PDF(Pubmed)
Abstract：
White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.
摘要：
白腐真菌使用分泌的碳水化合物活性酶（CAZymes）以及活性氧（ROS），像过氧化氢（H2O2），降解木材中的木质纤维素。H2O2在初始衰变阶段充当关键氧化还原酶的共底物。虽然CAZymes对木质纤维素的降解是有据可查的，ROS对分泌蛋白氧化的影响尚不清楚，氧化蛋白的身份是未知的。甲硫氨酸（Met）可以被氧化成Met亚砜（MetO）或Met砜（MetO2），抗氧化剂，或调节作用。其他残留物,像脯氨酸（Pro），可以进行羰基化。用白杨木上生长的白腐朱砂，我们结合H218O2和深度鸟枪蛋白质组学分析了分泌蛋白的Met含量及其对氧化的敏感性。引人注目的是，与细胞内蛋白质（2.1％）相比，它们的整体Met含量显着降低（1.4％），在真菌中但在后生动物或植物中不保守的特征。我们证明了过氧化氢酶，广泛存在于白腐真菌中，保护分泌的蛋白质免受氧化。我们的氧化还原蛋白质组学方法允许在少数分泌蛋白质中鉴定49个可氧化的Met和40个可氧化的Pro残基。主要是CAZymes。有趣的是,他们中的许多人在热点地区有几个氧化残留物。有些人遇到了，包括GH7纤维二糖水解酶，被氧化高达47%，有相当比例的砜(13%)。这些Met在真菌同源物中是保守的，建议重要的功能角色。我们的发现表明，白腐真菌通过最大程度地减少其Met含量，清除ROS和精确定位CAZymes中的氧化还原活性残基来保护其分泌的蛋白质。重要意义真菌降解木质纤维素的研究对于了解木材腐烂的生态和工业影响至关重要。虽然碳水化合物活性酶（CAZymes）在木质纤维素降解中起着公认的作用，过氧化氢（H2O2）对分泌蛋白的影响尚不清楚。本研究旨在评估H2O2对分泌蛋白的影响，专注于蛋氨酸的氧化（Met）。使用白杨木上生长的白腐真菌Pycnoporuscinnabarinus模型，我们表明，真菌通过减少其Met含量并利用分泌的过氧化氢酶清除外源H2O2来保护其分泌的蛋白质免受氧化。研究确定了分泌的CAZymes中关键的可氧化Met。重要的是,一些Met,像GH7纤维二糖水解酶一样，经历了大量的氧化水平，表明在木质纤维素降解中的重要作用。这些发现突出了白腐真菌在木材腐烂过程中保护其分泌蛋白的适应性机制，并强调了这些过程在木质纤维素分解中的重要性。
