| 1. |
- Cansby, Emmelie, 1984, et al.
(författare)
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Silencing of STE20-type kinase STK25 in human aortic endothelial and smooth muscle cells is atheroprotective
- 2022
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor to atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigate its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduces lipid accumulation and suppresses activation of inflammatory and fibrotic pathways as well as lowering oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hinders the shift from a contractile to a synthetic phenotype. Together, we provide several lines of evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease.
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| 2. |
- Cuéllar, J., et al.
(författare)
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The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3: CCT-Gelsolin interaction may affect actin dynamics
- 2022
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 434:5
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Tidskriftsartikel (refereegranskat)abstract
- The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca2+. This activated form of gelsolin binds to, but is not a folding substrate of the molecular chaperone CCT/TRiC. Here we demonstrate that in vitro, gelsolin is protected from cleavage by caspase-3 in the presence of CCT. Cryoelectron microscopy and single particle 3D reconstruction of the CCT:gelsolin complex reveals that gelsolin is located in the interior of the chaperonin cavity, with a placement distinct from that of the obligate CCT folding substrates actin and tubulin. In cultured mouse melanoma B16F1 cells, gelsolin co-localises with CCT upon stimulation of actin dynamics at peripheral regions during lamellipodia formation. These data indicate that localised sequestration of gelsolin by CCT may provide spatial control of actin filament dynamics. © 2021 The Authors
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| 3. |
- Echbarthi, Meriem, et al.
(författare)
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Interactions between monomeric CCTS and p150(Glued): A novel function for CCTS at the cell periphery distinct from the protein folding activity of the molecular chaperone CCT
- 2018
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827. ; 370:1, s. 137-149
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Tidskriftsartikel (refereegranskat)abstract
- Chaperonin containing tailless complex polypeptide 1 (CCT) is a molecular chaperone consisting of eight distinct protein subunits, that when oligomeric is essential for the folding of newly synthesized tubulin and actin. In addition to folding, CCT activity includes functions of individual subunits in their monomeric form. For example, when CCTS monomer levels are increased in cultured mammalian cells, numerous cell surface protrusions are formed from retraction fibres, indicating that an underlying function for the CCTS monomer exists. Here, using a yeast two-hybrid screen we identify the dynactin complex component p150Glued as a binding partner for CCT8 and show by siRNA depletion that this interaction is required for the formation of CCT8-induced cell surface protrusions. Intact microtubules are necessary for the formation of the protrusions, consistent with microtubule minus end transport driving the retraction fibre formation and depletion of either p150Glued or the dynactin complex-associated transmembrane protein dynAP prevents the previously observed localization of GFP-CCT8 to the plasma membrane. Wound healing assays reveal that CCTS monomer levels influence directional cell migration and together our observations demonstrate that in addition to the folding activity of CCT in its oligomer form, a monomeric subunit is associated with events that involve the assembled cytoskeleton.
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| 4. |
- Fletcher, John S., et al.
(författare)
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Correlated fluorescence microscopy and multi-ion beam secondary ion mass spectrometry imaging reveals phosphatidylethanolamine increases in the membrane of cancer cells over-expressing the molecular chaperone subunit CCT delta
- 2021
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 413, s. 445-453
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Tidskriftsartikel (refereegranskat)abstract
- Changes in the membrane composition of sub-populations of cells can influence different properties with importance to tumour growth, metastasis and treatment efficacy. In this study, we use correlated fluorescence microscopy and ToF-SIMS with C-60(+) and (CO2)(6k)(+) ion beams to identify and characterise sub-populations of cells based on successful transfection leading to over-expression of CCT delta, a component of the multi-subunit molecular chaperone named chaperonin-containing tailless complex polypeptide 1 (CCT). CCT has been linked to increased cell growth and proliferation and is known to affect cell morphology but corresponding changes in lipid composition of the membrane have not been measured until now. Multivariate analysis of the surface mass spectra from single cells, focused on the intact lipid ions, indicates an enrichment of phosphatidylethanolamine species in the transfected cells. While the lipid changes in this case are driven by the structural changes in the protein cytoskeleton, the consequence of phosphatidylethanolamine enrichment may have additional implications in cancer such as increased membrane fluidity, increased motility and an ability to adapt to a depletion of unsaturated lipids during cancer cell proliferation. This study demonstrates a successful fluorescence microscopy-guided cell by cell membrane lipid analysis with broad application to biological investigation. [GRAPHICS] .
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| 5. |
- Vallin, Josefine, 1987, et al.
(författare)
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Functional assessment of the V390F mutation in the CCT delta subunit of chaperonin containing tailless complex polypeptide 1
- 2021
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Ingår i: Cell Stress & Chaperones. - : Springer Science and Business Media LLC. - 1355-8145 .- 1466-1268. ; 26, s. 955-964
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Tidskriftsartikel (refereegranskat)abstract
- The chaperonin containing tailless complex polypeptide 1 (CCT) is a multi-subunit molecular chaperone. It is found in the cytoplasm of all eukaryotic cells, where the oligomeric form plays an essential role in the folding of predominantly the cytoskeletal proteins actin and tubulin. Both the CCT oligomer and monomeric subunits also display functions that extend beyond folding, which are often associated with microtubules and actin filaments. Here, we assess the functional significance of the CCT delta V390F mutation, reported in several cancer cell lines. Upon transfection into B16F1 mouse melanoma cells, GFP-CCT delta(V390F) incorporates into the CCT oligomer more readily than GFP-CCT delta. Furthermore, unlike GFP-CCT delta, GFP-CCT delta(V390F) does not interact with the dynactin complex component, p150(Glued). As CCT delta has previously been implicated in altered migration in wound healing assays, we assessed the behaviour of GFP-CCT delta(V390F) and other mutants of CCT delta, previously used to assess functional interactions with p150(Glued), in chemotaxis assays. We developed the assay system to incorporate a layer of the inert hydrogel GrowDex (R) to provide a 3D matrix for chemotaxis assessment and found subtle differences in the migration of B16F1 cells, depending on the presence of the hydrogel.
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| 6. |
- Vallin, Josefine, 1987, et al.
(författare)
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Sequestration of the Transcription Factor STAT3 by the Molecular Chaperone CCT: A Potential Mechanism for Modulation of STAT3 Phosphorylation.
- 2021
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 433:13
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Tidskriftsartikel (refereegranskat)abstract
- Chaperonin Containing Tailless complex polypeptide 1 (CCT) is an essential molecular chaperone required for the folding of the abundant proteins actin and tubulin. The CCT oligomer also folds a range of other proteins and participates in non-folding activities such as providing assembly support for complexes of the von Hippel Lindau tumor suppressor protein and elongins. Here we show that the oncogenic transcription factor STAT3 binds to the CCT oligomer, but does not display the early binding upon translation in rabbit reticulocyte lysate typical of an obligate CCT folding substrate. Consistent with this, depletion of each of the CCT subunits by siRNA targeting indicates that loss of CCT oligomer does not suppress the activation steps of STAT3 upon stimulation with IL-6: phosphorylation, dimerisation and nuclear translocation. Furthermore, the transcriptional activity of STAT3 is not negatively affected by reduction in CCT levels. Instead, loss of CCT oligomer in MCF7 cells leads to an enhancement of STAT3 phosphorylation at Tyr705, implicating a role for the CCT oligomer in the sequestration of non-phosphorylated STAT3. Thus, as CCT is dynamic oligomer, the assembly state and also abundance of CCT oligomer may provide a means to modulate STAT3 phosphorylation.
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| 7. |
- Vallin, Josefine, 1987
(författare)
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The molecular chaperone CCT: Functions beyond folding
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The chaperonin-containing tailless complex polypeptide 1 (CCT) is a eukaryotic ~1 MDa barrel shaped molecular chaperone, built up by eight distinct subunits and is required for the folding of the abundant cytoskeletal proteins actin and tubulin. CCT exists as an assembled oligomer, micro-complexes and as individual subunits in the cell. In addition to folding, CCT is connected to a variety of cellular processes that involve both assembled CCT oligomer and individual monomeric CCT subunits. Monomeric CCTδ translocates to the plasma membrane when overexpressed, indicating a function for CCTδ when not incorporated into the oligomer. One aim of this thesis is to understand the underlying monomeric function CCTδ. The dynactin complex component p150Glued was identified as a binding partner for monomeric CCTδ and together with the transmembrane protein dynAP, p150Glued;CCTδ creates an inward movement of the plasma membrane along microtubules, resulting in a curved membrane. We used fluorescence imaging and ToF-SIMS in cells overexpressing CCTδ and detected an increase in phosphatidylethanolamines (PE), lipids often found in membranes with high curvature. We show that one point-mutation in CCTδ can affect the assembly state of CCT, increasing the CCT oligomer pool and abolishes the binding of CCTδ to p150Glued. Oligomeric CCT is known to interact with the transcription factor STAT3. Here, we show that STAT3 does not behave as an obligate folding substrate. Instead, IL-6 induced tyrosine phosphorylation is increased when CCT levels are reduced. Furthermore, CCT depletion does not affect other stages of STAT3 activation. Therefore it is possible that CCT regulates STAT3 phosphorylation levels by acting as a sequestering protein for STAT3. CCT oligomer can also interact with the capping and severing protein gelsolin and by super resolution microscopy we detect CCT and gelsolin co-localised at the edge of protruding lamellipodia where extensive microfilament rearrangement occurs. By cryo-EM imaging using purified gelsolin:CCT complexes gelsolin is seen located deep in the CCT chaperonin cavity, suggesting a sequestering role for CCT. Consistently, in the presence of CCT, gelsolin is protected from caspase-3 cleavage. Taken together, we have identified the mechanism behind monomeric CCTδ at the plasma membrane and two possible sequestering protein interactions for CCT oligomer. Therefore, the work in this thesis extends the understanding of the non-folding properties of CCT.
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| 8. |
- Vallin, Josefine, 1987, et al.
(författare)
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The role of the molecular chaperone CCT in protein folding and mediation of cytoskeleton-associated processes: implications for cancer cell biology.
- 2019
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Ingår i: Cell stress & chaperones. - : Springer Science and Business Media LLC. - 1466-1268 .- 1355-8145. ; 24:1, s. 17-27
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Forskningsöversikt (refereegranskat)abstract
- The chaperonin-containing tailless complex polypeptide 1 (CCT) is required in vivo for the folding of newly synthesized tubulin and actin proteins and is thus intrinsically connected to all cellular processes that rely on the microtubule and actin filament components of the cytoskeleton, both of which are highly regulated and dynamic assemblies. In addition to CCT acting as a protein folding oligomer, further modes of CCT action mediated either by the CCT oligomer itself or via CCT subunits in their monomeric forms can influence processes associated with assembled actin filaments and microtubules. Thus, there is an extended functional role for CCT with regard to its major folding substrates with a complex interplay between CCT as folding machine for tubulin/actin and as a modulator of processes involving the assembled cytoskeleton. As cell division, directed cell migration, and invasion are major drivers of cancer development and rely on the microtubule and actin filament components of the cytoskeleton, CCT activity is fundamentally linked to cancer. Furthermore, the CCT oligomer also folds proteins connected to cell cycle progression and interacts with several other proteins that are linked to cancer such as tumor-suppressor proteins and regulators of the cytoskeleton, while CCT monomer function can influence cell migration. Thus, understanding CCT activity is important for many aspects of cancer cell biology and may reveal new ways to target tumor growth and invasion.
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