UBXN1 Regulates Inflammasome Signaling

脓毒症是一种威胁生命的疾病，由于人体对感染的极端炎症反应。那里 在美国，每年有150万败血症病例和25万与败血症相关的死亡。然而， 目前还没有有效的治疗方法。过度活跃的炎症体参与了脓毒症的发病机制和 可能是治疗上的目标，以拯救生命。炎性小体是一种大的胞浆多蛋白复合体 形成于对感染和细胞应激的反应，这些感染和细胞应激驱动炎性半胱氨酸酶的自动激活 (caspase-1，-4，-5)，炎症介质的产生[IL-1和-18]，以及下垂，一种形式的 细胞死亡。典型的炎症体由模式识别受体(PRR)、适配器ASC组装而成 (可选)和Caspase-1。非典型炎症体通过细菌内毒素的直接结合而激活 脂多糖与Caspase-4/5(小鼠Caspase-11)结合，寡聚并裂解Gasdermin D (GSDMD)。活性GSDMD可诱导质膜形成孔洞和细胞死亡。尽管有很多 人们已经了解了典型炎症体的调节机制，但对非典型炎症体的调节机制知之甚少 炎症性小体。为了解决这一差距，我们最近发现了一个泛素调节X(UBX)结构域，其中包含 蛋白1(UBXN1)参与非典型炎症小体介导的脓毒症，并认为验证其作用 在这一过程中将揭示新的治疗策略。为了开始揭示它的功能，我们生成了第一个 可诱导的全球Ubxn1基因敲除小鼠模型-一个关键的工具，因为早期的全球基因敲除在胚胎上是致命的。 使用该模型，我们发现Ubxn1-/-小鼠对致死性内毒素血症和盲肠毒素血症具有高度的抵抗力。 结扎穿刺术(CLP)诱发的多菌败血症与性别和年龄匹配的Ubxn1+/+的比较 一窝产仔。相应地，Ubxn1-/-的IL-1、IL-18和GSDMD/Caspase-11活化水平降低 老鼠。值得注意的是，在原发Ubxn1-/-中，GSDMD和Caspase-11的激活对内毒素的反应减少 巨噬细胞也是。这种UBXN1功能在人类细胞中是保守的。Caspase-4/GSDMD激活 在干扰素-γ诱导的、脂多糖转染组的UBXN1-/-HeLa细胞中，上睑下垂受到损害。此外，UBXN1 与脂多糖和Caspase-4相互作用。这些结果支持了我们的假设，即UBXN1正向调节非 典型的炎症体信号转导，从而参与脓毒症的发病机制。我们将通过精确定位来证明这点 UBXN1作用于Caspase-4/11-内毒素的分子机制 UBXN1在CLP败血症中的作用 UBXN1蛋白在啮齿动物和人类之间高度保守(93%相同)，其功能也是如此 炎症小体信号。通过使用CLP方法，我们的研究应该提供相关的临床前证据 人类败血症。R21的成功完成将为更深入的机制奠定坚实的基础 并在未来的R01应用中进行功能研究。

Sepsis is a life-threatening illness due to the human body’s extreme inflammatory response to infection. There are >1.5 million cases of sepsis and >250,000 sepsis-related deaths each year in the United States. However, there are no effective therapies. Hyperactive inflammasomes are involved in the pathogenesis of sepsis and could be therapeutically targeted to save lives. Inflammasomes are large cytosolic multiprotein complexes formed in response to infections and cellular stresses that drive auto-activation of inflammatory caspases (Caspase-1, -4, -5), production of inflammatory mediators [interleukin (IL) -1 and -18)], and pyroptosis, a form of cell death. Canonical inflammasomes are assembled by a pattern recognition receptor (PRR), an adaptor ASC (optional) and Caspase-1. Non-canonical inflammasomes are activated by direct binding of a bacterial endotoxin lipopolysaccharide (LPS) to Caspase-4/5 (mouse Caspase-11), which oligomerizes and cleaves Gasdermin D (GSDMD). Active GSDMD induces pore formation in the plasma membrane and cell death. Although much has been learned about the regulatory mechanisms for canonical inflammasomes, little is known for non-canonical inflammasomes. To address this gap, we recently identified an ubiquitin regulatory X (UBX) domain-containing protein 1 (UBXN1) involved in non-canonical inflammasome-mediated sepsis, and believe that validating its role in this process will reveal new treatment strategies. To begin uncovering its function, we generated the first inducible global Ubxn1 knockout mouse model—a critical tool, as early global knockout is embryonically lethal. Using this model, we found that Ubxn1-/- mice were highly resistant to lethal LPS endotoxemia and cecal- ligation-and-puncture (CLP)-elicited polymicrobial sepsis, compared to sex- and age-matched Ubxn1+/+ littermates. Accordingly, the levels of IL-1, IL-18, and GSDMD/Caspase-11 activation were reduced in Ubxn1-/- mice. Of note, GSDMD and Caspase-11 activation in response to LPS was reduced in primary Ubxn1-/- macrophages as well. This UBXN1 function is conserved in human cells. Both Caspase-4/GSDMD activation and pyroptosis were impaired in IFN-γ-primed, LPS-transfected UBXN1-/- HeLa cells. Moreover, UBXN1 interacted with LPS and Caspase-4. These results support our hypothesis that UBXN1 positively regulates non- canonical inflammasome signaling and thus contributes to sepsis pathogenesis. We will prove this by pinpointing the molecular mechanism of UBXN1 action on the Caspase-4/11-LPS, and by characterizing the physiological role of UBXN1 in CLP sepsis. UBXN1 protein is highly conserved between rodents and humans (93% identical), so is its function in inflammasome signaling. By using the CLP method, our studies should provide pre-clinical evidence relevant to human sepsis. Successful completion of this R21 will lay a solid foundation for more in-depth mechanistic and functional studies in a future R01 application.