Study On The Mechanism Of ATG5 Regulating C-Myc Protein Degradation

BackgroundAutophagy is a cellular self-protection mechanism,which can remove the abnormal folded proteins or aging organelles in the cell to maintain the homeostasis.Autophagy is essential for cell survival,especially when cells are exposed to a number of stressful environments,such as starvation,or various physiological and pathological conditions,including hypoxic ischemia,toxicity,immune and oxidative damage.Autophagy is beneficial to cell survival by providing necessary raw materials through degradation of intracellular substrates.More and more evidences showed that the maladjustment of autophagy is closely related to many types of diseases,such as the occurrence of tumors and neurodegenerative diseases.Autophagy-related proteins(ATGs)participate in the whole process of autophagy,of which APG/ATG5 is indispensable in both canonical and non-canonical autophagy.ATG5 is a key protein in autophagy.As autophagy occurring,ATG5 is involved in formatting the bilayer of autophagosome.Autophagy is significantly inhibited,while ATG5 was knocked out,this indicating that ATG5 plays a central role in the process of autophagy.In human cells,ATG5 is encoded by ATG5 gene,which is located on chromosome 6q21,and its classical biological function is mainly to form covalent coupling between lysine residues of ATG5 and glycine residues on C-terminal of ATG12 in cells,forming ATG5-ATG12 complex to participate in the formation of autophagy vesicles.Myc is an oncogene,which can act as a transcription factor to regulate the expression of its downstream target genes.Myc is also a short-lived protein,it’s half-life of protein and m RNA is about30 min.Myc must first bind with Max,then plays its function.Max is a protein which containing 160 amino acids,and a similar sequence to the C-terminal of c-Myc.Max is relatively stable and is more abundant than Myc molecules in many cells,They could bind closely to the E-box sequence of DNA to further regulate the transcription of target genes,only while they formed a Myc/Max dimer.Myc contains three members: c-Myc,N-Myc and L-Myc.C-Myc is one of the important members of Myc gene family,and it can influence cell differentiation and proliferation by participating in the regulation of various pathways,such as Wnt pathway,MAPK pathway and PI3 K pathway.C-Myc is one of the most common proto-oncogenes,which is involved in the regulation of about 20% of all human cancers,and affects hundreds of thousands of cancer patients to death every year.In normal cells,c-Myc is expressed at very low levels.Multiple mutations of c-Myc have been found in many human malignancies,and those mutants are usually expressed at a higher levels than normal cells.Over-expression of c-Myc often affects the process of cell cycle and cell apoptosis,so it is necessary to inhibit the expression of c-Myc in cancer cells to block the occurrence and development of tumors.Therefore,it becomes particularly important to study the mechanism of c-Myc expression change during cell differentiation.ObjectiveFor a long time,studies on ATG5 focused on its canonical role in autophagy process,but there were few studies on the "non-autophagy" function of ATG5.The stimulation of external environment,such as starvation,hypoxia,metabolic pressure and aging,can easily lead to the increase of intracellular autophagy level.However,under normal physiological conditions,the intracellular autophagy is only maintained at the most basic level,but autophagy related proteins such as ATG5 can also be expressed normally.Since ATG5 is less involved in autophagy under normal conditions,will it be involved in other biological processes? Thus,it is of great significance to study the "non-autophagy" function of ATG5 under normal conditions without external stimulation.Because c-Myc can promote cell division,so that cells can obtain immortalized function and infinite proliferation ability.Therefore,the study on the regulatory mechanism of c-Myc by ATG5 is introduced into the behavioral changes of tumors,will provide a new target for future tumor prevention and treatment.Methods1.The effects of ATG5 on the behavior of He La cells in normal culture and serum starvation culture were detected by CCK-8 cell proliferation test and Transwell test.2.RT-PCR assay and Westernblot assay were used to detect whether the regulation of c-Myc by ATG5 occurred at transcriptional level or protein level in normal culture and serum starvation culture.3.The interaction between ATG5 and c-Myc in normal culture and serum starvation culture was detected by Co-IP test and immunofluorescence test.4.The interaction between ATG5 and c-Myc was detected by Pulldown experiment in vitro.5.Through the construction of ATG5K130 R mutant plasmid and ATG5 knockout experiments,it was proved that the regulation of c-Myc by ATG5 in normal culture and serum starvation culture is independent on autophagy.Results1.Under normal culture conditions,the proliferation of He La cells was significantly promoted by knocking out of ATG5,and over-expression of ATG5 inhibited the proliferation and migration of He La cells.2.Under normal culture conditions,over-expression ATG5K130 R mutants or autophagy inhibitors were used to block autophagy,which did not affect the regulation of ATG5 on cell proliferation.3.Under normal culture conditions,after changing the expression of ATG5,the change of c-Myc expression was detected,and it was found that the influence of ATG5 on c-Myc expression occurred at the protein level rather than the m RNA level.4.Under normal culture conditions,it was found that the regulation of c-Myc protein level by ATG5 was mediated by ubiquitin-proteasome while adding autophagy inhibitors or proteasome inhibitors,and this mechanism independent of autophagy.5.Under normal culture conditions,over-expression of ATG5 can significantly increased the degradation rate of c-Myc in He La and HEK293 T cells,and knockout of ATG5 could inhibit this degradation of c-Myc.6.Under normal culture conditions,the ubiquitination level of c-Myc was significantly increased after overexpression of ATG5.7.Under normal culture conditions,Co-IP experiment was used to verify the interaction between ATG5 and c-Myc in both forward and reverse directions.8.Under normal culture conditions,the co-localization signals of ATG5 and c-Myc could be observed by immunofluorescence technology.9.In vitro,the induced expression of GST-ATG5 in Escherichia coli BL21 was mixed with His-c-Myc protein through pull down experiment,which further confirmed the interaction between ATG5 and c-Myc.10.Under normal culture conditions,through Co-IP experiment,it was found that both ATG5 and ATG5K130 R could promote ubiquitination degradation of c-Myc,further proving that the regulatory effect of ATG5 on c-Myc is independent of autophagy.ConclusionAccording to all the above results,we can draw the following conclusions: under normal culture conditions,ATG5 can directly interact with c-Myc protein,promoting the degradation of c-Myc protein through ubiquitin proteasome-mediated protein degradation pathway,and then affect expression of downstream target genes of c-Myc and proliferation of He La cells,and this regulation is independent of autophagy.However,during serum starvation,ATG5 may be mainly involved in autophagy to make cells cope with hunger environment,thus weakening the interaction between ATG5 and c-Myc proteins,and in this process,the effect of ATG5 on the proliferation of He La cells is also weakened.Therefore,the study on the non-autophagy function of ATG5 under normal culture conditions provides a solid theoretical basis for tumor prevention and treatment.