pFLAG-CMV2 vector

The nuclear protein HMGB1 is secreted in response to various stimuli and functions as a danger-associated molecular pattern.However,the mechanism of HMGB1 secretion remains largely unknown.Here,we investigated the role of secretory autophagy machinery and vesicle trafficking in HMGB1 secretion.The researchers observed that HSP90AA1(heat shock protein 90αfamily class A member 1),a stress-induced protein,regulates HMGB1 translocation from the nucleus to the cytoplasm and its secretion through direct interactions.Furthermore,geldanamycin,an HSP90AA1 inhibitor,reduced HMGB1 secretion.Secretion of HMGB1 is inhibited by early autophagy inhibitors and is reduced in ATG5-deficient cells even when GORASP2 is overexpressed.In contrast,late autophagy inhibitors increased HMGB1 secretion under the same conditions.The multivesicular body formation inhibitor GW4869 significantly reduces HMGB1 secretion under HMGB1 secretion-inducing conditions.Therefore,this study demonstrates that secretory autophagy and multivesicular body formation mediate HMGB1 secretion.

In this study,the pFLAG-CMV2 vector purchased from Creative Biogene was involved in the subcloning of HSP90AA1.cDNA of HSP90AA1 was subcloned into the pFlag CMV2 plasmid(Creative Biogene,VET1147).The HSP90AA1 domain mutants NTD(a.a.1–271),MD(a.a.272–629),and CTD(a.a.630–732)were inserted into the pFlag TOPO plasmid.

Figure 1.HSP90AA1 interacts with HMGB1.(Kim Y H,et al.,2021)