains relatively unexplored, and we welcome the contribution of Boothby and colleagues (Boothby et al., 2017), especially in replicating many previous findings including the requirement of preconditioning for successful anhydrobiosis in Hypsibius dujardini (Kondo et al., 2015), that CAHS proteins are intrinsically unstructured (Yamaguchi et al., 2012) and that CAHS proteins are constitutively highly abundant (Yamaguchi et al., 2012). Full-Text PDF Open Archive
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