Effect of Inhibition of Proteasome-Mediated Proteolysis on Ligninolytic Activities of White-Rot Fungi

Abstract

It has recently been established that most short- and long-lived cellular proteins (80-90%) are degrad- ed by a highly selective non-lysosomal pathway that requires ATP and a large (~2.5 MDa) multisubunit, multicatalytic proteinase complex known as the 26S proteasome. It degrades many important proteins involved in signaling pathway, in cell cycle control, and in general metabolism, including transcription fac- tors and key metabolic enzymes. Here, we demonstrated all distinct proteasome activities: chymotrypsin- like, trypsin-like, and caspase-like (peptidylglutamyl-peptide hydrolyzing) in mycelial extracts of the white-rot fungi Trametes versicolor and Phlebia radiata by monitoring cleavage of three different fluoro- genic peptide substrates: Suc-LLVY-MCA, Z-GGR-MCA, Z-LLE-βNA, respectively. We also found that this cleavage was ATP-dependent. Reagents that inhibit proteasome-mediated protein degradation in intact cells have recently become available, including substrate-related peptide aldehydes. These inhibitors are useful tools to demonstrate that a process exhibits proteasome-dependent biochemical regulation. In the present study, we report that in vivo Cbz-LLLal treatment strongly inhibited all tested proteasome activi- ties and affected ligninolytic activities in nutrient deprived cultures of both fungi.