Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

Abstract

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation

“…These gene families, CAHS, SAHS, and MAHS, were previously identified in a proteomic analysis of tardigrades (Tanaka et al, 2015; Yamaguchi et al, 2012). On the basis of heat solubility experiments and circular dichroism spectropolarimetry, all three families are thought to encode IDPs (Tanaka et al, 2015; Yamaguchi et al, 2012).…”

“…These observations, coupled with the fact that TDPs are induced by drying in H. dujardini , suggests that they play a role in tardigrade stress tolerance (Yamaguchi et al, 2012; Tanaka et al, 2015). However, until now no studies have been conducted to directly examine the effect of environmental conditions on the expression of genes encoding TDPs or their involvement in tardigrade desiccation tolerance.…”

“…On the basis of heat solubility experiments and circular dichroism spectropolarimetry, all three families are thought to encode IDPs (Tanaka et al, 2015; Yamaguchi et al, 2012). Previous bioinformatic studies suggest that not all members of disordered protein families are natively unfolded (Hundertmark and Hincha, 2008).…”

“…These data, combined with heat solubility, circular dichroism, and bioinformatics approaches, show that many, if not all, tardigrade CAHS, and likely MAHS and SAHS proteins, are disordered. We refer to these families (CAHS, SAHS, MAHS) of tardigrade-specific IDPs as TDPs to distinguish them from other IDPs, because, at the sequence level, no homologs of TDPs are found outside the phylum tardigrade (Tanaka et al, 2015; Yamaguchi et al, 2012). …”

“…Several families of IDPs, such as late embryogenesis abundant (LEA) proteins and hydrophilins, have known or suspected roles in stress tolerance in organisms spanning all kingdoms of life (Chakrabortee et al, 2012; Dang et al, 2014; Garay-Arroyo et al, 2000), and a recent study demonstrated slightly improved hyperosmotic tolerance in cells heterologously expressing an MAHS protein (Tanaka et al, 2015). These observations, coupled with the fact that TDPs are induced by drying in H. dujardini , suggests that they play a role in tardigrade stress tolerance (Yamaguchi et al, 2012; Tanaka et al, 2015).…”