TrypOx, a Novel Eukaryotic Homolog of the Redox-Regulated Chaperone Hsp33 in Trypanosoma brucei

Abstract

ATP-independent chaperones are widespread across all domains of life and serve as the first line of defense during protein unfolding stresses. One of the known crucial chaperones for bacterial survival in a hostile environment (e.g., heat and oxidative stress) is the highly conserved, redox-regulated ATP-independent bacterial chaperone Hsp33. Using a bioinformatic analysis, we describe novel eukaryotic homologs of Hsp33 identified in eukaryotic pathogens belonging to the kinetoplastids, a family responsible for lethal human diseases such as Chagas disease as caused by Trypanosoma cruzi, African sleeping sickness caused by Trypanosoma brucei spp., and leishmaniasis pathologies delivered by various Leishmania species. During their pathogenic life cycle, kinetoplastids need to cope with elevated temperatures and oxidative stress, the same conditions which convert Hsp33 into a powerful chaperone in bacteria, thus preventing aggregation of a wide range of misfolded proteins. Here, we focused on a functional characterization of the Hsp33 homolog in one of the members of the kinetoplastid family, T. brucei, (Tb927.6.2630), which we have named TrypOx. RNAi silencing of TrypOx led to a significant decrease in the survival of T. brucei under mild oxidative stress conditions, implying a protective role of TrypOx during the Trypanosomes growth. We then adopted a proteomics-driven approach to investigate the role of TrypOx in defining the oxidative stress response. Depletion of TrypOx significantly altered the abundance of proteins mediating redox homeostasis, linking TrypOx with the antioxidant system. Using biochemical approaches, we identified the redox-switch domain of TrypOx, showing its modularity and oxidation-dependent structural plasticity. Kinetoplastid parasites such as T. brucei need to cope with high levels of oxidants produced by the innate immune system, such that parasite-specific antioxidant proteins like TrypOx-which are depleted in mammals-are highly promising candidates for drug targeting.

The Cys Sense: Thiol Redox Switches Mediate Life Cycles of Cellular Proteins

“…One such redox-regulated chaperone, which uses protein plasticity for its activation, is a highly conserved ATP-independent holdase chaperone, Hsp33. Hsp33 is a predominantly bacterial chaperone, also found in unicellular pathogens such as Trypanosoma and Leishmania [ 122 ], which protects microbes against a wide range of oxidants similar to those applied by the host immune system [ 21 ]. Exposure to oxidants or chlorine species (e.g., HOCl) triggers large conformational changes and exposure of hydrophobic regions involved in the anti-aggregation activity [ 91 , 123 , 126 , 127 ].…”

The Central Role of Redox-Regulated Switch Proteins in Bacteria

“…Around 20 years ago, the Hsp33 chaperone was discovered as a first line of defense chaperone protecting bacterial proteins against aggregation during oxidative stress in E. coli (Hoffmann et al, 2004). Since then, additional homologues of Hsp33 were identified and characterized in other bacterial species as well as in unicellular algae (Segal and Shapira, 2015) and pathogens (Trypanosoma and Leishmania) (Aramin et al, 2020). This highlights Hsp33 as a promising new drug target against bacterial and Trypanosoma pathogens.…”

“…Hsp33 is one of the crucial ATP-independent holdases (or holding chaperones), which is activated under conditions that lead to protein misfolding and accumulation of toxic aggregates, such as oxidative unfolding. Hsp33 "senses" the presence of oxidants or chlorine species (e.g., HOCl) through a highly reactive Zn center, comprising of four completely conserved cysteines forming CXCX and CXXC motifs harboring one Zn 2+ ion in the inactive, reduced form (Ilbert et al, 2006;Aramin et al, 2020). Oxidation triggers Zn release, formation of two disulfide bonds and rapid unfolding of almost half of the protein, which exposes hydrophobic regions involved in the antiaggregation activity of Hsp33 (Rimon et al, 2017).…”

A dynamic redox switch turns TRC40 into a chaperone protecting human cells against ATP-depleting, oxidative stress

“…The oxidative activation of ATP-independent chaperones serves as a first line of defense to prevent wide-spread protein aggregation [10][11][12] . Since the discovery of Hsp33 as the first redox-regulated chaperone in E. coli 13 , stress-activated chaperones have been shown to be essential for mediating resistance towards environmental challenges in various organisms [14][15][16][17][18] .…”