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- Svanström, Andreas
(author)
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The Extended Role of the Molecular Chaperone CCT
- 2016
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Doctoral thesis (other academic/artistic)abstract
- The oligomeric chaperone CCT is a large ATP-dependent chaperonin that consists of two rings placed back-to-back with eight different paralogous subunits with a size of ~ 55 kDa that sit in each of the two rings. The function of CCT is mainly to fold the abundant proteins actin and tubulin, components of the cytoskeleton. However, several studies have shown that CCT has a wide diversity of low-abundant substrates. In addition, CCT and monomeric subunits of CCT have been shown to influence cytoskeletal organization and processes that the cytoskeleton mediates. The aim of this thesis was to study the role of CCT beyond the folding of proteins. We have overexpressed the subunits of CCT as monomers and demonstrated that monomeric CCTδ has an unknown function at the plasma membrane. The overexpression of monomeric CCTδ mainly induced lamellipodia retraction fibres and the function of monomeric CCTδ at the plasma membrane was shown to be dependent on a wild-type ATP-binding site and a wild-type apical domain of CCTδ. By reducing the levels of individual subunits of CCT, we report in a second study a function of CCTε to regulate the activity of the transcription factor SRF, which controls the transcription of cytoskeletal genes such as actin, via the transcription activator MRTF-A. Cells depleted of CCTε have an increased SRF-mediated transcription in an SRF-luciferase gene reporter system. Monomeric CCTε was shown to interact directly with MRTF-A and the interaction site was identified as the apical domain of CCTε and the cterminal half of MRTF-A. Consistent with an increased SRF-transcription upon the reduction of CCTε levels, the overexpression of monomeric CCTε delayed the translocation of MRTFA to the nucleus in serum-stimulated cells. In our final study, we addressed the possibility of CCT to affect the number of actin filaments via the interaction between CCT and the actin filament severing protein gelsolin. We showed that CCT binds to the activated severing conformation of gelsolin and that CCT inhibits activated gelsolin to sever actin filaments. Taken together, we present several studies that independently identify the CCT oligomer, or its individual subunits, to affect processes related to the cytoskeleton. Thus, there is a close interplay between CCT and the cytoskeleton that extends beyond the dependency of actin and tubulin to be folded by CCT.
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| 2. |
- Svanström, Andreas, et al.
(author)
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The molecular chaperone CCT modulates the activity of the actin filament severing and capping protein gelsolin in vitro
- 2016
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In: Cell Stress & Chaperones. - : Springer Science and Business Media LLC. - 1355-8145 .- 1466-1268. ; 21:1, s. 55-62
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Journal article (peer-reviewed)abstract
- The oligomeric molecular chaperone CCT is essential for the folding of the highly abundant protein actin, which in its native state forms actin filaments that generate the traction forces required for cell motility. In addition to folding proteins, CCT can provide a platform for protein complex assembly and binds actin filaments assembled in vitro. Some individual subunits of CCT, when monomeric, have been shown to be functionally active, and in particular, the CCTepsilon subunit is involved in the serum response factor pathway that controls actin transcription. Thus, there is a complex interplay between CCT and actin that extends beyond actin folding. CCT has recently been shown to bind gelsolin, an actin filament severing protein that increases actin dynamics by generating filament ends for further actin polymerization. However, the biological significance of the CCT:gelsolin interaction is unknown. Here, using a co-immunoprecipitation assay, we show that CCT binds directly to gelsolin in its calcium-activated, actin-severing conformation. Furthermore, using actin filaments retained from fixed and permeabilized cells, we demonstrate that CCT can inhibit the actin filament severing activity of gelsolin. As our work and that of others shows gelsolin is not folded by CCT, the CCT:gelsolin interaction represents a novel mode of binding where CCT may modulate protein activity. The data presented here reveal an additional level of interplay between CCT and actin mediated via gelsolin, suggesting that CCT may influence processes depending on gelsolin activity, such as cell motility.
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