Modulation of intracellular proteolytic pathways in neoplastic and neurodegenerative diseases

The ubiquitinâ€proteasome system (UPS) and autophagy, the two major intracellular protein degradation systems, play a critical role in the regulation and maintenance of cellular homeostasis. The proteasome is known to degrade the majority of intracellular proteins, including cyclins, metabolic enzymes, antigen, transcription factors, and tumour suppressor proteins. Autophagy, or selfâ€eating, is a lysosomal degradation pathway in charge of recycling dysfunctional organelles and aggregated proteins. Impairments in the functionality of proteolytic pathways favour the accumulation of misfolded and abnormal proteins, resulting in the deposition of toxic aggregates that characterize diverse pathologic conditions such as cancer and neurodegenerations. For many years UPS and autophagy have been thought as separated pathways whereas an increasing number of data recently elucidated their intimate correlation. This PhD thesis is focused on understanding the role of UPS and autophagy in such diseases aimed at better clarifying their interplay. The potential of natural occurring compounds as proteasome modulators in the treatment and prevention of cancer is widely documented, with epigallocatechinâ€3â€gallate (EGCG) being the most studied polyphenol. In particular, owing to EGCG instability under physiological conditions, its degradation pattern was monitored and an equally active metabolite has been isolated and evaluated for its ability to modulate both proteasome functionality and apoptotic pathways. Furthermore, the marine sponge metabolite petrosaspongiolide M (PM), a natural proteasome inhibitor, has been considered, describing the molecular mechanism of interaction of PM with the immunoproteasome, a proteasome isoform with a prevalent role in immune response. This natural compound was also able to impair autophagy, with p62 serving as the link between the two proteolytic processes. Among polyphenolsâ€based anticancer approaches, curcumin represents a promising but poor bioavailable compound. Considering that metal complexes offer an opportunity for the design of bioactive compounds with anticancer properties, and that ruthenium is a valid non toxic alternative to Cisplatin, three ruthenium(II)curcumin complexes containing different arene moieties have been synthesized and analyzed for their ability to modulate proteasome functionality, comparing their efficacies with that of free curcumin in isolated proteasome complexes and in cultured colon cancer cells. A stable and effective curcumin derivative, in terms of proteasome inhibitory ability, antioxidant capacity, DNA binding ability has been identified, thus proving that the complexation of curcumin with ruthenium(II) is a good starting point for the development of curcuminâ€based anticancer drugs. Successively, a series of ruthenium(II) arene complexes with the 4â€(biphenylâ€4â€carbonyl)â€3â€methylâ€1â€phenylâ€5â€pyrazolonate ligand, and different ancillary ligands have been synthesized and characterized, identifying the hexamethylbenzene−ruthenium complexes as the most efficacious, in terms of antiproliferative activity in four human cancer cell lines, through the induction of apoptosis. Finally, regarding the interplay of UPS and autophagy in cancer, the effect of the hunger hormone ghrelin on both proteasome and autophagy has been analysed. Interestingly, this endogenous compound triggers apoptosis in colon cancer cells, via proteasome inhibition and autophagy induction, with p53 protein having an interactive role. Aging and neurodegenerative conditions, including Alzheimer disease (AD) are characterized by alterations in the normal cellular homeostasis with deregulation of the proteolysis. UPS and autophagy interplay in AD has been studied using human SHâ€SY5Y neuroblastoma cells stably transfected either with wildâ€type βâ€amyloid precursor protein (AβPP) gene or mutant Val717Gly AβPP gene as experimental model. The APPmut clone produces and releases significantly higher amounts of Aβ42, the amyloid peptide which is more prone to aggregation. The overâ€expression of the APP correlated with an increase in oxidative stress and with a reorganization of the cellular proteolytic machineries. Additionally, HDAC6â€increased expression has been identified as the cellular attempt to activate compensatory autophagy in such altered scenario. The existence of an amyloid (Aβ42) threshold level beyond which proteasomeâ€dependent proteolysis becomes definitely dysfunctional has been proved. Moreover, these cellular models have been used to demonstrate a role of the APP in affecting the downstream effects of proteolysis inhibition. The occurrence of APP wildâ€type form or the APP Val717Gly mutated form was observed to impair both proteasome or autophagy activities upon treatment with proteasome or autophagy inhibitors. Collectively, our results provide evidences on the key role of UPS and autophagy in both neoplastic and neurodegenerative diseases and gain insight into the interplay between the two pathways in proteinopathies, thus representing a real contribution in the development of new strategies to modulate the two pathways for therapeutic purposes