Biochemical and functional characterization of a novel anti-inflammatory biogenic amine

摘要 色氨酸(Trp)分解代谢是一种复杂的代谢途径，以细胞特有的方式产生超过50种代谢物。 色氨酸除了是5-羟色胺和褪黑素的前体外，还会产生一系列已知的代谢物 以犬尿氨酸的形式。犬尿氨酸代谢物被认为是最强大的免疫介体之一。 监管。这一应用的主旨源于我们最初的观察：(I)淋巴管内皮细胞 (LEC)和树突状细胞(DC)分泌一种以前未知的生物胺3HKA，它来自一种 色氨酸分解代谢的侧向途径，其功能目前尚不清楚；(Ii)3HKA具有明显的抗 通过抑制STAT1/NF-κΒ通路在小鼠和人树突状细胞(DC)中的炎性变化 随之而来的促炎趋化因子和细胞因子的释放减少；最值得注意的是，IL-6， IL12p70和肿瘤坏死因子α；(Iii)在体内，3HKA通过以下途径对实验性银屑病模型起到保护作用 减少皮肤厚度、红斑、结垢和皱纹。在一个肾毒性狼疮模型中，3HKA改善了 蛋白尿和血清尿素氮，全面改善免疫介导性肾小球肾炎和肾脏 功能障碍。因此，本申请的最终目标是全面表征3HKA的生物活性。 在目标1中，将使用一系列生化和生物物理分析来确定负责的酶 并研究其在生理和病理条件下的合成调节 条件。在目标2中，通过使用3HKA的生物素化衍生物，以及一系列生化和生物物理 实验中，我们将确定3HKA的受体靶点及其表达谱。在目标3中，使用目标和 非靶向磷酸化蛋白质组学方法、激酶文库筛选及CRISPR/Cas9基因敲除 策略我们将描述3HKA信号转导途径。此外，我们还将分析 3HKA对T细胞活化和分化为TH1、TH2、TH17表型、幼稚与效应型和记忆性T的影响 细胞转化和3HKA对不同APC(DC、PDC、MΦ)成熟/激活的影响。免疫表型 将在体外和体内进行，使用牛皮癣或肾毒性狼疮作为动物模型。最后，在AIM中 4我们利用我们对3HKA的了解，产生了3HKA类化合物，预测具有抗- 发炎作用。这些类似物将通过一系列体外和体内试验进行筛选，以充分评估它们的 潜在的新型免疫调节分子

Summary Tryptophan (Trp) catabolism is a complex pathway that generates over fifty metabolites in a cell-specific manner. Besides being the precursor of serotonin and melatonin, tryptophan generates a cascade of metabolites known as kynurenines. Kynurenine metabolites are regarded as one of the most powerful mediators of immune regulation. The thrust of this application stems from our original observations that: (i) lymphatic endothelial cells (LEC) and dendritic cells (DCs) secrete a previously unidentified biogenic amine, 3HKA, which derives from a lateral pathway of Trp catabolism, whose function is currently unknown; (ii) 3HKA exhibits a clear anti- inflammatory profile by inhibiting the STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines; most notably, IL-6, IL12p70 and TNFα; (iii) in vivo, 3HKA exerts protective effects in the experimental model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring. In a model of nephrotoxic lupus, 3HKA improved proteinuria and serum urea nitrogen, overall ameliorating the immune-mediated glomerulonephritis and renal dysfunction. As such, the ultimate goal of this application is to fully characterize the biological activity of 3HKA. In Aim 1, a series of biochemical and biophysical analyses will be employed to identify the enzyme responsible for 3HKA production and investigate the regulation of its synthesis under physiological and pathological conditions. In Aim 2, by using biotinylated derivatives of 3HKA, and a series of biochemical and biophysical experiments, we will identify the receptor target of 3HKA and its expression profile. In Aim 3, using targeted and untargeted phosphoproteomic approaches, screening of kinase libraries and CRISPR/Cas9 knock down strategies we will characterize the 3HKA signal transduction pathway. Additionally, we will analyze the effect of 3HKA on T cell activation and differentiation into a TH1, TH2, TH17 phenotype, naïve vs effector and memory T cell transition and 3HKA effect on maturation/activation of different APC (DC, pDC, MΦ). Immunophenotyping will be performed both in vitro and in vivo, using psoriasis or nephrotoxic lupus as animal models. Finally, in Aim 4 we leveraged our knowledge on 3HKA and generated 3HKA-like compounds, predicted to have an anti- inflammatory effect. These analogs will be screened in a series of in vitro and in vivo assay to fully assess their potential as novel immunomodulatory molecules