Redox regulation of CLIC1 and CLIC4 protein-protein interactions

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dc.contributor.authorFaerch, Olga Maria Magdalena (Mia)
dc.date.accessioned2021-12-18T11:44:19Z
dc.date.available2021-12-18T11:44:19Z
dc.date.issued2021
dc.descriptionA thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2021en_ZA
dc.description.abstractThe anionic channels CLIC1 and CLIC4 appear soluble in the cytoplasm and are capable of reversible membrane insertion. The cellular redox state regulates the distribution and function of these proteins. This includes oxidation induced CLIC1 dimerisation, which augments membrane insertion, and S-nitrosylation of CLIC4 promoting nuclear import through the binding of a nuclear localisation sequence (NLS) to Impα. In the event of cellular oxidative stress, these processes exhibit a role in tumourigenesis and Aβ-induced neurotoxicity. This study aimed to assess the effect of redox regulation on both CLIC1 dimerisation, as well as whether the CLIC1 and CLIC4 NLSs can bind truncated hImpα isoform 1, omitting the IBB domain (hImpα1ΔIBB), in the event of oxidation induced exposure of the NLS regions. Reduced CLIC1 only appears as a monomer, whereas incubation with 2 mM H2O2 results in a mixture of dimeric and monomeric oxidised CLIC1 separable through SEC. Far-UV CD confirmed that all three redox species exhibited isolated α-helical coils. Fluorescence spectroscopy showed a 2 nm hypsochromic shift and 13 % decrease in emission intensity of the CLIC1 oxidised dimer compared to the monomeric forms. This indicates that the two monomeric forms display similar conformations, which differ from that of the oxidised dimer, where Trp35 appears more buried and quenched. Thermal unfolding indicated greater instability of the CLIC1 oxidised dimer compared to the monomeric forms. Finally, LC-MS/MS showed that Cys24 and Cys59 in CLIC1 form a stable intramolecular disulfide bond in the oxidised dimer, while undergoing significant disulfide bond shuffling in the monomeric forms. Peptides of the NLSs of CLIC1 (Pep1) and CLIC4 (Pep4) were designed. Investigating the interactions of Pep1, Pep1_A (Pep1 C/A mutant), Pep1_S (Pep1 C/S mutant) and Pep4 to ImpαΔIBB through molecular docking indicated that the core NLS region (KKYR) of these peptides form a similar binding pattern to both truncated mouse Impα isoform 1, omitting the IBB domain (mImpα1ΔIBB), and hImpα1ΔIBB. Fluorescence quenching demonstrated that Pep1_S (Kd ≈ 237 μM) and Pep4 (Kd ≈ 317 μM) bind hImpα1ΔIBB weakly. ITC confirmed the weak binding interaction between Pep4 and hImpα1ΔIBB (Kd ≈ 130 μM), as well as the presence of a proton linked effect. In conclusion, CLIC1 dimerisation appears to stabilise the oxidative conformational changes and both the CLIC1 and CLIC4 NLSs likely bind a different hImpα isoform due to the weak binding detected toward hImpα1ΔIBB.en_ZA
dc.description.librarianTL (2021)en_ZA
dc.facultyFaculty of Scienceen_ZA
dc.identifier.urihttps://hdl.handle.net/10539/32432
dc.language.isoenen_ZA
dc.phd.titlePHDen_ZA
dc.schoolSchool of Molecular and Cell Biologyen_ZA
dc.titleRedox regulation of CLIC1 and CLIC4 protein-protein interactionsen_ZA
dc.typeThesisen_ZA
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