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CNOT2 promotes degradation of p62/SQSTM1 as a negative regulator in ATG5 dependent autophagy.

ABSTRACT: Though CNOT2 is involved in regulation of adipogenic differentiation, apoptotic cell death and metastasis, the underlying autophagic mechanism of CNOT2 was unknown until now. Thus, in the present study, the critical role of CNOT2 in autophagy was elucidated in association with p62/SQSTM1 signaling. CNOT2 depletion induced p62/SQSTM1 accumulation and LC3B-II conversion, and also increased the number of puncta with impaired autophagic flux. In contrast, CNOT2 overexpression induced downregulation and ubiquitination of p62/SQSTM1 in HEK293 QBI. Furthermore, ubiquitination of p62/SQSTM1 was blocked by autophagy inhibition. Interestingly, CNOT2 was correlated with p62/SQSTM1 in HEK293 QBI cells and also was colocalized with p62/SQSTM1 in H1299 cells. Additionally, ATG5 was upregulated in CNOT2-depleted H1299 cells, while degradation of p62/SQSTM1 by CNOT2 was detected in ATG5+/+ MEF cells but not in ATG5-/- MEF cells. Of note, CNOT2 induced degradation of p62/SQSTM1 in HEK293 QBI cells co-transfected with Myc-ΔLIR/KIR or Myc-ΔUBA, but not with Myc-ΔPB1. Sub G1 population was increased in CNOT2-depleted H1299 cells by late autophagy inhibitors, ammonium chloride and chloroquine compared to 3-methyladenine. Overall, these findings provide novel insight into the critical role of CNOT2 as a negative regulator in ATG5 dependent autophagy.

SUBMITTER: Jeong K

PROVIDER: S-EPMC5542246 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Project description:SERPINA1/AAT/?-1-antitrypsin (serpin family A member 1) deficiency (SERPINA1/ AAT-D) is an autosomal recessive disorder characterized by the retention of misfolded SERPINA1/AAT in the endoplasmic reticulum (ER) of hepatocytes and a significant reduction of serum SERPINA1/AAT level. The Z variant of SERPINA1/AAT, containing a Glu342Lys (E342K) mutation (SERPINA1E342K/ATZ), the most common form of SERPINA1/AAT-D, is prone to misfolding and polymerization, which retains it in the ER of hepatocytes and leads to liver injury. Both proteasome and macroautophagy/autophagy pathways are responsible for disposal of SERPINA1E342K/ATZ after it accumulates in the ER. However, the mechanisms by which SERPINA1E342K/ATZ is selectively degraded by autophagy remain unknown. Here, we showed that ER membrane-spanning ubiquitin ligase (E3) SYVN1/HRD1 enhances the degradation of SERPINA1E342K/ATZ through the autophagy-lysosome pathway. We found that SYVN1 promoted SERPINA1E342K/ATZ, especially Triton X 100-insoluble SERPINA1E342K/ATZ clearance. However, the effect of SYVN1 in SERPINA1E342K/ATZ clearance was impaired after autophagy inhibition, as well as in autophagy-related 5 (atg5) knockout cells. On the contrary, autophagy induction enhanced SYVN1-mediated SERPINA1E342K/ATZ degradation. Further study showed that SYVN1 mediated SERPINA1E342K/ATZ ubiquitination, which is required for autophagic degradation of SERPINA1E342K/ATZ by promoting the interaction between SERPINA1E342K/ATZ and SQSTM1/p62 for formation of the autophagy complex. Interestingly, SYVN1-mediated lysine 48 (K48)-linked polyubiquitin chains that conjugated onto SERPINA1E342K/ATZ might predominantly bind to the ubiquitin-associated (UBA) domain of SQSTM1 and couple the ubiquitinated SERPINA1E342K/ATZ to the lysosome for degradation. In addition, autophagy inhibition attenuated the suppressive effect of SYVN1 on SERPINA1E342K/ATZ cytotoxicity, and the autophagy inducer rapamycin enhanced the suppressive effect of SYVN1 on SERPINA1E342K/ATZ-induced cell apoptosis. Therefore, this study proved that SYVN1 enhances SERPINA1E342K/ATZ degradation through SQSTM1-dependent autophagy and attenuates SERPINA1E342K/ATZ cytotoxicity.

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CNOT2 promotes degradation of p62/SQSTM1 as a negative regulator in ATG5 dependent autophagy.

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