The Role Of E3 Ubiquitin Ligase NEDD4 In Autophagy

Background:Autophagy is a lysosome-mediated cellular degradation process and plays essential roles in regulation of metabolic and oxidative stress and multiple pathological processes.Autophagy is activated by many cellular signals,mainly nutritional starvation,endoplasmic reticulum?ER?stress and oxidative stress signals.Abnormal autophagy process has been linked to a variety of human diseases.Early studies have shown that autophagy is a non-selective degradative pathway.In recent years,it has been found that in many autophagy processes,such as autophagic clearance of damaged mitochondria,peroxides and denatured protein aggregates,the autophagic cargos are labeled by ubiquitination.The ubiquitinated cargos are recognized by autophagy receptors and recruited to autophagosomes through the binding of autophagy receptors to the autophagosomal protein LC3,then transported to lysosomes for degradation.This type of autophagic process mediated by autophagy receptor is defined as selective autophagy.So far,most of the studies on ubiquitination in selective autophagy have been focused on the role of ubiquitination in recognition of autophagic cargos.How ubiquitination involved in regulation of autophagy receptor and autophagosomal proteins,initiation and activation of selective autophagy remains poorly understood.Therefore,investigation of regulation of autophagy receptors and autophagosomal proteins by ubiquitination will provide an insight into the mechanism by which the selective autophagy is regulated.NEDD4?NEDD4-1?,a member of the HECT E3 ubiquitin ligase family,is originally identified in the early embryonic mouse central nervous system,which is frequently overexpressed in many types of human cancerous tissues.Many ubiquitination substrates of NEDD4 have been identified by an in vitro substrate screening assay,and NEDD4 is inclined to ubiquitinate receptor tyrosine kinases and endocytic sorting proteins.NEDD4 is involved in regulation of ion channel activity,receptor endocytic trafficking,viral budding and neural development process by ubiquitination of different substrates.Preliminary studies have been shown that NEDD4 might play a role in autophagy.However,how NEDD4 regulates autophagy as an E3 ubiquitin ligase remains unknown.We found that NEDD4 has 4 regions containing a putative LC3-binding?WXXL/I?motif,suggesting that NEDD4 may directly interact with the autophagosomal protein LC3 and regulate autophagy.Therefore,further investigation of the role of NEDD4-mediated ubiquitination in autophagy will help us to gain a new view on ubiquitination-mediated regulation of autophagy.Research purposes:This study is to define the role of NEDD4 as an E3 ubiquitin ligase in regulation of autophagy through interaction with and ubiquitination of the autophagosomal protein LC3 and autophagy receptor SQSTM1.Through the study,we hopefully gain a new insight into the role of ubiquitination in selective autophagy,and provide new therapeutic targets for the autophagy-related diseases.Materials and Methods:1.Cell culture and treatmentHEK293T,HEK293A and A549 cells were purchased from ATCC.Cells were maintained in Dulbecco's modified Eagle's medium with 10%heat-inactived fetal bovine serum?FBS?,1%penicillin and streptomycin at 37°C with 5%CO₂.According to related experiments,cells were treated with Starvation or 1uM Rapamycin for 18hours or lysosome inhibitor Chloroquine?50uM?for 18 hours.In the ubiquitination assay of SQSTM1 and NBR1 associated experiments,cells were pretreated with10uM MG132 or 10uM Bortezomib for 18 hours to inhibit protein degradation.2.Western blotWestern blot was used to detect NEDD4,LC3,SQSTM1,BECN1,PDCD6IP and NBR1.Bands were detected by enzyme-linked chemiluminescence.3.Coimmunoprecipitation?CO-IP?and GST-LC3B pulldown assayProtein binding to NEDD4 was immunoprecipitated using CO-IP or GST-LC3B pulldown assay and detected the binding protein through Western blot.4.Lentiviral vector-loaded systemThe shNEDD4 knockdown and RNA-resistant NEDD4 overexpression sequences were inserted into the pLKO.1 or pFUW vector respectively to created knockdown or overexpression plasmid.Lentiviral vector-loaded GFP-LC3 and Lentiviral vector-loaded GFP-SQSTM1 were used to trace autophagy.5.Transmission electron microscope?TEM?Transmission electron microscope was used to detect the formation of autophagosomes and autolysosomes for evaluating the effect of NEDD4 knockdown on autophagy.6.Immunofluorescence stainingImmunofluorescence staining was performed to analyze the process of autophagy,cellular localization and morphology of autophagosomes.7.Cyto-ID autophagy assay kitTo determine live autophagic processes,the Cyto-ID autophagy detection kit was used to label autophagosomes in living cells as instructed by the manufacturer.The labeled autophagosomes were visualized under a Nikon Eclipse TE2000 inverted fluorescent microscope or confocal fluorescence microscope and the Cyto-ID positivity was quantified using a Beckman Coulter Cytomics FC500 cytometer by measuring the fluorescent density from 10⁴ cells in each sample.8.Detection of ubiquitinated proteinsCells were lysed with RIPA buffer,and the ubiquitinated proteins were detected either by immunoprecipitation with the primary antibody followed by immunoblotting with an anti-Ub antibody,or by affinity precipitation with GST-Uba-conjugated glutathione beads followed by immunoblotting with an anti-SQSTM1 antibody.9.In vitro E3 ubiquitin ligase activity assayNEDD4 was overexpressed in HEK293T cell and isolated by immunoprecipitation.The immunoprecipitated NEDD4 was then incubated with or without purified LC3B on ice.The ubiquitin ligation reaction was initiated by addition of monoubiquitin to the reaction mixture.The polyubiquitin was separated by SDS-PAGE and detected by immunoblotting with an anti-Ub antibody.The polyubiquitin product conjugated from monoubiquitin was used for evaluating the ligase activity of NEDD4.Results:1.CO-IP and GST-LC3B pulldown assay confirmed NEDD4 is an LC3-interactive protein and identified the LIR2 in NEDD4 as the LC3 binding site.2.Knockdown of NEDD4 caused defective autophagy,elevated the SQSTM1protein level,and impaired Starvation-or Rapamycin-induced activation of autophagy.3.Depletion of NEDD4 impaired process of autophagosomes and caused aberrant distribution and aggregation of the LC3-positive phagophores and autophagosomes,and knockdown of NEDD4 caused aggregates of the LC3 puncta co-localized with endoplasmic reticulum membrane markers.4.Transmission electron microscope observed gigantic deformed mitochondria in NEDD4 knockdown cells,suggesting that NEDD4 might function in mitophagy.5.LC3 is not a ubiquitination substrate of NEDD4,but an activator of NEDD4ligase activity.6.NEDD4 directly binds to the PB1 domain of SQSTM1 via its HECT domain,and polyubiquitinates SQSTM1 through K63 chain conjugation to the PB1 domain,and the ubiquitination does not cause proteasomal degradation;another autophagy receptor NBR1,is neither a binding protein nor an ubiquitinated substrate of NEDD4.7.Depletion of NEDD4 or overexpression of the ligase defective mutant of NEDD4 produced a much more severe defect in autophagy-mediated removal of cellular inclusion bodies,which induced accumulation of aberrant enlarged SQSTM1-positive inclusion bodies that were co-localized with the ER marker CANX.Conclusions:The key autophagosomal protein LC3 interacts with NEDD4 and activates the ligase activity of NEDD4.Activated NEDD4 interacts with and ubiquitinates autophagy receptor SQSTM1,which regulates the SQSTM1-mediated inclusion body autophagy.NEDD4 might be an effective therapeutic target for SQSTM1-mediated selective autophagy-related diseases.