Archaeal Hsp14 drives substrate shuttling between small heat shock proteins and thermosome: insights into a novel substrate transfer pathway

Abstract

Heat shock proteins maintain protein homeostasis and facilitate the survival of an organism under stress. Archaeal heat shock machinery usually consists of only sHsps, Hsp70, and Hsp60. Moreover, Hsp70 is absent in thermophilic and hyperthermophilic archaea. In the absence of Hsp70, how aggregating protein substrates are transferred to Hsp60 for refolding remains elusive. Here, we investigated the crosstalk in the heat shock response pathway of thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In the present study, we biophysically and biochemically characterized one of the small heat shock proteins, Hsp14, of S. acidocaldarius. Moreover, we investigated its ability to interact with Hsp20 and Hsp60 to facilitate the substrate proteins' folding under stress conditions. Like Hsp20, we demonstrated that the dimer is the active form of Hsp14, and it forms an oligomeric storage form at a higher temperature. More importantly, the dynamics of the Hsp14 oligomer are maintained by rapid subunit exchange between the dimeric states, and the rate of subunit exchange increases with increasing temperature. We also tested the ability of Hsp14 to form hetero-oligomers via subunit exchange with Hsp20. We observed hetero-oligomer formation only at higher temperatures (50 °C-70 °C). Furthermore, experiments were performed to investigate the interaction between small heat shock proteins and Hsp60. We demonstrated an enthalpy-driven direct physical interaction between Hsp14 and Hsp60. Our results revealed that Hsp14 could transfer sHsp-captured substrate proteins to Hsp60, which then refolds them back to their active form.

Minimal Yet Powerful: The Role of Archaeal Small Heat Shock Proteins in Maintaining Protein Homeostasis

“…This phenomenon has been observed in Mycobacterium butanii , Pyrococuus furiosus, and also in Sulfolobus acidocaldarius ( Laksanalamai et al, 2006 ; Laksanalamai et al, 2009 ; Luo et al, 2009 ; Roy et al, 2021 ). It has been observed that excess addition of sHsp from Thermococcus sp .…”

“…Furthermore, in hyperthermophilic and some thermophilic archaea, Hsp70 is also absent ( Usui et al, 2004 ). Interestingly, it has recently been demonstrated that in the absence of Hsp70, sHsps of these organisms could directly transfer the associated substrate protein to Hsp60 for refolding ( Laksanalamai et al, 2006 ; Laksanalamai et al, 2009 ; Roy et al, 2021 ). Studies have shown that Pyrococcus furiosus Hsp60 refolds heat-inactivated Taq polymerase five-fold more efficiently in the presence of sHsp ( Laksanalamai et al, 2006 ).…”

“…Studies have shown that Pyrococcus furiosus Hsp60 refolds heat-inactivated Taq polymerase five-fold more efficiently in the presence of sHsp ( Laksanalamai et al, 2006 ). In Sulfolobus acidocaldarius , Hsp14 can bind to the unfolded substrate and is also involved in shuttling the unfolded substrate to Hsp60 for final ATP-dependent refolding ( Figure 3 ) ( Roy et al, 2021 ). Other than thermophilic archaea, transfer of the unfolded substrate from sHsp to Hsp60 was also reported in Methanococcoides burtonii , a cold adaptive archaeon ( Laksanalamai et al, 2009 ).…”

“…However, the exact mechanism of substrate shuttling between sHsps and Hsp60 is not yet clear. Recently, a direct physical interaction between Hsp14 and Hsp60 is reported in Sulfolobus acidocaldarius ( Roy et al, 2021 ). In bacteria, a substrate transfer between Hsp60 and sHsp was also demonstrated in Deinococcus radiodurans , however, unlike archaea, substrate transfer between sHsp and Hsp70 remains the predominant refolding pathway ( Bepperling et al, 2012 ).…”

“…In E. coli , two sHsps, IbpA and IbpB, form a co-complex that gets associated with unfolded substrate proteins and recruits Hsp70 for downstream substrate processing ( Zwirowski et al, 2017 ). In archaea, only single evidence of such hetero-oligomer formation is reported to date in Sulfololobus acidocaldarius , where Hsp20 and Hsp14 can dynamically associate with each other to form a co-complex ( Roy et al, 2021 ). Moreover, the hetero-oligomer formation between Hsp14 and Hsp20 has been shown to occur at temperatures beyond 50°C when the subunit exchange rate crosses a certain threshold ( Roy et al, 2021 ).…”