Target enzymes are stabilized by AfrLEA6 and a gain of α-helix coincides with protection by a group 3 LEA protein during incremental drying

LeBlanc, Blase M.; Hand, Steven C.

doi:10.1016/j.bbapap.2021.140642

Cited by 13 publications

(12 citation statements)

References 82 publications

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“…varieornatus was also reported to form granules in response to hyperosmotic stress in human cultured cells [ 37 ], and these stress-dependent granule condensation by CAHS8 and CAHS1 resembled the stress-granule formation in mammalian cells that occurs through phase separation to create protective membrane-less compartments against stress [ 42 , 43 ]. A recent study revealed that another desiccation tolerance protein, AfrLEA6, which is a group 6 LEA protein of Artemia franciscana , also undergoes phase separation to form granules in insect cells [ 14 ] and protects enzyme activity from desiccation stress in vitro [ 44 ]. Like stress granules and AfrLEA6 granules, CAHS8 granules exhibited certain sensitivity against 1,6-hexanediol treatment ( S9 Fig ).…”

Section: Discussionmentioning

confidence: 99%

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel

et al. 2022

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Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed “TFE-Dependent ReversiblY condensing Proteins (T-DRYPs).” Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

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Section: Discussionmentioning

confidence: 99%

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel

et al. 2022

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“…Stressdependent granule condensation by CAHS8 resembled the stress-granule formation in mammalian cells that occurs through phase separation to create protective membrane-less compartments against stress [34,35]. A recent study revealed that another desiccation tolerance protein, AfrLEA6, which is a group 6 LEA protein of Artemia franciscana, also undergoes phase separation to form granules in insect cells [14] and protects enzyme activity from desiccation stress in vitro [36]. Like stress-granules Our isolation scheme (Fig 1A ) successfully identified CAHS proteins that reversibly condensed to filaments or granules in a stress-dependent manner from anhydrobiotic tardigrades.…”

Section: Discussionmentioning

confidence: 97%

“…Recently, CAHS1 protein from R. varieornatus was also reported to form granules in response to hyperosmotic stress in human cultured cells [34], and these stress-dependent granule condensation by CAHS8 and CAHS1 resembled the stress-granule formation in mammalian cells that occurs through phase separation to create protective membrane-less compartments against stress [39,40]. A recent study revealed that another desiccation tolerance protein, AfrLEA6, which is a group 6 LEA protein of Artemia franciscana, also undergoes phase separation to form granules in insect cells [14] and protects enzyme activity from desiccation stress in vitro [41]. Like stress-granules and AfrLEA6 granules, CAHS8 granules exhibited certain sensitivity against 1,6-hexanediol treatment (Fig 2C).…”

Section: Discussionmentioning

confidence: 99%

Stress-dependent cell stiffening by tardigrade tolerance proteins through reversible formation of cytoskeleton-like filamentous network and gel-transition

Tanaka

Nakano

Watanabe

et al. 2021

Preprint

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Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective cytoplasmic-abundant heat-soluble (CAHS) proteins which are essential for the anhydrobiotic survival of tardigrades. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "dehydration-induced reversibly condensing proteins (DRPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRPs, 3 of which were major components of DRPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro, which increases the mechanical strength of cell-like microdroplets. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and contribute to the exceptional stability of dehydrated tardigrades.

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“…Molecular shield proteins have been proposed to bind partially unfolded proteins during desiccation to limit contact with other denatured proteins and the formation of toxic aggregates (3,6). Late embryogenesis abundant (LEA) proteins from desiccation-tolerant organisms are one family of protectants that have been shown to fulfill this role (4,(7)(8)(9)(10)(11). A limited number of other proteins have also been shown to act as molecular shields, but there are likely many other proteins that carry out a similar function to prevent desiccation-induced aggregation and loss of protein function (6,12).…”

Section: Introductionmentioning

confidence: 99%

“…These results suggest that sHSPs, classical chaperones, provide a mechanism of general stress resistance that can also be deployed to support survival during anhydrobiosis.formation of toxic aggregates (3,6). Late embryogenesis abundant (LEA) proteins from desiccation-tolerant organisms are one family of protectants that have been shown to fulfill this role (4,(7)(8)(9)(10)(11). A limited number of other proteins have also been shown to act as molecular shields, but there are likely many other proteins that carry out a similar function to prevent desiccation-induced aggregation and loss of protein function (6,12).Small heat shock protein (sHSPs) are small (12-43 kDa) proteins that contain an alphacrystallin domain flanked by disordered N-and C-terminal sequences (13,14).…”

mentioning

confidence: 99%

Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation

Hibshman¹,

Carra

Goldstein

2022

Preprint

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Small heat shock proteins (sHSPs) are chaperones known for their response to heat stress. While sHSPs have well-characterized roles in heat tolerance, potential roles for sHSPs in desiccation tolerance have not been as thoroughly explored. We identified nine sHSPs from the genome of the tardigrade Hypsibius exemplaris, each containing a conserved alpha-crystallin domain flanked by disordered regions. Many of these sHSPs are highly expressed and in some cases are upregulated during desiccation. We found that tardigrade sHSPs were sufficient to improve desiccation tolerance when expressed in E. coli. Purification and subsequent analysis of two sHSPs, HSP21 and HSP24.6, revealed that these proteins can form large complexes in vitro, similar to oligomeric assemblies documented for other sHSPs. These proteins limited heat-induced aggregation of the model enzyme citrate synthase. Heterologous expression of HSP24.6 improved bacterial heat shock survival, and the protein significantly reduced heat-induced aggregation of soluble bacterial protein. Thus, HSP24.6 likely chaperones against protein aggregation to promote survival of heat stress. Furthermore, HSP21 and HSP24.6 also limited desiccation-induced aggregation and loss of function of citrate synthase. This suggests a mechanism by which tardigrade sHSPs promote desiccation tolerance: by limiting desiccation-induced protein aggregation, thereby maintaining proteostasis and supporting survival. These results suggest that sHSPs, classical chaperones, provide a mechanism of general stress resistance that can also be deployed to support survival during anhydrobiosis.

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Target enzymes are stabilized by AfrLEA6 and a gain of α-helix coincides with protection by a group 3 LEA protein during incremental drying

Cited by 13 publications

References 82 publications

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel

Stress-dependent cell stiffening by tardigrade tolerance proteins through reversible formation of cytoskeleton-like filamentous network and gel-transition

Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation

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