CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control.
- MeSH
- alosterická regulace MeSH
- exprese genu MeSH
- interferonový regulační faktor 1 genetika metabolismus MeSH
- kinetika MeSH
- lidé MeSH
- lymfocyty cytologie metabolismus MeSH
- mapování interakce mezi proteiny MeSH
- molekulární modely MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteiny tepelného šoku HSP70 chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- terciární struktura proteinů MeSH
- ubikvitinace MeSH
- ubikvitinligasy chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20-40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106-140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or "docking" of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase.
- MeSH
- aminokyselinové motivy MeSH
- interferonový regulační faktor 1 * chemie metabolismus MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- nádorové buněčné linie MeSH
- nádorové supresorové proteiny * chemie metabolismus MeSH
- peptidové fragmenty metabolismus MeSH
- proteiny tepelného šoku HSP70 metabolismus MeSH
- reakce na tepelný šok MeSH
- sekvence aminokyselin MeSH
- terciární struktura proteinů MeSH
- těžké kovy toxicita MeSH
- ubikvitin konjugující enzymy metabolismus MeSH
- ubikvitinace * účinky léků MeSH
- ubikvitinligasy * metabolismus MeSH
- vazba proteinů účinky léků MeSH
- Check Tag
- lidé MeSH