Department of Biochemistry and Biotechnology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.
Int J Mol Sci. 2020 Apr 2;21(7):2463. doi: 10.3390/ijms21072463.
The 26S proteasome is an ATP-dependent protease complex (2.5 MDa) that degrades most cellular proteins in Eukaryotes, typically those modified by a polyubiquitin chain. The proteasome-mediated proteolysis regulates a variety of critical cellular processes such as transcriptional control, cell cycle, oncogenesis, apoptosis, protein quality control, and stress response. Previous studies conducted in our laboratory have shown that 26S proteasomes are involved in the regulation of ligninolytic enzymes (such as laccase) in white-rot fungi in response to nutrient starvation, cadmium exposure, and ER stress. Laccases are useful biocatalysts for a wide range of biotechnological applications. The goal of the current study was to determine the effect of ferulic acid (4-hydroxy-3-methoxycinnamic acid), a phenolic compound known to induce some ligninolytic enzymes, on proteasomes isolated from mycelia of the wood-decomposing basidiomycete . The peptidase activities of 26S proteasomes were assayed by measuring the hydrolysis of fluorogenic peptide substrates specific for each active site: Suc-LLVY-AMC, Z-GGR-AMC and Z-LLE-AMC for chymotrypsin-like, trypsin-like, and caspase-like site, respectively. Ferulic acid affected all peptidase activities of the 26S fungal proteasomes in a concentration-dependent manner. A possible inhibitory effect of ferulic acid on peptidase activities of the 26S human proteasomes was tested as well. Moreover, the ability of ferulic acid to inhibit (at concentrations known to induce laccase activity in white-rot fungi) the rate of 26S proteasome-catalyzed degradation of a model full-length protein substrate (β-casein) was demonstrated by a fluorescamine assay and by a gel-electrophoretic analysis. Our findings provide new insights into the role of ferulic acid in lignin-degrading fungi. However, the detailed molecular mechanisms involved remain to be elucidated by future studies.
26S 蛋白酶体是一种依赖于 ATP 的蛋白酶复合物（2.5 MDa），可降解真核生物中大多数细胞蛋白，通常是那些被多泛素链修饰的蛋白。蛋白酶体介导的蛋白水解调节多种关键的细胞过程，如转录控制、细胞周期、癌变、细胞凋亡、蛋白质质量控制和应激反应。我们实验室之前的研究表明，26S 蛋白酶体参与了白腐真菌中木质素降解酶（如漆酶）的调节，以响应营养饥饿、镉暴露和内质网应激。漆酶是广泛生物技术应用的有用生物催化剂。本研究的目的是确定酚酸化合物阿魏酸（4-羟基-3-甲氧基肉桂酸）对木质素降解真菌菌丝体分离的 26S 蛋白酶体的影响，阿魏酸已知能诱导一些木质素降解酶。通过测量荧光肽底物对每个活性位点的水解来测定 26S 蛋白酶体的肽酶活性：Suc-LLVY-AMC、Z-GGR-AMC 和 Z-LLE-AMC 分别用于胰凝乳蛋白酶样、胰蛋白酶样和半胱氨酸蛋白酶样位点。阿魏酸以浓度依赖的方式影响 26S 真菌蛋白酶体的所有肽酶活性。还测试了阿魏酸对 26S 人蛋白酶体肽酶活性的可能抑制作用。此外，通过荧光胺测定法和凝胶电泳分析，证明了阿魏酸在已知浓度下抑制（诱导白腐真菌漆酶活性）模型全长蛋白底物（β-酪蛋白）的 26S 蛋白酶体催化降解的能力。我们的研究结果为阿魏酸在木质素降解真菌中的作用提供了新的见解。然而，详细的分子机制仍有待未来研究阐明。
