DEFA1Interacts With BECN1to Modulate The Process Of Macroautophagy

Objective:In the cause of observing the relationship between macroautophagy and antimicrobial peptide, since both of them play key roles in protecting cells from infection by microbe. The DEFA1was chosen as a representative of antimicrobial peptide to study any physical and/or functional interaction with the macroautophagy-related protein (BECNl).Methods:Immunoprecipitation, western blotting and confocal microscopy were used to study this research. The methods that had been reported as induction or inhibition of macroautophagy were also used in different time window to observe the change in the localization and protein stability of BECN1, DEFA1and the macroautphagy. The plasmids of BECN1-myc and DEFA1-flag were transfected into HEK293cells for immunoprecipitation and western blotting experiments, the plasmids with different tags of BECN1and DEFA1were transfected to Hela cells for confocal microscopy experiment. The western blotting results were quantified for statistical analysis, in which P values less than0.05were regarded as statistically significant.Results:1. BECN1interacted with DEFA1in vitro.2. Both BECNl and DEFA1were targeted to lysosome compartment, and made lysosomes to be restricted to near peri-nuclear compartment. BECN1appeared to make more lysosomes targeted to DEFA1.3. During the starvation-induced macroautophagy for12hrs, BECN1did not show any significant alteration in its localization, DEFA1localization changed from widespread cytoplasmic to peri-nuclear over the time course, but the significant change happened at2hrs. LC3aggregation increased at2hrs time point, whereas all of the scattered LC3disappeared and instead became aggregated4hrs time point throughout12hrs time point. The localization patterns of BECN1-LC3and DEFA1-LC3seemed similar to those of non-starved ones. At4hrs starvation time point, DEFA1appeared to have an effect on the localization of LC3. However, LC3localization pattern almost disappeared when three of them were co-expressed together in normal growth culture media. There were more fluorescent signals of the proteins when the cell was subjected to2hrs starvation, but when starvation went on to4hrs, DEFA1and LC3patterns changed similar to those without BECN1.4. BECN1and/or DEFA1increased the ratio of LC3-Ⅱ/LC3-Ⅰ. After2hrs and4hrs starvation, DEFA1seemed to increase the degradation of BECN1. At the same time, BECN1protected the degradation of DEFA1. The ration of LC3-II/LC3-I in cells overexpressed with BECN1did not increase compared to the others.5. Bafilomycin A1was found to protect DEFA1and weakly protect BECN1from lysosomal degradation. DEFA1synergized with Bafilomycin A1to increase the stability of BECN1, whereas BECN1helped the Bafilomycin A1to protect the DEFA1especially at200μM compared to that of100μM. In the presence of Bafilomycin Al, BECN1was apparently protected from the lysosomal degradation regardless of the expression of DEFA1. On the other hand, the overall stability of DEFA1was increased by BECN1and also Bafilomycin Al. visibly at12hrs time point.6. BECN1was better protected than DEFA1from the lysosomal degradation by these lysosomal inhibitors (E64d/Pepstatin A) only at12hrs time point. However, DEFA1reduced the protective function of E64d/Pepstatin A for BECN1. BECN1induced the protective function of E64d/Pepstatin A for DEFA1stability.7. Proteasomal inhibitor-MG132treatment clearly inhibited the degradation of both BECN1and DEFA1proteins.Conclusion:DEFA1can induce macroautophagy. also participate in and modulate the process of macroautophagy by interacting with BECN1and LC3proteins. Proteasomal degradation also contributes to both BECN1and DEFA1proteins stability.