Ubiquitin ligase SYVN1/HRD1 facilitates degradation of the SERPINA1 Z variant/alpha-1-antitrypsin Z variant via SQSTM1/p62-dependent selective autophagy

SERPINA1/AAT/alpha-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 (SERPINA1^E342K/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 SERPINA1^E342K/ATZ after it accumulates in the ER. However, the mechanisms by which SERPINA1^E342K/ATZ is selectively degraded by autophagy remain unknown. Here, we showed that ER membrane-spanning ubiquitin ligase (E3) SYVN1/HRD1 enhances the degradation of SERPINA1^E342K/ATZ through the autophagy-lysosome pathway. We found that SYVN1 promoted SERPINA1^E342K/ATZ, especially Triton X 100-insoluble SERPINA1^E342K/ATZ clearance. However, the effect of SYVN1 in SERPINA1^E342K/ATZ clearance was impaired after autophagy inhibition, as well as in autophagy related 5 (<i>atg5</i>) knockout cells. On the contrary, autophagy induction enhanced SYVN1-mediated SERPINA1^E342K/ATZ degradation. Further study showed that SYVN1 mediated SERPINA1^E342K/ATZ ubiquitination, which is required for autophagic degradation of SERPINA1^E342K/ATZ by promoting the interaction between SERPINA1^E342K/ATZ and SQSTM1/p62 for formation of the autophagy complex. Interestingly, SYVN1-mediated lysine 48 (K48)-linked polyubiquitin chains that conjugated onto SERPINA1^E342K/ATZ might predominantly bind to the ubiquitin-associated (UBA) domain of SQSTM1 and couple the ubiquitinated SERPINA1^E342K/ATZ to the lysosome for degradation. In addition, autophagy inhibition attenuated the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ cytotoxicity, and the autophagy inducer rapamycin enhanced the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ-induced cell apoptosis. Therefore, this study proved that SYVN1 enhances SERPINA1^E342K/ATZ degradation through SQSTM1-dependent autophagy and attenuates SERPINA1^E342K/ATZ cytotoxicity.

SERPINA1/AAT/α1-抗胰蛋白酶（serpin家族A成员1，SERPINA1/AAT/alpha-1-antitrypsin）缺乏症（简称SERPINA1/AAT-D）是一种常染色体隐性遗传病，其特征为错折叠的SERPINA1/AAT在肝细胞内质网（endoplasmic reticulum, ER）中滞留，且血清SERPINA1/AAT水平显著降低。SERPINA1/AAT的Z型变异体携带Glu342Lys（E342K）突变（SERPINA1^E342K/ATZ），是SERPINA1/AAT-D最常见的致病类型，该变异体易发生错折叠与聚合，进而滞留于肝细胞内质网并引发肝损伤。蛋白酶体（proteasome）与巨自噬/自噬（macroautophagy/autophagy）通路均负责清除在内质网中蓄积的SERPINA1^E342K/ATZ，但目前尚不明确自噬选择性降解SERPINA1^E342K/ATZ的具体机制。

本研究证实，跨内质网膜泛素连接酶（E3，ubiquitin ligase (E3)）SYVN1/HRD1可通过自噬-溶酶体通路促进SERPINA1^E342K/ATZ的降解。研究发现，SYVN1可促进SERPINA1^E342K/ATZ的清除，尤其是曲拉通X-100（Triton X-100）不溶性的SERPINA1^E342K/ATZ。然而，自噬抑制剂处理或自噬相关5（autophagy related 5, ATG5）基因敲除细胞会削弱SYVN1对SERPINA1^E342K/ATZ的清除作用。反之，自噬诱导可增强SYVN1介导的SERPINA1^E342K/ATZ降解。进一步研究显示，SYVN1可介导SERPINA1^E342K/ATZ的泛素化，这一过程可促进SERPINA1^E342K/ATZ与SQSTM1/p62的相互作用以形成自噬复合体，是SERPINA1^E342K/ATZ通过自噬降解的必要条件。有趣的是，结合在SERPINA1^E342K/ATZ上的、由SYVN1介导的赖氨酸48（K48）连接型多泛素链，可主要结合SQSTM1/p62的泛素相关（UBA）结构域（ubiquitin-associated (UBA) domain），进而将泛素化的SERPINA1^E342K/ATZ靶向至溶酶体进行降解。此外，自噬抑制会削弱SYVN1对SERPINA1^E342K/ATZ细胞毒性的抑制作用，而自噬诱导剂雷帕霉素（rapamycin）可增强SYVN1对SERPINA1^E342K/ATZ诱导的细胞凋亡的抑制效果。综上，本研究证明SYVN1可通过依赖SQSTM1/p62的自噬通路促进SERPINA1^E342K/ATZ的降解，并减轻其细胞毒性。