Ubiquitin Ligase Hrd1 Facilitates Degradation Of The Z Variant α1-antitrypsin And Enhances Cell Survival

Alpha-1-antitrypsin (AAT) is mainly synthesized in the hepatocytes and secreted into the blood. It is a major plasma serum protease inhibitor that protects tissues from enzymes, especially elastase digestion. AAT deficiency is an autosomal-recessive disorder that is characterized by the retention of malfolded AAT in the endoplasmic reticulum (ER) of hepatocytes and by a significant diminution in the serum levels of AAT, which subsequently causes liver and lung diseases. It is caused by the mutations in genes encoding AAT. The most common mutation that causes more than 95% clinical cases of severe AAT deficiency is the Z allele. The mutated gene encodes AAT Z variant (ATZ). Previous studies have demonstrated that the ubiquitin-proteasome pathway is involved in the degradation of ATZ. However, detailed mechanisms of ATZ degradation are not fully understood. We have also found gp78, an E3 with ring-finger domain, enhances ATZ degradation. In present study, we investigated whether another ring-finger domain E3, ER membrane ubiquitin ligase Hrd1 facilitated the removal of ATZ through ER-associated degradation (ERAD).Objective:To investigate whether ER membrane ubiquitin ligase Hrd1 facilitates the removal of ATZ through ER-associated degradation (ERAD) and the effect of Hrd1 on the function of cells expressing ATZ.Methods:To construct the eukaryotic expresson vectors for ATZ, wt Hrd1, E3 activity abolished mutant Hrd1C1A, etc. Small interference RNA for Hrd1 was systhesized from Genepharm. Co. LTD. The eukaryotic expresson vectors or siRNA were transfected to HEK 293T cells or HepG2 cells. Reverse transcription polymerase chain reaction (RT-PCR) and immune blot (IB) were used to detect mRNA and protein level respectively. Flow cytometry was used to monitor the GFP fluorescence intensity. The mechanisms for Hrd1 mediated influences on ATZ level were explored by cycloheximide chase (CHX chase) and proteasome activity inhibition followed by IB. Cell morphologies, intracellular protein localization and quantitation were examed by immunocytochemistry and immunofluorescence. Co-immunoprecipitation (Co-IP) and protein fragment complementation assay (PCA) were employed to determine the direct interaction between Hrd1 and ATZ. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to find the apopototic cells. Results:1.Hrd1 decreases intracellular ATZ levelHEK 293T cells were co-transfected with ATZ and Hrd1 cDNA. Co-tansfection of ATZ and empty vector was used as control. Twenty four hours after transfection, cells were collected and lysed in Triton X-100 buffer containing protease inhibitors. After centrifugation, the supernatant was processed for IB AAT. ATZ in the supernatant (Triton X-100-soluble fraction) was not markedly reduced after co-transfection with Hrd1, but the high molecular weight (MW) ATZ was decreased, suggesting that Hrd1 may inhibit the formation of insoluble ATZ aggregates. To test this possibility, the transfected cells were collected and lysed with 0.1% SDS. The supernatant and the pellet were processed for IB respectively. We found that the ATZ level was significantly decreased in the pellet, though it was slightly increased in the supernatant. As a result, the total level of ATZ was decreased by Hrd1.2.Knockdown of endogenous Hrd1 with siRNA stabilized intracellular ATZ levelThe synthetic siRNA-Hrd1 was co-transfected with ATZ into HEK 293T cells. IB AAT was followed 24 hrs after transfection. The results indicated knockdown of Hrd1 with siRNA stabilized ATZ especially in the detergent-insoluble fractions.3.E3 activity is required for Hrd1-mediated reduction of intracellular ATZ levelHrd1 belongs to ring-finger domain E3 family. Its E3 activity relies on the integrity of the ring-finger domain. To assess the effect of Hrd1 E3 activity on ATZ level, the Hrd1C1A mutant that is deficient in E3 activity by the mutation of zinc ligand residue at the first cysteine site was tested. Immunocytochemistry revealed a negative relationship between intracellular ATZ and wt Hrd1 but not Hrd1C1A level. Western blotting demonstrated that the Hrd1C1A mutant did not decrease the level of ATZ in both the SDS-soluble fraction and the pellet, compared with wild type Hrd1. These results suggest that the reduction of ATZ retention by Hrd1 depends on the intact zinc ligand residues of Hrd1 and its E3 activity.4.Hrd1 facilitates ATZ degradationIntracellular protein level is determined by the balance of protein synthesis and degradation. Either blocking protein synthesis or increasing protein degradation will lead to the reduction of cellular protein levels. To determine whether the reduction of intracellular ATZ is due to the enhancement of ATZ degradation, CHX (100μg/ml) was used to inhibit protein synthesis. CHX was added to the cells 24 hrs after transfection and the cells were collected at different time intervals. The results show that Hrd1 mainly accelerated ATZ clearance in the SDS-insoluble fraction, not in the supernatant. On the contrary, Hrd1 increased the amount of ATZ in the supernatant. These data indicate that Hrd1 increases the solubility of ATZ and facilitates ATZ degradation.5.E3 activity is required for Hrd1-mediated ATZ degradationTo further determine whether Hrd1 E3 activity is involved in ATZ degradation, Hrd1C1A and ATZ were co-transfected into HEK 293T cells. CHX chase was conducted to detect protein degradation. The results indicate Hrd1C1A mutant stabilized ATZ in the cells, especially in the supernatant compared to wt Hrd1. This result suggests that one cysteine residue mutation in the ring-finger domain does not affect the function of Hrd1 on ATZ solubility, but attenuates its activity on ATZ degradation.6.Hrd1 increases ATZ ubiquitinationThere are two major protein degradation pathways for intracellular protein, namely the ubiquitin-proteasome pathway (UPP) and the lysosomal pathway. ATZ was shown to be degraded by both of them. Since Hrd1 is a ring-finger domain E3 and degrades ATZ dependently on its intact ring-finger domain, we reason that Hrd1 targets ATZ for degradation at least in part through UPP. To test this hypothesis, we first investigated whether ubiquitination was required for Hrd1-mediated ATZ degradation. The plasmid encoding ubiquitin was co-transfected into 293T cells to enhance ATZ ubiquitination. We found that Hrd1 decreased soluble polyubiquitinated ATZ. After inhibition of proteasome activity by MG132, the high MW ATZ was present in the cells co-expressing Hrd1, but not Hrd1C1A. These results indicate that the presence of the high MW ATZ depends on Hrd1 E3 activity and proteasome inhibition, and that Hrd1 mediates degradation of polyubiquitinated ATZ.7 . valosin-containing protein (VCP) is required for Hrd1-mediated ATZ degradationATZ is an endoplasmic reticulum (ER) lumen protein and its degradation for proteasome requires retrotranslocation from the ER into the cytosol. Therefore, VCP, together with its cofactors, must play an essential role in the retrotranslocation of ATZ for UPP. To test this possibility, VCPQQ, a mutant form of p97/VCP that does not have ATPase activity and does not function in ERAD was used. The result showed that VCPQQ stabilized ATZ, especially in the SDS-soluble fraction, suggesting that soluble ATZ degradation was blocked by VCPQQ. This result indicates that VCP is required for Hrd1-mediated soluble ATZ degradation and also provides additional evidence that Hrd1 increases the solubility of ATZ.8. Lysosomal pathway maybe involved in Hrd1-mediated ATZ degradationTo clarify whether lysosomal pathway is involved in Hrd1-mediated ATZ degradation. Ammonium chloride was used as lysosome inhibitor. The result indicated that ammonium chloride increased the high molecular weight ATZ level. For more intuitive observation of Hrd1 on the level of non-ER localized intracellular ATZ, we constructed GFP-tagged ATZ expression vector and put GFP before the signal peptide of ATZ to change the intracellular localization of ATZ. GFP-ATZ and Hrd1 were expressed in HEK 293T cells. The GFP fluorenscence intensity was detected by both fluorenscencent microscope and FCS 24 hrs after transfection. Hrd1 significantly reduced the GFP fluorescence intensity of GFP-ATZ expressing cells compared to the control. By contrast, in the control cells expressing GFP, Hrd1 had no effect on the level of GFP either in supernatant or in pellet, suggesting the effect of Hrd1 on ATZ is specific.9.Hrd1 interacts with ATZThe previous data indicates that Hrd1 targets ATZ degradation through UPP. We next explore whether Hrd1 interacts with ATZ. In the cells co-transfected with FLAG-tagged Hrd1 and ATZ, double immunostaining with anti-AAT and anti-FLAG antibodies revealed that Hrd1 and ATZ were strongly co-localized. Immunoprecipitation assay was also conducted to confirm the interaction of Hrd1 and ATZ. The results indicated that ATZ was co-immunmoprecipitated with both wt Hrd1 and Hrd1C1A, suggesting this interaction did not depend on the intact ring-finger domain. We got further proof for this interaction from PCA. We fused C (G1) and N terminal (G2) of luciferase to N terminal of Hrd1 and ATZ respectively and the two constructs were co-transfected to HEK 293T cells. The relative hGluc activity significantly increased in the cells co-transfected with Hrd1-G1 and ATZ-G2 compared with Hrd1-G1 or ATZ-G2 alone.10.Hrd1 relieves the toxicity of ATZIntracellular accumulation of ATZ is toxic to hepatocytes. We wondered whether the removal of the accumulated ATZ by Hrd1 promoted cell survival. Double immunofluorescent labeling was conducted to show the over-expressed proteins. In both HEK 293T and HepG2 cells, ATZ-expressing resulted in abnormal morphologies. The cells became round, detached, decreased in size, and shortened in cell processes. The nuclei were especially reduced in ATZ-positive cells. However, the amount of ATZ was decreased and the proportion of ATZ-positive cells with normal morphologies increased after wt Hrd1 co-transfection. Moreover, Hrd1C1A mutant did not improve the abnormal morphologies of the ATZ-positive cells. In situ cell death detection assay indicated wt Hrd1 but not Hrd1C1A reduced cell apoptosis. These datas indicate Hrd1 relieves ATZ toxicity and it requires its E3 activity.Conclusions:Our findings demonstrated that Hrd1 acts as an E3 that mainly decreases the retention of detergent-insoluble ATZ in ER by ERAD and increases its solubility. Accordingly, Hrd1 relieves ATZ toxicity and promotes cell survival, which implies a potential target in the treatment of AAT deficiency liver complications.