High mobility group box 1 (HMGB1) is a multifunctional regulator that plays different roles in various physiological and pathological processes including cell development, autophagy, inflammation, tumor metastasis, and cell death based on its cellular localization. Unlike mammalian HMGB1, two HMGB1 paralogues (HMGB1a and HMGB1b) have been found in fathead minnow and other fish species and its function as an inflammatory cytokine has been well investigated. However, the role of fish HMGB1 in autophagy regulation has not been well clarified. In the present study, we generated HMGB1 paralogues single (HMGB1a and HMGB1b) and double knockout (DKO) epithelioma papulosum cyprini (EPC) cells from fathead minnow by CRISPR/Cas9 system, and the knockout efficiency of these genes was verified at both gene and protein levels. In this context, the effects of HMGB1 gene knockout on the protein expression of microtubule-associated protein 1 light chain 3 II (LC3-II), an autophagy marker, were determined, showing that single knockout of two HMGB1 paralogues significantly decreased the expression of LC3-II, and these inhibitory effects were further amplified in HMGB1 DKO cells under both basal and rapamycin treatment conditions, indicating the role of two HMGB1 paralogues in fish autophagy. In agreement with this notion, overexpression of HMGB1a or HMGB1b with Flag-tag markedly upregulated LC3-II protein expression. Interestingly, overexpressing two paralogues distributed in both cytoplasm and nucleus. Finally, the role of HMGB1-mediated autophagy was further explored, finding that HMGB1 could interact with Beclin1, a key initiation factor of autophagy. Taken together, these findings highlighted the role of HMGB1 paralogues as the autophagy regulator and increased our understanding of autophagic machinery in teleost.

中文翻译：

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两个 HMGB1 旁系同源物的功能鉴定提供了对硬骨鱼自噬机制的见解

高迁移率族蛋白1（HMGB1）是一种多功能调节因子，根据其细胞定位，在细胞发育、自噬、炎症、肿瘤转移和细胞死亡等多种生理和病理过程中发挥不同的作用。与哺乳动物 HMGB1 不同，在黑头鲦鱼和其他鱼类中发现了两种 HMGB1 旁系同源物（HMGB1a 和 HMGB1b），并且其作为炎症细胞因子的功能已得到充分研究。然而，鱼类HMGB1在自噬调节中的作用尚未得到很好的阐明。在本研究中，我们通过CRISPR/Cas9系统从黑头呆鱼中产生了HMGB1旁系同源物单（HMGB1a和HMGB1b）和双敲除（DKO）上皮瘤鲤鱼（EPC）细胞，并在基因和基因组上验证了这些基因的敲除效率。蛋白质水平。在这种情况下，确定了 HMGB1 基因敲除对自噬标记物微管相关蛋白 1 轻链 3 II (LC3-II) 蛋白表达的影响，结果表明，两个 HMGB1 旁系同源物的单一敲除显着降低了LC3-II，并且这些抑制作用在基础和雷帕霉素处理条件下的 HMGB1 DKO 细胞中进一步放大，表明两个 HMGB1 旁系同源物在鱼类自噬中的作用。与这一观点一致，带有 Flag 标签的 HMGB1a 或 HMGB1b 的过表达显着上调了 LC3-II 蛋白的表达。有趣的是，过表达两个旁系同源物分布在细胞质和细胞核中。最后，进一步探讨了HMGB1介导的自噬的作用，发现HMGB1可以与自噬的关键启动因子Beclin1相互作用。总而言之，这些发现强调了 HMGB1 旁系同源物作为自噬调节剂的作用，并增加了我们对硬骨鱼自噬机制的理解。