| Proteins are delivered to degradation in the lysosome mainly by autophagosomes and multivesicular bodies (MVBs). The selective recruitment of proteins to these vesicles depends on ubiquitination and on adaptor proteins, yet the exact mechanisms remain only partly defined. In the first part of my PhD I utilized the lysine-less 7KR-Ub mutant, fused to GFP at its N-terminus, to study the delivery of Ub-proteins to the lysosome. I demonstrated that the 7KR-tagged proteins, predicted to be mostly conjugated by monoUb or short polyUb chains, translocated to lysosomes faster than WT Ub-proteins, and that a mutation in Lys27 was sufficient to affect Ub-protein localization. Inhibition of autophagy resulted in the appearance of membranal structures containing p62 and Ub, which were dependent on high level of p62 for their formation; thus, the fine-tuning of p62 level determines the subcellular localization and consequently the fate of Ub-proteins. Impediment of the ESCRT machinery resulted in the accumulation of Ub-containing late endosomes and delayed autophagic activity. We therefore propose that membranes associated with Ub and p62 may play a key role in the formation of insoluble protein aggregates. Such aggregates often lead to neurodegenerative disease, previously associated with aberrations in autophagy and the ESCRT machinery. Collectively, these results illustrate a functional interaction between autophagy and the ESCRT machinery, which governs trafficking of Ub-substrates.;In the second part of my PhD I found that extrinsic apoptotic stimuli caused proteasome-mediated p62 cleavage. Using mass spectrometry techniques, I identified the truncation occurred at Asp329, a predicted cleavage site of caspase-8, thereby separating the autophagy-related LIR, KIR and UBA domains from the rest of the protein. Such cleavage may inhibit selective autophagy during programmed cell death. I therefore propose that such cleavage plays an important role in the cellular response to the induction of apoptosis.;Finally, as part of a collaborative effort with the group of Prof. Doron Lancet, we identified a recessive mutation in the TECPR2 gene, which encodes an Atg8-binding protein, as the cause for Hereditary Spastic Paraparesis in Jewish Bukharian families. In my work I demonstrated that cells from HSP patients with mutant TECPR2, as well as cells in which TECPR2 was knocked down, exhibit aberrant autophagy. These results demonstrate the deleterious outcomes of impaired TECPR2 and autophagy on neuronal health. |
