br Conclusion Four cysteine peptidases

Conclusion Four cysteine peptidases, from B. xylophilus secretome, were further characterised using the previously determined cDNA or genomic DNA sequences and bioinformatics approaches. From these, BxCP3 and BxCP11 were identified as cathepsin L-like proteins and BxCP7 and BxCP8 proteins as cathepsin B proteins. Only BxCP8 had high homology with another B. xylophilus cathepsin B present on GenBank database, all the others differ from the closer proteins deposited in this database. In silico three-dimensional structures of BxCP3 and BxCP11 suggest that these proteins, like other cathepsin L-like proteins, are pro-enzymes that become active when the pro-peptide is cleaved. BxCP7 and BxCP8 predicted structures suggested that these are pro-enzymes activated after removal of the N-terminal pro-peptide and revealed the presence of an occluding loop that occludes the active site cleft, typical of cathepsin B proteases. These four cysteine proteases are functional, highly secreted by B. xylophilus and putatively related to its pathogenicity to pine trees. Our findings contribute to increase the knowledge of these essential protein groups and will be supportive for further research on the development of alternative biologic strategies toward this nematode-specific proteases inhibition and B. xylophilus pathogenicity control.
Acknowledgments This research was supported by Foundation for Science and Technology (FCT), Portugal, within the PT2020 Partnership Agreement and COMPETE 2020 under the project UID/BIA/04004/2013 and Instituto do Ambiente, Tecnologia e Vida, Portugal. Joana M.S. Cardoso is funded by post-doctoral fellowship (BPD9) financed by the Project ReNATURE-CENTRO-01-0145-FEDER-000007-Valorization of the Natural Endogenous Resources of the Centro Region, Portugal. Luís Fonseca is funded by post-doctoral fellowship (SFRH/BPD/101325/2014) financed by MEC national funding (Portugal) and by the European Social Fund through POCH (Programa Operacional Capital Humano).
Introduction Sphingolipids are ubiquitous membrane components of all eukaryotic GPCR Compound Library synthesis belonging to a group of complex lipids. The basic structure of sphingolipids is composed of a sphingoid long chain base (LCB) linked to a fatty acid via an amide bond to form a ceramide [1]. In animals, sphingolipids play a key role in cell death regulation [2]. Indeed, sphingosine (Sph, d18:1), which is the most abundant free LCB in animals, induces apoptosis in many cell types in response to death physiological activators [2,3]. In plants, it becomes now evident that, similarly to animals, sphingolipids are cell death mediators. For instance, spontaneous cell death phenotypes are observed in Arabidopsis mutants such as accelerated cell death 5 (acd5) and accelerated cell death 11 (acd11) impaired in a ceramide kinase or a LCB transport protein [4,5]. In addition, the mycotoxins fumonisin B1 (FB1) and Alternaria alternata lycopersici (AAL) toxin, two natural mimics of LCBs produced by the necrotrophic fungi Fusarium moniliforme and Alternaria alternata respectively, interfere with plant sphingolipid metabolism by inhibiting the activity of ceramide synthase. This enzyme inhibition leads to plant cell death by a subsequent accumulation of dihydrosphingosine (DHS, d18:0) and phytosphingosine (PHS, t18:0), which are the two major free LCBs in Arabidopsis [[6], [7], [8]]. The FB1 resistant11-1 (fbr11-1) Arabidopsis mutant is impaired in the gene encoding the long-chain base 1 (LCB1) subunit of serine palmitoyltransferase (SPT). This enzyme catalyzes the first rate-limiting step of de novo sphingolipid synthesis. The mutant presents a lower accumulation of free LCBs in response to FB1 than the wild type and fails to initiate programmed cell death (PCD) when challenged with FB1 [8]. Interestingly, inoculation of Arabidopsis plants with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato (avrRpm1), that typically induces a localized PCD termed the hypersensitive response (HR), leads to a rapid and sustained increase in PHS, due to de novo synthesis from DHS [9]. A similar response was observed with the necrotrophic fungus Sclerotinia sclerotiorum indicating that fungal pathogens as well as bacteria can trigger PHS increases. These data suggest a crucial involvement of sphingolipid metabolism in plant immunity.