Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade

Abstract

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades’ response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol–gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells

“…For those proteins with no visible condensates, the experimental conditions may be insufficient to support phase separation. PARRC CAHS2 exhibited the most dramatic phenotype, which is consistent with recent work describing the gel forming capabilities of CAHS proteins [44][45] (Figure 4E&F). ApoE variants were of interest, based on the puncta observed in the top performing ApoE constructs.…”

“…While we observe condensate formation in many of our top performing hits, we also observe condensate formation in targets like HYPDU CAHS3 and PARRC CAHS2, which do not reduce apoptosis very much. Additionally, some proteins like PARRC CAHS1 (101-189) are truncated in ways predicted to reduce condensate formation 44,[46][47] , but they still perform well in apoptosis protection. These observations suggest yet-to-be-discovered mechanisms are likely responsible for apoptosis protection in these cases.…”

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

“…This prediction was also supported by the previous circular dichroism (CD) spectroscopy of CAHS1 protein of R. varieornatus, another member of the CAHS family [15]. During the review of this manuscript, two related papers were published [34,35, which reported that two other CAHS proteins, i.e., CAHS1 of R. varieornatus and CAHS8 of H. exemplaris, formed fibrous structure and gel in a concentration-dependent manner in vitro, and the enriched helix structure in the C-terminal regions in either CAHS proteins were demonstrated by elaborate NMR analyses and/or CD spectroscopy under the condition forming filaments or gels. These recent structural analyses are in a good agreement with our structural predictions (Fig 5A and S13 Fig), although the necessity of such helix structure for filament/gel formation was not demonstrated.…”

“…In contrast to filament-forming CAHS3 and CAHS12, CAHS8 alone formed granule-like condensates in both human and insect cells under a hyperosmotic condition (Fig 2A and S8 Fig). 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). Two well-known desiccation-tolerance protein families, LEA proteins and CAHS proteins, are mutually unrelated in the primary sequence, but both become helix-rich structure from unstructured state by dehydration [12,34]. TFE also induces similar conformational changes of both protein families 18 [15,27] and thereby might mimic dehydration stress.…”