Isorhamnetin, a 3’-methoxylated flavonol, enhances the lysosomal proteolysis in J774.1 murine macrophages in a TFEB-independent manner.
Isorhamnetin, a 3’-methoxylated flavonol, enhances the lysosomal proteolysis in J774.1 murine macrophages in a TFEB-independent manner.
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DOI：
10.1080/09168451.2020.1727309
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发表时间：
2020-02
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通讯作者：
Maiko Sakai;K. Ohnishi;Masashi Masuda;Hirokazu Ohminami;H. Yamanaka-Okumura;Taichi Hara;Y. Taketani
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作者：
Maiko Sakai;K. Ohnishi;Masashi Masuda;Hirokazu Ohminami;H. Yamanaka-Okumura;Taichi Hara;Y. Taketani
溶酶体是细胞大分子最终降解的主要细胞器，这些大分子通过内吞作用、吞噬作用和自噬传递。溶酶体功能已被发现受到脂肪食物和衰老的损害，更重要的是，巨噬细胞溶酶体功能障碍已被报道为动脉粥样硬化发展的危险因素。在这项研究中，我们寻找具有促进小鼠巨噬细胞样细胞系J774.1溶酶体降解活性的膳食多酚。利用DQ-BSA酶切法进行筛选，鉴定出异鼠李素（3′- o -甲基槲皮素）为活性化合物。有趣的是，与无活性黄酮醇的结构比较表明，异鼠李素b环部分的化学结构是其溶酶体增强活性的主要决定因素。出乎意料的是，异鼠李素未能抑制mTORC1-TFEB信号，这是溶酶体生物发生和功能的主要调节因子。我们的数据表明，其他分子机制可能对巨噬细胞中溶酶体的调节至关重要。缩写：ANOVA：方差分析；ApoE：载脂蛋白E；ATP6V0D2：转运V0亚基d2的ATPase H+；BAF：巴菲霉素A1；BODIPY：二硝基甲烷硼；牛血清白蛋白；CTSD：组织蛋白酶D；CTSF：组织蛋白酶F；DMEM: Dulbecco改良eagle培养基；DMSO：二甲基亚砜；EGCG: epigallocatechin-3-gallate;胎牛血清；GAPDH：甘油醛-3-磷酸脱氢酶；HPLC：高效液相色谱法；LAMP1：溶酶体相关膜蛋白1；LAMP2A：溶酶体相关膜蛋白2A；LC-MS/MS：液相色谱串联质谱法；MITF：小眼相关转录因子；MRM：多反应监测；mTORC1：雷帕霉素复合物1的机制靶点PBS：磷酸盐缓冲盐水；PPARγ：过氧化物酶体增殖物激活受体γ；RT-qPCR：逆转录定量聚合酶链反应；SDS：十二烷基硫酸钠；SNARE：可溶性n -乙基马来酰亚胺敏感因子附着蛋白受体；TBS: tris缓冲盐水；TFA：三氟乙酸；TFE3：结合IGHM增强子3的转录因子；TFEB：转录因子EB；TFEC：转录因子EC；v - atp酶：液泡型质子atp酶。对56种膳食多酚进行筛选，发现异鼠李素是小鼠J774.1巨噬细胞样细胞系溶酶体的激活剂。
ABSTRACT Lysosome is the principal organelle for the ultimate degradation of cellular macromolecules, which are delivered through endocytosis, phagocytosis, and autophagy. The lysosomal functions have been found to be impaired by fatty foods and aging, and more importantly, the lysosomal dysfunction in macrophages has been reported as a risk of atherosclerosis development. In this study, we searched for dietary polyphenols which possess the activity for enhancing the lysosomal degradation in J774.1, a murine macrophage-like cell line. Screening test utilizing DQ-BSA digestion identified isorhamnetin (3ʹ-O-methylquercetin) as an active compound. Interestingly, structural comparison to inactive flavonols revealed that the chemical structure of the B-ring moiety in isorhamnetin is the primary determinant of its lysosome-enhancing activity. Unexpectedly isorhamnetin failed to inhibit mTORC1-TFEB signaling, a master regulator of lysosomal biogenesis and function. Our data suggested that the other molecular mechanism might be critical for the regulation of lysosomes in macrophages. Abbreviations: ANOVA: analysis of variance; ApoE: apolipoprotein E; ATP6V0D2: ATPase H+ transporting V0 subunit d2; BAF: bafilomycin A1; BODIPY: boron dipyrromethene; BSA: bovine serum albumin; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco’s modified eagle medium; DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LC-MS/MS: liquid chromatography tandem mass spectrometry; MITF: microphthalmia-associated transcription factor; MRM: multiple reaction monitoring; mTORC1: mechanistic target of rapamycin complex 1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor γ; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SDS: sodium dodecyl sulfate; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TBS: Tris-buffered saline; TFA: trifluoroacetic acid; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcriptional factor EB; TFEC: transcription factor EC; V-ATPase: vacuolar-type proton ATPase. Graphical abstract Screening of 56 kinds of dietary polyphenols identified isorhamnetin as an activator of lysosome in J774.1 mouse macrophage-like cell line.