UBXN1 Regulates Inflammasome Signaling

脓毒症是一种危及生命的疾病，由于人体的极端炎症反应感染。那里 在美国，每年有> 150万例败血症和> 250，000例败血症相关死亡。然而，在这方面， 没有有效的治疗方法。过度活跃的炎性小体参与脓毒症的发病机制， 可以通过治疗来挽救生命。炎性小体是大的胞质多蛋白复合物 在感染和细胞应激反应中形成，这些应激反应驱动炎性半胱天冬酶的自动激活 （半胱天冬酶-1，-4，-5），炎症介质[白细胞介素（IL）-1和-18）]的产生，和焦亡，一种形式的 细胞死亡典型的炎性小体由模式识别受体（PRR）、适配器ASC （任选的）和胱天蛋白酶-1。非典型炎性小体被细菌内毒素直接结合激活 脂多糖（LPS）转化为Caspase-4/5（小鼠Caspase-11），其寡聚化并切割Gasdermin D （GSDMD）。活性GSDMD诱导质膜中的孔形成和细胞死亡。虽然很多 关于经典炎性小体的调节机制，人们对非经典炎性小体的调节机制知之甚少。 炎性小体为了解决这一问题，我们最近发现了一个含有泛素调节X（UBX）结构域的 蛋白1（UBXN 1）参与非典型炎性小体介导的脓毒症，并认为验证其作用 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 诱导型Ubxn 1基因全敲除小鼠模型-一种重要工具，因为早期全敲除是胚胎致死的。 使用该模型，我们发现Ubxn 1-/-小鼠对致死性LPS内毒素血症和盲肠炎具有高度抵抗性。 与性别和年龄匹配的Ubxn 1 +/+相比，结扎和穿刺（CLP）引起的多微生物败血症 同窝出生的因此，在Ubxn 1-/-中IL-1、IL-18和GSDMD/Caspase-11活化水平降低。 小鼠值得注意的是，在原发性Ubxn 1-/- 巨噬细胞也是。这种UBXN 1功能在人类细胞中是保守的。Caspase-4/GSDMD激活 在IFN-γ致敏的LPS转染的UBXN 1-/- HeLa细胞中，此外，UBXN 1 与LPS和Caspase-4相互作用。这些结果支持了我们的假设，即UBXN 1正调控非- 典型的炎性体信号传导，因此有助于脓毒症发病机制。我们将通过精确定位 UBXN 1作用于Caspase-4/11-LPS的分子机制，并通过表征其生理学特性， UBXN 1在CLP脓毒症中的作用 UBXN 1蛋白在啮齿类动物和人类之间高度保守（93%相同），其功能也是如此。 炎性小体信号传导。通过使用CLP方法，我们的研究应提供相关的临床前证据 人类败血症本次R21的顺利完成将为更深入的机械化奠定坚实的基础 以及未来R 01应用中的功能研究。

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.