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  • Inflammation is critical in the pathobiology

    2020-07-11

    Inflammation is critical in the pathobiology of atherosclerosis. The inflammatory mediators impacted by FBXO3 described herein i.e. NF-κB, IL-1β, and TNF-α, and IL-8 have purported causal roles in atherosclerosis. For example, NF-κB activation is increased in mononuclear cells in human plaques [29] and in peripheral blood mononuclear cells in patients with unstable 84 9 [30], and NF-κB inhibition attenuates atherosclerosis in hyperlipidemic mice [31]. IL-1β is a classic pro-inflammatory cytokine that has been linked to atherosclerosis and has been extensively studied [32]. Antagonizing IL1-β with a monoclonal antibody reduces atherosclerotic events in humans [3]. IL-8 causes rolling monocytes to adhere to endothelial cells, is mitogenic and chemotactic for vascular smooth muscle cells, and is increased within unstable atherosclerotic plaques [33,34]. TNF-α is a potent stimulator of several of the matrix metalloproteinases and of plasminogen activator inhibitor-1 [35] and has been identified as a player in several of the complications of atherosclerosis. Inhibition of TNF-α reduces atherosclerosis in apolipoprotein E knockout mice [35,36]. Hence, NF-κB antagonism, and reduced production of IL1-β, TNF-α, and IL-8 by upstream molecular inputs within the ubiquitin apparatus such as FBXO3 depletion would be expected to impair the development of atherosclerosis. Our study is not the first to implicate genetic variants in the FBXO3 gene as modulators of atherosclerosis risk. A genome wide association study that included over 5000 African-American participants and examined 2.5 million SNPs identified two variants in FBXO3 (rs11825259 and rs12291756) as among the top 67 SNPs associated with atherosclerosis risk. Unlike the SNP that was genotyped in our cohort, little is known about how rs11825259 and rs12291756 may alter function of FBXO3 [37]. Nonetheless, these independent findings corroborate the potential role of FBXO3 in regulating atherosclerosis in humans. There are multiple E3 ligases that can impact the pathobiology of atherosclerosis [38,39]. For example, HECT domain E3 ligases HUWE1 and NEDD4–1 control stability of the ATP-binding cassette transporter ABCG1 and ABCG4, both critical for cholesterol homeostasis [40]. In addition, the E3 ubiquitin ligase IDOL triggers lysosomal degradation of the low-density lipoprotein receptor [41]. Also, the ubiquitin ligase von Hippel-Lindau controls stability of hypoxia-inducible factor (HIF)-1a that in turn transcriptionally controls vascular endothelial growth factor (VGEF), an important regulator of neovascularization of atherosclerotic lesions [42]. Further, the E3 ubiquitin ligase ITCH modulates lipid metabolism and atherosclerosis by ubiquitination of SIRT6 and SREBP2, while genetic variants in E3 ligase RNF213 that regulates non-canonical Wnt signaling pathway have been associated with intracranial atherosclerosis [43,44]. While these studies demonstrate how the ubiquitin proteasome system can regulate atherosclerosis, no ubiquitin based therapies for atherosclerosis have been demonstrated as yet, nor have any SCF F-box protein E3 ligases been implicated in atherosclerosis to our knowledge. Modulating protein stability, specifically inhibiting ubiquitin E3 ligases, provides potential advantages over other druggable targets given the presence of biochemically multiple, unique pharmacophores within complexes that can bind a variety of small molecules [12]. The FBXO3 inhibitors used here were generated using in silico design and have been shown to ameliorate severity of inflammation in several preclinical models of cytokine-driven inflammation [10]. The FBXO3 inhibitor BC-1215 is a tool compound that binds within the ApaG domain of the protein. A Pan labs screen demonstrated that it exhibits 15/109 off-target hits mainly in the serotonin and adrenergic pathways (data not shown). The mechanistic centerpiece for targeting of FBXO3 rests upon its ability to upregulate NF-κB signaling through polyubiquitination and degradation of a constitutively active inhibitor of TRAF proteins, FBXL2 [10]. These results provide relevant pharmacodynamic and pharmacokinetic associations with biologic relevance that validate FBXO3 as a potential therapeutic target in preclinical models.