Abstract:Prefoldin is a hetero-hexameric ATP-independent chaperone, shared by eukaryotes and archaea, which binds non-native proteins preventing them from aggregation. We report the identification and characterization in vivo and in vitro of the first prefoldin from a crenarchaeon, the hyperthermophile Sulfolobus solfataricus. A functional complex was obtained either co-expressing the alpha- and beta-prefoldin subunits in Escherichia coli, or incubating at high temperature the separately expressed subunits. In S. solfa… Show more
“…As shown in Figure 1B, this was the case, thus suggesting that, in cell extracts, reverse gyrase, PolY and SSB are present in binary and/or ternary complexes. As a specificity control, we used an antibody directed against the β-subunit of the chaperone prefoldin (28), a protein not involved directly in DNA metabolism, which revealed no bands immunoprecipitating with PolY. Figure 1.Physical interaction of reverse gyrase and PolY.…”
Section: Resultsmentioning
confidence: 99%
“…Total and fractionated extracts were analyzed using the Amersham ECL-Plus kit and a VersaDoc apparatus (BioRad). Recombinant TopR1 purified from E. coli was used to raise custom polyclonal antibodies in rabbit; other antibodies were as follows: anti-PolY ( 27 ); anti-β-subunit of S. solfataricus prefoldin ( 28 ); anti- S. solfataricus SSB [gift of M. F. White, St Andrews University, UK ( 29 )].…”
Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.
“…As shown in Figure 1B, this was the case, thus suggesting that, in cell extracts, reverse gyrase, PolY and SSB are present in binary and/or ternary complexes. As a specificity control, we used an antibody directed against the β-subunit of the chaperone prefoldin (28), a protein not involved directly in DNA metabolism, which revealed no bands immunoprecipitating with PolY. Figure 1.Physical interaction of reverse gyrase and PolY.…”
Section: Resultsmentioning
confidence: 99%
“…Total and fractionated extracts were analyzed using the Amersham ECL-Plus kit and a VersaDoc apparatus (BioRad). Recombinant TopR1 purified from E. coli was used to raise custom polyclonal antibodies in rabbit; other antibodies were as follows: anti-PolY ( 27 ); anti-β-subunit of S. solfataricus prefoldin ( 28 ); anti- S. solfataricus SSB [gift of M. F. White, St Andrews University, UK ( 29 )].…”
Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.
“…The observed transcriptional upregulation of the Pfdα and Pfdβ genes after 30 minutes of HS treatment (Figure 4; Supplementary Table S2) was not found for the homologs in S. solfataricus (Tachdjian & Kelly, 2006;D'Amaro et al, 2008). Although their upregulation was less pronounced than that of Thα and Thβ, it might be indirectly linked, keeping in mind that the function of PFD is intertwined with the chaperone functioning of the thermosome complex (Vainberg et al, 1998;Okochi et al, 2004;Ohtaki et al, 2010;Lemmens et al, 2018).…”
Section: Transcriptional Expression Of Important Hsps Upon Hsmentioning
confidence: 87%
“…In addition, sHSP-1 stabilizes membrane lipids, counteracting the effect of temperature on membrane permeability (Roy et al, 2018). Similar to sHSPs, PFDs are ATP-independent chaperones that bind a large variety of denaturing proteins and typically transfer these to the thermosome complex for refolding (Vainberg et al, 1998;Siegert et al, 2000;Okochi et al, 2004;D'Amaro et al, 2008;Ohtaki et al, 2010;Sahlan et al, 2018). The set of HSPs present in Sulfolobales is relatively well conserved, in terms of protein sequence identity and of gene synteny and genomic location of the respective genes (Supplementary Figure S1).…”
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“…Some β PFDN subunits are also able to form tetrameric complexes, such as the β1 PFDN from Thermococcus strain KS-1 ( Kida et al., 2008 ). Other archaea, nevertheless, seem to only present a pair of α and β PFDN genes, like in Sulfolobus solfataricus ( D'Amaro et al., 2008 ).…”
Summary
Prefoldins (PFDNs) are evolutionary conserved co-chaperones, initially discovered in archaea but universally present in eukaryotes. PFDNs are prevalently organized into hetero-hexameric complexes. Although they have been overlooked since their discovery and their functions remain elusive, several reports indicate they act as co-chaperones escorting misfolded or non-native proteins to group II chaperonins. Unlike the eukaryotic PFDNs which interact with cytoskeletal components, the archaeal PFDNs can bind and stabilize a wide range of substrates, possibly due to their great structural diversity. The discovery of the unconventional RPB5 interactor (URI) PFDN-like complex (UPC) suggests that PFDNs have versatile functions and are required for different cellular processes, including an important role in cancer. Here, we summarize their functions across different species. Moreover, a comprehensive analysis of PFDNs genomic alterations across cancer types by using large-scale cancer genomic data indicates that PFDNs are a new class of non-mutated proteins significantly overexpressed in some cancer types.
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