Expression, Functional Characterization, and Preliminary Crystallization of the Cochaperone Prefoldin from the Thermophilic Fungus Chaetomium thermophilum

Abstract

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin–chaperonin system.

Novel Proteome and N-Glycoproteome of the Thermophilic Fungus Chaetomium thermophilum in Response to High Temperature

“…Protein misfolding is the main feature of heat stress ( Jacob et al, 2017 ). Heat shock proteins have a complex protective mechanism against heat stress, which is important in folding peptide chains and the degradation and removal of denatured proteins to maintain protein homeostasis and cell physiological functions ( Voellmy and Boellmann, 2007 ; Venkatesh and Suzuki, 2017 ; Morita et al, 2018 ). As shown in the Venn diagram of differentially expressed proteins ( Figure 8 ), five Hsps G0S4G4, G0SAF6, G0SCQ6, G0SH15, and G0S5V3 were upregulated in T50/T30 and T55/T30, which correspond to XP_006692738, XP_006694613, XP_006696127, XP_006697122, and XP_006693811 in NCBI; four Hsps G0RYB3, G0S8C8, G0S4L1, and G0SBY8 were upregulated in T50/T30, T55/T30, and T55/T50, which correspond to XP_006691141, XP_006692599, XP_006693582, and XP_006695859 in NCBI; and one Hsp G0RYP6 (XP_006691274) was upregulated in T55/T50 and T55/T30, which correspond to XP_006691274 in NCBI.…”

Bridging human chaperonopathies and microbial chaperonins

“…Another experimental model recently developed uses the thermophilic fungus Chaetomium thermophilum : the entire CCT complex from this microbe has recently been expressed in recombinant form, which allowed the study of ring closure at single-molecule scale 48 , 49 . Proteins from the thermophilic eukaryote C. thermophilum , like those from thermophilic archaea such as P. furiosus , exhibit structural stability compared with those from mesophilic organisms and are, therefore, convenient to study the effect of mutations.…”

“…Eukaryotic prefoldin is a multisubunit chaperone, with a hexameric complex assembled from six different subunits 61 , 62 . Recently, a hexameric prefoldin complex from C. thermophilum (CtPFD) was successfully reconstituted 49 . The interaction between CtPFD and CCT from C. thermophilum (CtCCT) was analyzed by surface plasmon resonance and the K D value is similar to that of the archaeal counterparts 49 .…”

“…Recently, a hexameric prefoldin complex from C. thermophilum (CtPFD) was successfully reconstituted 49 . The interaction between CtPFD and CCT from C. thermophilum (CtCCT) was analyzed by surface plasmon resonance and the K D value is similar to that of the archaeal counterparts 49 . The system will be used to analyze the effects of pathogenic mutations of CCT/PFD folding pathways affecting the maturation of specific client proteins such as actin and tubulin.…”

A comprehensive analysis of prefoldins and their implication in cancer

“…Unlike archaeal PFDNs, it was not able to prevent citrate synthase aggregation, supporting the idea that eukaryotic PFDN has more specific functions than a regular chaperone as happens in archaea. Nevertheless, eukaryotic PFDN is also able to bind actin and tubulin ( Morita et al, 2018 ), and genetic ablation of the prefoldin genes in yeast alters the microtubules phenotype ( Chesnel et al., 2020 ; Geissler et al., 1998 ).…”