hRpn13 binds epigenetic transcriptional regulators HDAC8 and PADI4 with its loss causing a reconfigured proteome and transcriptome

hRpn13 is an established proteasome substrate receptor that also exists off proteasomes. We demonstrate that its deletion by gene-editing leads to correlated proteomic and transcriptomic changes and that these effects are cell type-specific. The hRpn13 Pru region, which binds proteasomes and ubiquitin, directly interacts with two regulators of protein expression; namely, the histone deacetylase HDAC8, which we find to be inhibited by hRpn13 in a catalytic assay and to interact with hRpn13 off proteasomes, and bone marrow-specific protein arginine deimidase PADI4, which is depleted from multiple myeloma cells by either hRpn13 loss or proteasome inhibition. The NF-kB p105 precursor protein is partially proteolyzed by the proteasome to yield transcription activator p50; we find hRpn13 deletion or loss by PROTAC targeting to reduce p50 levels and that loss of hRpn13 or NF-kB inhibition reduces PADI4 and HDAC8 protein abundance, with their mRNA depleted by proteasome inhibition. Altogether, we discover a multi-layered regulatory network that connects hRpn13 to NF-kB activity and epigenetic regulators of arginine deimination and histone deacetylation.

hRpn13是一种公认的蛋白酶体（proteasome）底物受体，同时也可游离于蛋白酶体之外存在。本研究证实，通过基因编辑敲除hRpn13会引发相关性的蛋白质组学与转录组学变化，且此类效应具有细胞类型特异性。hRpn13的Pru结构域可结合蛋白酶体与泛素（ubiquitin），可直接与两种蛋白质表达调控因子发生相互作用：其一为组蛋白去乙酰化酶HDAC8（histone deacetylase 8），本研究通过催化实验发现，hRpn13可抑制HDAC8的活性，且二者可在游离于蛋白酶体之外的状态下相互结合；其二为骨髓特异性蛋白精氨酸脱亚胺酶PADI4（protein arginine deimidase PADI4），无论是敲除hRpn13还是抑制蛋白酶体活性，均可使多发性骨髓瘤细胞内的PADI4表达量显著下调。核因子κB（NF-κB）p105前体蛋白可被蛋白酶体部分水解为转录激活因子p50；本研究发现，敲除hRpn13或通过蛋白降解靶向嵌合体（PROTAC）介导的hRpn13降解均可降低p50的表达水平，而敲除hRpn13或抑制NF-κB活性则会减少PADI4与HDAC8的蛋白丰度，且蛋白酶体抑制可使二者的mRNA水平显著下调。综上，本研究揭示了一个多层级调控网络，该网络将hRpn13与NF-κB活性以及精氨酸脱亚胺和组蛋白去乙酰化的表观遗传调控因子联系起来。