Structural and Functional Analysis of Peptides Derived from KEX2-Processed Repeat Proteins in Agaricomycetes Using Reverse Genetics and Peptidomics

Bioactivities of fungal peptides are of interest for basic research and therapeutic drug development. Some of these peptides are derived from KEX2-processed repeat proteins (KEPs), a recently defined class of precursor proteins that contain multiple peptide cores flanked by KEX2 protease cleavage sites. Genome mining has revealed that KEPs are widespread in the fungal kingdom. Their functions are largely unknown. Here, we present the first in-depth structural and functional analysis of KEPs in a basidiomycete. We bioinformatically identified KEP-encoding genes in the genome of the model agaricomycete Coprinopsis cinerea and established a detection protocol for the derived peptides by overexpressing the C. cinerea KEPs in the yeast Pichia pastoris. Using this protocol, which includes peptide extraction and mass spectrometry with data analysis using the search engine Mascot, we confirmed the presence of several KEP-derived peptides in C. cinerea, as well as in the edible mushrooms Lentinula edodes, Pleurotus ostreatus, and Pleurotus eryngii. While CRISPR-mediated knockout of C. cinerea kep genes did not result in any detectable phenotype, knockout of kex genes caused defects in mycelial growth and fruiting body formation. These results suggest that KEP-derived peptides may play a role in the interaction of C. cinerea with the biotic environment and that the KEP-processing KEX proteases target a variety of substrates in agaricomycetes, including some important for mycelial growth and differentiation.

Automated summary

Fungal peptides derived from KEPs have potential bioactivities and are of interest for drug development. This study provides the first detailed analysis of KEPs in a basidiomycete, showing their presence in different species of mushrooms and suggesting their role in fungal interactions with the environment and in mycelial growth and differentiation.