Structural and functional characterization of CENP-V: a novel glutathione-binding protein
Creators: name: Di Pompeo, Lorenza
Publisher: name: The University of Edinburgh
Year: 2023
Subjects: glutathione-binding protein
cell division
centromere protein V (CENP-V).
glutathione (GSH)-dependent formaldehyde-activating (GFA)
nuclear magnetic resonance (NMR)
CENP-V-like (CENPVL) paralogous genes
CENP-Vfl

description: The ultimate goal of cell division is to distribute the genetic information equally and accurately between the newly forming daughter cells. The establishment of stable microtubule-kinetochore attachments during mitosis is crucial to achieve this. A proteomic analysis of isolated chromosomes originally identified centromere protein V (CENP-V). In human cells, CENP-V depletion was reported to affect the compaction of mitotic chromosomes and to perturb the achievement of chromosome alignment in mitosis, thus affecting cell division. CENP-V overexpression caused a remarkable condensation of the chromatin throughout interphase cell nuclei. More recently, CENP-V was shown to maintain spindle integrity and ensure chromosome-microtubule attachment during meiosis in mouse oocytes. How CENP-V exerts its function(s) remains unknown. Sequence analysis suggests that CENP-V contains a glutathione (GSH)-dependent formaldehyde-activating (GFA) domain involved in formaldehyde metabolism in bacteria. Due to its chemical reactivity, formaldehyde readily forms nucleic acids and protein adducts, DNA crosslinks and DNA-protein crosslinks, which makes this compound highly toxic for cells. Nevertheless, formaldehyde is also endogenously produced as a by-product of cellular metabolism. Within the nucleus, demethylation reactions of histones and nucleic acids release formaldehyde. By using a combination of biochemical, structural and cell biology approaches, this study aimed to investigate whether CENP-V is a GFA-like enzyme and whether its cellular function might be associated with glutathionedependent formaldehyde metabolism. To this purpose, the crystal structures of apo CENP-V141-275 (fragment encompassing the whole putative GFA-domain) and CENP-V141-275:glutathione complex were solved at high resolution. Together the two structures confirm that CENP-V is characterised by a C-terminal GFA-domain and is able to bind glutathione. Glutathione binding induces structural rearrangements around the catalytic pocket, particularly stabilising the CENP-V C-terminal region that results in a tight channelling of the ligand. Despite maintaining the glutathione binding property of bacterial GFA, CENP-V does not seem to catalyse the reaction of glutathione and formaldehyde to form S-hydroxymethylglutathione, as observed by nuclear magnetic resonance (NMR). However, saturation transfer difference (STD) NMR suggests that CENP-V is able to bind both glutathione and Shydroxymethylglutathione. Aside from canonical CENP-V, human cells have three CENP-V-like (CENPVL) paralogous genes. In human epithelial cells, canonical CENP-V exhibited a bipartite localization, associating with both DNA and spindle microtubules during mitosis. The microtubule binding property of full-length CENP-V (CENP-Vfl) was also confirmed in vitro. Interestingly, CENP-V in complex with glutathione not only maintains the ability to bind microtubules but it also shows a marginal increase in apparent binding affinity. To further explore CENP-V function, cells inducible for CENP-V depletion in combination or not with the deletion of two out of the three CENPVL genes were established. My preliminary in vivo studies reveal that removal of canonical CENP-V does not impact the growth of cells or their tolerance for formaldehyde. However, one of the three CENPVL genes could not be removed, which may be sufficient to account for the maintenance of a normal cell growth. This will need to be further investigated. This thesis presents a novel, multi-disciplinary characterization of CENP-V. Altogether my results indicate that human CENP-V is a glutathione-binding and microtubule-interacting protein. CENP-V apparently does not catalyse the formation of S-hydroxymethylglutathione, but it might influence this reaction via a different mechanism than that proposed for PnGFA. The recruitment of CENP-V to the mitotic spindle and the spindle mid-body during mitosis might suggest a protective role of CENP-V against formaldehyde during this delicate process of the cell cycle.