ABSTRACT

Nuclear protein HMGB1 is secreted in response to various stimuli and functions as a danger-associated molecular pattern. Extracellular HMGB1 induces inflammation, cytokine production, and immune cell recruitment via activation of various receptors. As HMGB1 does not contain an endoplasmic reticulum-targeting signal peptide, HMGB1 is secreted via the endoplasmic reticulum-Golgi independently via an unconventional secretion pathway. However, the mechanism underlying HMGB1 secretion remains largely unknown. Here, we investigated the role of secretory autophagy machinery and vesicular trafficking in HMGB1 secretion. We observed that HSP90AA1 (heat shock protein 90 alpha family class A member 1), a stress-inducible protein, regulates the translocation of HMGB1 from the nucleus to the cytoplasm and its secretion through direct interaction. Additionally, geldanamycin, an HSP90AA1 inhibitor, reduced HMGB1 secretion. GORASP2/GRASP55 (golgi reassembly stacking protein 2), ARF1^Q71L (ADP ribosylation factor 1), and SAR1A^T39N (secretion associated Ras related GTPase 1A), which promoted unconventional protein secretion, increased HMGB1 secretion. HMGB1 secretion was inhibited by an early autophagy inhibitor and diminished in ATG5-deficient cells even when GORASP2 was overexpressed. In contrast, a late autophagy inhibitor increased HMGB1 secretion under the same conditions. The multivesicular body formation inhibitor GW4869 dramatically decreased HMGB1 secretion under HMGB1 secretion-inducing conditions. Thus, we demonstrated that secretory autophagy and multivesicular body formation mediate HMGB1 secretion.

摘要

核蛋白 HMGB1 是响应各种刺激而分泌的，并作为与危险相关的分子模式起作用。细胞外 HMGB1 通过激活各种受体诱导炎症、细胞因子产生和免疫细胞募集。由于 HMGB1 不含内质网靶向信号肽，因此 HMGB1 通过非常规分泌途径独立地通过内质网-高尔基体分泌。然而，HMGB1 分泌的机制仍然很大程度上未知。在这里，我们研究了分泌自噬机制和囊泡运输在 HMGB1 分泌中的作用。我们观察到 HSP90AA1（热休克蛋白 90 α 家族 A 类成员 1）是一种应激诱导蛋白，通过直接相互作用调节 HMGB1 从细胞核到细胞质的易位及其分泌。此外，格尔德霉素是一种 HSP90AA1 抑制剂，可减少 HMGB1 的分泌。GORASP2/GRASP55（高尔基体重组堆叠蛋白 2），ARF1^{促进非常规蛋白质分泌的Q71L}（ADP 核糖基化因子 1）和 SAR1A ^T39N（分泌相关的 Ras 相关 GTPase 1A）增加了 HMGB1 的分泌。HMGB1 的分泌受到早期自噬抑制剂的抑制，并且在 ATG5 缺陷细胞中减少，即使 GORASP2 过表达也是如此。相反，晚期自噬抑制剂在相同条件下增加了 HMGB1 的分泌。在 HMGB1 分泌诱导条件下，多泡体形成抑制剂 GW4869 显着降低了 HMGB1 分泌。因此，我们证明了分泌性自噬和多泡体形成介导了 HMGB1 的分泌。