The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKK kinases prior to complex assembly
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ABSTRACT:
PROVIDER: PRJNA761401 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15743179.fastq.gz | Fastqsanger.gz | |||
| SRR15743180.fastq.gz | Fastqsanger.gz | |||
| SRR15743181.fastq.gz | Fastqsanger.gz | |||
| SRR15743182.fastq.gz | Fastqsanger.gz | |||
| SRR15743183.fastq.gz | Fastqsanger.gz |
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Project description:PIKKs are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work identified an Hsp90 cochaperone, the TTT complex, which specifically stabilizes PIKKs. Here we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we found that they do not engage in cotranslational assembly with their partners. Rather, we accumulated evidence that TTT protects nascent PIKK polypeptides from misfolding and degradation and that PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.
2021-09-10 | GSE183610 | GEO
Project description:PIKKs are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work identified an Hsp90 cochaperone, the TTT complex, which specifically stabilizes PIKKs. Here we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we found that they do not engage in cotranslational assembly with their partners. Rather, we accumulated evidence that TTT protects nascent PIKK polypeptides from misfolding and degradation and that PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.
2021-09-10 | PXD028351 | Pride
Project description:Cdc37 is a core cochaperone of the Hsp90 machinery. It is involved in an early stage of the chaperone cycle to activate nascent forms of protein kinases as well as to stabilize mature forms of a subset of protein kinases. Overall, the chaperone cycle is quite complex and involves several steps. Progression, through these steps is regulated by the incorporation of several other cochaperones, ATP hydrolysis and posttranslational modifications on both Cdc37 and Hsp90. We show that phosphorylation of Cdc37 at Y298 by Yes kinase results in partial unfolding of its C-terminal domain, without affecting its ability to form binary or ternary complexes with kinases and Hsp90. Unfolding, unmasks a phosphopeptide sequence that exhibits high affinity for SH2 domains of non-receptor tyrosine kinases (nRTKs), resulting in their recruitment in the chaperone complex. In turn, the high local concentration of nRTKs potentiates Hsp90 phosphorylation at Y197, which results in dissociation of this early kinase-recruitment complex
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Project description:Hsp90 is a molecular chaperone involved in the regulation and maturation of kinases and transcription factors. In Caenorhabditis elegans, it contributes to the development of fertility, maintenance of muscle structure, the regulation of heat-shock response and dauer state. To understand the consequences of Hsp90-depletion, we studied Hsp90 RNAi-treated nematodes by DNA microarrays and mass spectrometry. We find that upon development of phenotypes the levels of chaperones and Hsp90 cofactors are increased, while specific proteins related to the innate immune response are depleted. In microarrays, we further find many differentially expressed genes related to gonad and larval development. These genes form an expression cluster that is regulated independently from the immune response implying separate pathways of Hsp90-involvement. Using fluorescent reporter strains for the differentially expressed immune response genes skr-5, dod-24 and clec-60 we observe that their activity in intestinal tissues is influenced by Hsp90-depletion. Instead, effects on the development are evident in both gonad arms. After Hsp90-depletion, changes can be observed in early embryos and adults containing fluorescence-tagged versions of SEPA-1, CAV-1 or PUD-1, all of which are downregulated after Hsp90-depletion. Our observations identify molecular events for Hsp90-RNAi induced phenotypes during development and immune responses, which may help to separately investigate independent Hsp90-influenced processes that are relevant during the nematode’s life and development. We used microarrays to detail the global programme of gene expression after RNAi-mediated Hsp90 knock-down.
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2019-03-06 | PXD009777 | Pride
Project description:Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case of Hsp70 and Hsp90, which direct their bound substrate to degradation through ubiquitination in concert with the cochaperone CHIP –an E3 ligase. We have generated complexes between the chaperone (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). The two ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinate the substrate, and this is done with a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, show the substrate located between the chaperone and the cochaperone, which suggests an ubiquitination mechanism. Both complexes are extremely flexible, which is crucial for the ubiquitination process.
2019-03-07 | PXD009756 | Pride
Project description:Transcription initiation involves the coordinated activities of large multimeric complexes that are organized into functional modules. Little is known about the mechanisms and pathways that govern their assembly from individual components. We report here several principles governing the assembly of the highly conserved SAGA and NuA4 co-activator complexes. Using fission yeast, which contain two functionally non-redundant paralogs of the shared Tra1 subunit, we demonstrate that Tra1 contributes to scaffolding the entire NuA4 complex. In contrast, within SAGA, Tra1 specifically promotes the incorporation of the de-ubiquitination module (DUB), defining an ordered assembly pathway. Biochemical and functional analyses elucidated the mechanism by which Tra1 assemble differentially into SAGA or NuA4 and identified a small, conserved region of Spt20 that is both necessary and sufficient to anchor Tra1 within SAGA. Finally, we establish that Hsp90 and its cochaperone TTT are required for Tra1 de novo incorporation into both SAGA and NuA4, indicating that Tra1, a pseudokinase of the PIKK family, shares a dedicated chaperone machinery with its cognate kinases. Overall, our work brings mechanistic insights into the de novo assembly of transcriptional complexes through ordered pathways and reveals the contribution of dedicated chaperones to this process.
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Project description:A375 cells with inducible knockdown HSF1 with and without HSP90 inhibitor treatment A375 cells were seeded in 6-well plates for 3d with 100ng/ml doxycycline prior to treatment with 0.1% DMSO or 100nM HSP990 for 3h
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