HECT-domain E3 Ligase and Acute Lung Injury

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Acute Respiratory Distress Syndrome (ARDS) affects almost a quarter million patients annually and has a mortality rate of over 40%. Its primary causes are pneumonia and sepsis. Central to the pathophysiology of this lung injury is a sustained immune response. With an exaggerated immune response, NF-κB mediated cytokine release leads to the devastating effects of pulmonary edema, multi-organ failure, and shock. Recently, we discovered a novel pathway for inflammation through protein ubiquitination, a universal mechanism whereby ubiquitin E3 ligases target proteins for degradation. We discovered that a pro-inflammatory, orphan HECT- domain ubiquitin (Ub) E3 ligase, termed KIAA0317, is activated after microbial infection. This E3 ligase ubiquitinates a potent anti-inflammatory protein termed SOCS2 (Suppressor of Cytokine Signaling 2), thereby marking it for degradation in the proteasome. SOCS2 not only suppresses the activation of signal transducers and activators of transcription (STATs), but also prevents NF-κB-dependent gene activation. Maneuvers designed to selectively modulate the abundance of SOCS2 might serve as a novel strategy for therapeutic intervention. However, to date, very little is known regarding the molecular regulation of SOCS2 at the level of protein stability. Our preliminary data suggest that (i) bacteria activates KIAA0317, which is sufficient to ubiquitinate and mediate the degradation of the inflammatory repressor SOCS2, (ii) KIAA0317 is a pro- inflammatory protein in vivo and in vitro, (iii) the kinase PKCα phosphorylates SOCS2, thereby creating a unique molecular signal for KIAA0317 targeting, and (iv) a novel small molecule inhibitor of KIAA0317, BC-1365, attenuates LPS and P. aeruginosa-induced cytokine secretion in vivo. These data led to our novel hypothesis that transcriptionally upregulated KIAA0317 specifically targets SOCS2 for ubiquitination and degradation, thus inducing inflammation and tissue injury. We will determine how KIAA0317 is regulated transcriptionally by bacterial pathogens and the molecular basis of how SOCS2 is targeted by KIAA0317, thereby upregulating NF-κB mediated inflammation. We will also determine whether KIAA0317 and its genetic variants can be used as novel biomarkers for inflammatory diseases (Aim 1). We will test KIAA0317 knockout mice in various experimental lung injury models to confirm that KIAA0317 is a druggable target. Further, we will design an optimal KIAA0317 antagonist using a quantitative structure-activity relationship (qSAR) based design and test its toxicity, target engagement, and anti-inflammatory activity both in vitro and in vivo (Aim 2). Last, this proposal unveils a new molecular model of lung injury as it relates to cytokine signaling. Our preliminary data have uncovered a novel protein, KIAA0317, which is linked to cytokine response through SOCS2 protein signaling. These studies will be the first to elucidate the enzymatic behavior of KIAA0317, which appears to activate the NF-κB-cytokine axis. Execution of these studies will lay the groundwork for a fundamental, paradigm-changing therapeutic advance for regulating innate immunity and treating inflammatory diseases that will ultimately set the stage for a new translational initiative.