Dissertation committee:
Εμμανουήλ Γ. Φραγκούλης - Καθηγητής , Διδώ Βασιλακοπούλου - Αναπληρώτρια Καθηγήτρια (Επιβλέπουσα), Ευστάθιος Γκόνος - Διευθυντής Ερευνών Ινστιτούτου Βιολογικών Ερευνών και Βιοτεχνολογίας, Εθνικό Ίδρυμα Ερευνών, Εργ. Μοριακής και Κυτταρικής Γήρανσης
Summary:
Αgeing is a physiological, non-reversible phenomenon that occurs in all
organisms. Both genetic and environmental factors influence the ageing process.
In specific, ageing includes distinct, non-reversible changes in the anatomy,
physiology and adaptability of the multicellular organisms. Ageing also
involves different organs and tissues and finally results to progressive
degeneration of cellular homeostasis, which means the organismal ability to
maintain the structural and functional integrity.
Replicative senescence in human fibroblasts is accompanied by alterations in
various biological processes, including the impaired function of the
proteasome. The proteasome, the major cellular non-lysosomal threonine protease
and especially the ATP/ubiquitin-dependent intracellular protein degradation
pathway mediated by the 26S complex, is responsible for the removal of both
normal and damaged proteins. Due to its latter function, proteasome is also
considered as a representative secondary antioxidant cellular mechanism. The
function and the expression of the proteasome are decreased in replicative
senescence, while its overexpression delays the senescence in human
fibroblasts.
P53 protein is one of the most important transcriptional regulators that causes
cells to repair, arrest or die when stressed or damaged. Its transcriptional
targets include a variety of genes involved in cell cycle control, DNA repair,
apoptosis and cellular senescence. In mammalian cells, senescence induced by
p53 protein occurs either due to telomere shortening which triggers replicative
senescence, or due to detrimental cellular damage, like the stress-induced
cellular senescence. It is well established that p53 has a central role in
organismal ageing and is responsible for induction of the senescent phenotype,
but only a high level of the cyclin-dependent kinase inhibitor p16Ink4a is
capable of making this process irreversible. p53 is activated upon entry into
senescence and specifically, it is stabilized and differentially regulates its
transcriptional targets; with p21Cip1/Waf1 being the best characterised example
of transactivation related to senescence induction. Different theories
implicate p53 as the main regulator of organismal ageing, but its biological
function needs to be elucidated through the complicated network of proteins
that p53 interacts with.
In normal cells, p53 is kept at very low levels through a number of
post-translational modifications and the ubiquitin-dependent proteasomal
degradation has emerged as a fundamental mechanism of p53 regulation. Mdm2 is
considered the major p53 regulator that acts as an E3 ligase and ubiquitinates
p53. p53 transcriptionally activates the expression of Mdm2 in a negative
feedback loop. Mdm2 functions as an E3 ligase, covalently attaching ubiquitin
molecules to p53, which leads to both the export of p53 to the cytoplasm and
its proteasomal degradation. Augmenting the level of complexity of p53
regulation, a number of other p53 E3 ligases have been recently identified that
act independently of Mdm2. These include Pirh2, Cop1, TOPORS and CHIP
(C-terminus of Hsp70-interacting protein).
Given the large amount of E3 ligases responsible for p53 ubiquitination, the
particular research aimed at identifying specific E3 ligases that regulate p53
stability during the manifestation of replicative senescence in human
fibroblasts. For this purpose, we studied the expression levels of p53 and its
main ligases in replicative senescence, as also in premature senescence caused
by partial proteasome inhibition. Thus, we have demonstrated that CHIP is the
only E3 ligase, among the p53 ligases tested, that is up-regulated in mRNA and
protein level concomitant with a significant down-regulation of p53 in
senescent fibroblasts. In contrast, the continuous silencing of CHIP expression
leads to premature senescence of human fibroblasts. Moreover, we have
established in this study that CHIP partially translocates to the nucleus and
acquires elevated ubiquitination levels in senescent cells, suggesting an
enhanced ligase activity during senescence.
Furthermore, since Mdm2 was found to be down-regulated in senescent
fibroblasts, we studied its function in correlation with the activity of CHIP
ligase. Therefore, we examined the effect of either overexpression or silencing
the expression of each ligase, CHIP and Mdm2, as also the inhibition of Mdm2
function on p53 protein expression. Notably, CHIP overexpression in young
cells, to levels similar to those recorded during senescence, leads to p53
degradation bellow its basal levels. In addition, while Mdm2-dependent
regulation of p53 occurs in both young and senescent fibroblasts, CHIP ligase
participates in regulating p53 levels mainly in senescence.
Moreover, it is well established that CHIP is a bona fide interaction partner
of the major cytoplasmic chaperones, namely the heat shock proteins Hsp70/Hsp90
and the co-chaperone Hsp40 and also, the proper folding of p53 is regulated by
these chaperones. The protein levels of the above mentioned chaperones were
also examined and they were found to be significantly down-regulated in
senescent cells, suggesting attenuation in the molecular folding-refolding
machinery during senescence. The effect of chaperone inhibition on the p53
degradation profile was also analysed using a specific inhibitor of Hsp90
activity that blocks the proper foding of its substrates, including p53. The
inhibition of Hsp90 activity leads to p53 rapid degradation only in senescent
cells due to the specific up-regulation of the levels and the activity of the
chaperone-associated CHIP ligase. Taken together, the aforementioned data
reveal the involvement of CHIP ubiquitin ligase in the regulation of p53
pathway towards the manifestation of cellular senescence.
