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Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo., (© 2024 The Author(s). PROTEINS: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Allelopathy, the chemical interaction of plants by their secondary metabolites with surrounding organisms, profoundly influences their functional features. Lichens, symbiotic associations of fungi and algae and/or cyanobacteria, produce diverse secondary metabolites, among other usnic acid, which express to have potent biological activities. Mosses, i.e. Physcomitrium patens, share the habitat with other organisms including lichens, experiencing the allelopathic effects of their metabolites. In this study, we investigated the interference of usnic acid on P. patens as inferred by proteomics, shedding light on the physiological response of this moss. Our results revealed spreading inhibition of of P. patens, under usnic acid treatment (reduction of protonemal patches and enhanced gametophore growth), along with significant alterations in the moss proteome. The results showed that structural proteins and those involved in vital life function are stable or even increased under the treatments. Thus, proteins associated with photosynthesis, stress response, and defense mechanisms were up-regulated, while those involved in energy metabolism and protein biosynthesis were down-regulated. These findings enhance our understanding of moss responses to allelopathic stress and lay the groundwork for future investigations into the functional significance of specific proteins in moss adaptation to environmental challenges., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)