| Alpha-1-antitrypsin (AAT), a glycoprotein, is synthesized and secretedpredominantly by liver cells. Its major function is to inhibit destructive neutrophilproteases, including elastase, cathepsin G, and proteinase. The most common causalmutation is the replacement of Glu-342by Lys that characterizes the Z mutant of AAT(ATZ). This substitution leads to retention of this mutant protein in the ER, which cancause two major organ injury, destructive lung disease/emphysema and the chronic liverdisease. In contrast to the pathobiology of lung disease, liver disease is due to thehepatotoxic effects of retained ATZ. Because lack of ATZ is assoiciated with thepresence of hepatocellular disease, the mechanisms by which ATZ is degraded arethought to be particularly important.OBJECTIVE:To investigate whether ubiquitin ligase Hrd1facilitates the clearance of ATZ throughautophagy pathway. METHODS:Small interference RNA for Hrd1was systhesized from Genepharm. Co. LTD.Real-time PCR and western blot were used to detect mRNA and protein level,respectively. Immunofluorescence assay was used to monitor the localization andquantitation of protein. Cycloheximide, MG132(proteasome activity inhibitor) andBFA1(ysosome activity inhibitor) were used to explore the mechanisms forHrd1-mediated autopagic clearance of ATZ followed by immune blot.Co-immunoprecipitation (Co-IP) was employed to determine the interaction betweenATZ and p62.RESULTS:1. Autophagy is involved in Hrd1-mediated ATZ degradation1.1Hrd1promotes the colocalization of ATZ and LC3The mammalian autophagy protein LC3is a marker of autophagosomes. LC3-I issubsequently conjugated with phosphatidylethanolamine (PE) to become LC3-II(LC3-PE). In comparison to the cytoplasmic localization of LC3-I, LC3-II associatestightly with the autophagosome membrane. In order to investigate the localization ofATZ, Hrd1, and LC3, we performed double immunostaining assay. HEK293T wereco-transfected with ATZ and Hrd1and treated with NH4Cl24hrs after transfection.Four hrs later, cells were fixed and subjected to double labeled immunofluorescencewith monoclonal anti-ATZ (blue), monoclonal anti-myc(red) and polyclonal anti-LC3(green). After over-expressing ATZ and Hrd1, LC3puncta were observed in thecytoplasm. Hrd1promoted the colocalization of ATZ and LC3after treatment withNH4Cl.1.2Hrd1facilitates ATZ degradation partially through autophagy pathwayHEK293T cells were co-transfected with pcDNA3.1-zeo+-ATZ andpcDNA3.1-Hrd1-myc-His. Co-tansfection of ATZ and pcDNA3.1-zeo+-vector was used as control.Twenty four hrs after transfection, the cells were treated with autophagyinducer HBSS or autophagy inhibitor BFA1. Cells lyses were prepared for Western blotanalysis and ATZ protein was blotted. Compared with the DMSO control, Hrd1significantly reduced ATZ protein level under starvation condition. Howerver, BFA1inhibited Hrd1–mediated reduction of ATZ.1.3Knockdown of endogenous Hrd1with siRNA inhibited autophagic degradationof ATZHEK293T cells were co-transfected with Hrd1-siRNA and ATZ. Co-tansfection of ATZand NC-siRNA was used as control. NH4Cl was added to the medium24hrs aftertransfection.4hrs later, Cells were lysed and processed for detecting ATZ. The resultshowed that compared with the control, inhibiting the endogenous expression of Hrd1increased intracellular ATZ level.1.4Hrd1-mediated ATZ autophagic degradation depends on its E3activityTo further investigate whether Hrd1E3activity is required for ATZ autophagicclearance, Hrd1C1A and ATZ were co-transfected into HEK293T cells. Westernblotting demonstrated that in contrast to wild type Hrd1, the Hrd1C1A mutant did notdecrease the level of ATZ. This result indicates that the reduction of ATZ level by Hrd1depends on its E3activity.1.5Hrd1promotes autophagic degradation of insoluble ATZOur previous findings had demonstrated that Hrd1interacts with ATZ and mediates itsSDS-soluble part to the proteasome for clearance and Hrd1primarily alters the level ofinsoluble ATZ, we wonder whether this is related to its autophagic degradation. To testthis hypothesis, HEK293T cells were co-transfected with ATZ and Hrd1.Co-tansfection of ATZ and vector was used as control. Twenty four hrs after transfection, cells were treated with NH4Cl for4hrs. The transfected cells were collected and lysed,the supernatant and the pellet were performed for western blotting, respectively.Consistent with previous study, ATZ level was significantly decreased in the pellet.However, NH4Cl blocked Hrd1-meidated insoluble ATZ degradation.1.6Co-expression of ATZ and Hrd1elevated the mRNA level of autophagy relatedgenes Atg5, Atg12and Beclin1Beclin1is one of the Atg family proteins conserved from yeast to humans (it is themammalian orthologue of yeast Atg6), and is required for autophagosome formation.The Atg12-Atg5conjugation is also involved in the process.293T cells wereco-transfected with ATZ and Hrd1cDNA.Twenty hour hrs after transfection, cells wereharvested. Total RNA was extracted and relative mRNA levels were measured byreal-time PCR. We found that co-expression of ATZ and Hrd1increased the mRNAlevel of Atg5, Atg12, and Beclin1..2. Hrd1targets K48-linked polyubiquitin chains of ATZ for autophagicdegradationUbiquitination is a dynamic post-translational modification process that functions asdiverse cellular roles. Ubiquitin is a small protein of76amino acids that acts as amodifier by covalent attachment to substrates. Conjugation of ubiquitin is a cascade ofenzymatic reactions that requires ubiquitin-activating enzyme (E1), aubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Polyubiquitinationchains are formed though isopeptide bonds between the C-terminal glycine residue ofubiquitin and the ε-amino group of a lysine residue on the substrate protein. There areseven internal lysine residues (K6, K11, K27, K29, K33, K48, and K63) within theconjugation of ubiquitin molecules. It has been reported that K48-linkedpolyubiquitination chains target the substrate protein to proteasome for dgradationn,while K63-linked polyubiquitination chains or monoubiquitination represents a proteasome-independent pathway in several cellular processes. We wonder whichkind of polyubiquitin chains is involved in Hrd1-mediated autophagic ATZ degradation.Cells were co-transfected with pdsRed-Hrd1and pcDNA3.1zeo+/ATZ.24hrs aftertransfection, cells were fixed and stained with anti-AAT (blue) and anti-K48(green) oranti-K63(green). We found that Hrd1and ATZ were strongly co-localized with K48butnot K63. To provide more evidence for the K48linkage of Hrd1-mediatedpolyubiquitination of ATZ, we performed co-immunoprecipitation assay. We found thatATZ interacted with K48when treated with the proteasome inhibitor MG132or thelysosome inhibitor BFA1. This result suggests that K48-linked polyubiquitin is involvedin Hrd1-mediated ATZ degradation, including autophagic ATZ degradation.3. p62is involved in Hrd1-mediated ATZ degradationSequestosome1/p62protein is commonly found in inclusion bodies containing largecytoplasmic aggregates. p62protein is able to polymerize via its N-terminal Phox andBem1p(PB1) domain. It binds ubiquitin via its C-terminal UBA motif, and binding toLC3via LIR domain. Our previous study had demonstrated that Hrd1enhances thedegradation of polyubiquitinated-ATZ, we wonder whether p62binds topolyubiquitinated-ATZ and targets it into lysosome for degradation. Cells wereco-transfected with pcDNA3.1-Hrd1-myc-His and pcDNA3.1zeo+/ATZ and treatedwith NH4Cl to inhibit autophagy activity. Double immunostaining with anti-AAT (blue),anti-myc (red) and anti-p62(green) revealed that ATZ, Hrd1and p62were stronglyoverlapped. Immunoprecipitation assay was also conducted to confirm the interaction ofATZ, Hrd1and p62. The result indicated that ATZ binds to p62. As a key cargo adaptorprotein, our results suggest that p62interacted with ATZ to mediate its autophagicdegradation. CONCLUSIONS:Hrd1mediates K48-linked polyubiquitination of ATZ and targets it to lysosome forautophagic degradation via interacting with p62. |
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