Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double-or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.Autophagy is a highly conserved biological mechanism that is responsible for lysosome-dependent recycling of long-lived abnormal or misfolded proteins as well as dysfunctional or unnecessary organelles (such as depolarized mitochondria) (1). Under normal conditions, basal autophagy help maintain cellular homeostasis. Autophagy is rapidly up-regulated following stress, including nutrient deprivation, accumulation of misfolded proteins, mitochondrial depolarization, or exposure to toxic chemicals (2). Autophagy malfunctions were shown to contribute to several pathologies, such as neurodegenerative diseases, lysosomal storage disorders, and cancer (3).The process starts with the nucleation and elongation of double-membrane structures called "autophagosomes" or "autophagic vesicles." As they mature through fusion with late endosomes or lysosomes, vesicles give rise to "autolysosomes," a hybrid compartment in which vesicle contents are degraded by the action of lysosomal hydrolases (4). So far, around 33 different core autophagy proteins (ATGs) were described (5). Among them, two ubiquitination-like reactions are key to autophagic vesicle membrane elongation as follows: ATG12-ATG5-ATG16L1 and ATG8 (MAP1LC3 or shortly LC3 in mammals). The first ubiquitination-like reaction results in the covalent conjugation of a Lys-130 residue of the ATG5 protein to a ubiquitin-like protein, ATG12. Following addition of ATG16L1 to the ATG12-conjugated ATG5, a larger complex of around 669 -800 kDa forms (6). The ATG12-ATG5-ATG16L1 complex serves as an E3-like enzyme for the second ubiquitylation-like reaction. Here, the LC3 protein is covalently attached to a lipid molecule, generally to a phosphatidylethanolamine contributing to the elongation of autophagic membranes (7,8). Conversion of the free cytosolic form of LC3 (LC3-I) to the lipid-conjugated form (LC3-II) leads to its localization to dot-li...

Given the accumulated evidence on the effects of water-in-deep eutectic solvents (DESs) on the solvent nanostructure and the yield of lipase reactions, here we have used molecular dynamics (MD) simulations to delineate the structure and dynamics of thermoalkalophilic lipases in choline chloride/urea-based DES (reline) with varying hydration levels. Results indicated that pure reline almost froze the lipase backbone, while hydrated reline that showed a less ordered nanostructure than the pure form introduced some fluctuations to lipase structures, particularly to the lid domain. Although none of the solvents led to unfolding, solvation by 8 M urea or water when accompanied with elevated temperature caused the most significant loss of secondary structure. Experimental results indicated that lipase incubation in slightly hydrated reline [5% (v/v)] led to the highest level of residual activity, implying interfacial activation. Overall, we report that slightly hydrated reline activates thermoalkalophilic lipases, consistent with the particular MD observation showing enhanced mobility of the lid domain. These insights provided by this study contribute to designing efficient lipase applications in DES-based reaction media, giving cues for customizing water-in-DESs for optimal enzyme stability and activity.

Background Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. Methods We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. Findings Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. Interpretation Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. Funding This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.