Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly 82 of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ϳ45-fold higher K m(ATP) . We have also shown that Lys 103 is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX 17-21 K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.
Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparasitoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidase. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C. rubecula PDV-protein (Crp32).
Proteins with antifungal activity towards Rhynchosporium secalis conidia were isolated from the intercellular washing fluid (IWF) of barley leaves. The active components were purified by high-performance liquid chromatography under conditions that maintained biological activity. Five major barley IWF proteins deleterious to the cell wall of viable R. secalis conidia were isolated and identified by a combination of N-terminal amino acid sequencing, peptide mapping, and determination of mass and isoelectric point. They were a 32-kDa beta-1,3-glucanase (Pr32), a 25-kDa chitinase (Pr25), and three 22-kDa thaumatin-like (TL) proteins (Pr22-1, Pr22-2, and Pr22-3). Pr22-1 and Pr22-2 were similar to the protein R class of TL proteins, whereas Pr22-3 was more similar to the S class. Pr22-3 was shown to digest laminarin, indicating that this TL protein has glucanase activity. In addition, Pr22-3 was more active in the spore bioassay than Pr22-2. Various combinations of the five proteins had a greater effect on R. secalis spores than did the individual proteins. The extraction of proteins with antifungal activity from the IWF of barley leaves indicates their possible role in defense against leaf pathogens. A similar bioassay may be developed for other systems to identify particular isoforms of pathogenicity-related proteins that might have a role in plant disease resistance.
Introduction Plants are in constant contact with microorganisms, many of which are potentially pathogenic, but the infection is a relatively rare event that usually involves speciesspecific pathogens. Plant disease resistance is elicited by specific recognition of pathogen-derived molecules. The recognition then leads to an array of resistance responses including the hypersensitive reaction (HR) at the site of pathogen entry and induction of the systemic acquired resistance (SAR) immune response. SAR provides protection not only at the site of infection but also in distant uninfected plant parts against a wide range of pathogens and is correlated with the expression of pathogenesisrelated (PR) proteins, many with antimicrobial activities (Zhang et al., 2010). Pathogenesis-related proteins were first described based on their accumulation in tobacco leaves infected with Tobacco Mosaic Virus (TMV). A defensive role for these proteins in plant systems is suggested by their induction during the pathogen attack, and by their antifungal activity in vitro and in vivo. Genes encoding these proteins are now considered as an arsenal for the molecular breeding of pathogen-resistant plants. (Boccardo et al., 2019). PR proteins are classified into 17 families (PR-1 to PR-17) (Van loon et al., 2006). Amongst which, PR-5 (or thaumatin-like) proteins are a group of highly soluble strongly antifungal proteins that originally detected as the slowest moving band among several low-molecular-weight proteins resolved by polyacrylamide gel electrophoresis analyses of acidic extracts of tobacco leaves challenged with TMV. They were related to the intensely sweettasting protein, thaumatin, from the West African shrub, Thaumatococcus daniellii based on sequence analysis (Van der Wel and Loeve, 1972). Other members of the thaumatin-like proteins (TLPs) are known as permatins (Skadsen et al., 2000) and osmotins (Anzlovar and Dermastia, 2003). Mature TLPs fall into 2 size ranges, 1 group with a size range of 22 to 26 kDa (201 to 229 amino acids), and the other group with sizes around 16 kDa (148 to 151
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