16 research outputs found
iBench: A ground truth approach for advanced validation of mass spectrometry identification method
Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata
Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding
Current Allergy and Asthma Reports / Ligand Binding of PR-10 Proteins with a Particular Focus on the Bet v 1 Allergen Family
Purpose of Review
Pathogenesis-related class 10 (PR-10) proteins are highly conserved plant proteins, which are induced in response to abiotic and biotic stress factors. To date, no unique biological function could be assigned to them. Rather a more general role of PR-10 in plant development and defense mechanisms has been proposed. In addition, some PR-10 proteins act as allergens by triggering allergic symptoms in sensitized individuals. Regardless of the diversity of reported activities, all PR-10 proteins share a common fold characterized by a solvent-accessible hydrophobic cavity, which serves as a binding site for a myriad of small-molecule ligands, mostly phytohormones and flavonoids.
Recent Findings
Most of available data relate to the ligand binding activity of allergenic PR-10, particularly for those belonging to Bet v 1 family of allergens. Bet v 1 and its homologues were shown to bind flavonoids with high affinity, but the specificity appears to differ between homologues from different species. The flavonoid Q3O-(Glc)-Gal was shown to specifically bind to hazelnut Cor a 1 but not to Bet v 1. Similarly, Q3OS bound only to the major isoform Bet v 1.0101 and not to other closely related isoforms. In contrast, Bet v 1 and hazelnut Cor a 1 showed very similar binding behavior towards other flavonoids such as quercetin, genistein, apigenin, daidzein, and resveratrol.
Summary
Recent research findings highlighted the importance of more precise knowledge of ligand binding for understanding the functional diversification of PR-10 proteins.(VLID)515225
ACS Catalysis / Distinct roles of catalytic cysteine and histidine in the protease and ligase mechanisms of human legumain as revealed by DFT-based QM/MM simulations
The cysteine protease enzyme legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [Baker, E. N. J. Mol. Biol. 1980, 141, 441484; Baker, E. N.; J. Mol. Biol. 1977, 111, 207210; Drenth, J.; Biochemistry 1976, 15, 37313738; Menard, R.; J. Cell. Biochem. 1994, 137; Polgar, L. Eur. J. Biochem. 1978, 88, 513521; Storer, A. C.; Methods Enzymol. 1994, 244, 486500. Remarkably, legumain additionally exhibits ligase activity that prevails at pH > 5.5. The atomic reaction mechanisms including their pH dependence are only partly understood. Here we present a density functional theory (DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of the detailed reaction mechanism of both activities for human legumain in solution. Contrasting the situation in other papain-like proteases, our calculations reveal that the active site Cys189 must be present in the protonated state for a productive nucleophilic attack and simultaneous rupture of the scissile peptide bond, consistent with the experimental pH profile of legumain-catalyzed cleavages. The resulting thioester intermediate (INT1) is converted by water attack on the thioester into a second intermediate, a diol (INT2), which is released by proton abstraction by Cys189. Surprisingly, we found that ligation is not the exact reverse of the proteolysis but can proceed via two distinct routes. Whereas the transpeptidation route involves aminolysis of the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted ligation route was found. Given legumains important roles in immunity, cancer, and neurodegenerative diseases, our findings open up possibilities for targeted drug design in these fields.(VLID)223311
Endolysosomal Degradation of Allergenic Ole e 1-Like Proteins: Analysis of Proteolytic Cleavage Sites Revealing T Cell Epitope-Containing Peptides
Knowledge of the susceptibility of proteins to endolysosomal proteases provides valuable information on immunogenicity. Though Ole e 1-like proteins are considered relevant allergens, little is known about their immunogenic properties and T cell epitopes. Thus, six representative molecules, i.e., Ole e 1, Fra e 1, Sal k 5, Che a 1, Phl p 11 and Pla l 1, were investigated. Endolysosomal degradation and peptide generation were simulated using microsomal fractions of JAWS II dendritic cells. Kinetics and peptide patterns were evaluated by gel electrophoresis and mass spectrometry. In silico MHC (major histocompatibility complex) class II binding prediction was performed with ProPred. Cleavage sites were assigned to the primary and secondary structure, and in silico docking experiments between the protease cathepsin S and Ole e 1 were performed. Different kinetics during endolysosomal degradation were observed while similar peptide profiles especially at the C-termini were detected. Typically, the identified peptide clusters comprised the previously-reported T cell epitopes of Ole e 1, consistent with an in silico analysis of the T cell epitopes. The results emphasize the importance of the fold on allergen processing, as also reflected by conserved cleavage sites located within the large flexible loop. In silico docking and mass spectrometry results suggest that one of the first Ole e 1 cleavages might occur at positions 107–108. Our results provided kinetic and structural information on endolysosomal processing of Ole e 1-like proteins
ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease
Bottom-up mass spectrometry-based proteomics utilizes proteolytic enzymes with well characterized specificities to generate peptides amenable for identification by high-throughput tandem mass spectrometry. Trypsin, which cuts specifically after the basic residues lysine and arginine, is the predominant enzyme used for proteome digestion, although proteases with alternative specificities are required to detect sequences that are not accessible after tryptic digest. Here, we show that the human cysteine protease legumain exhibits a strict substrate specificity for cleavage after asparagine and aspartic acid residues during in-solution digestions of proteomes extracted from Escherichia coli, mouse embryonic fibroblast cell cultures, and Arabidopsis tlialiana leaves. Generating peptides highly complementary in sequence, yet similar in their biophysical properties, legumain (as compared to trypsin or GluC) enabled complementary proteome and protein sequence coverage. Importantly, legumain further enabled the identification and enrichment of protein N-termini not accessible in GluC- or trypsin-digested samples. Legumain cannot cleave after glycosylated Asn residues, which enabled the robust identification and orthogonal validation of N-glycosylation sites based on alternating sequential sample treatments with legumain and PNGaseF and vice versa. Taken together, we demonstrate that legumain is a practical, efficient protease for extending the proteome and sequence coverage achieved with trypsin, with unique possibilities for the characterization of post-translational modification sites
Structural and functional studies ofArabidopsis thalianalegumain beta reveal isoform specific mechanisms of activation and substrate recognition
The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest,e.g.for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure ofArabidopsis thalianalegumain isoform beta (AtLEG beta) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform gamma (AtLEG gamma) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEG beta is concentration independent. Additionally, in AtLEG beta the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEG gamma, resulting in significantly different pH stability profiles. The crystal structure of AtLEG beta revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEG beta exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEG beta is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development
International Journal of Molecular Sciences / Two Distinct Conformations in Bet v 2 Determine Its Proteolytic Resistance to Cathepsin S.
Birch pollen allergy affects more than 20% of the European allergic population. On a molecular level, birch pollen allergy can be linked to the two dominant allergens Bet v 1 and Bet v 2. Bet v 2 belongs to the profilin family, which is abundant in the plant kingdom. Importantly, the homologous plant profilins have a conserved cysteine motif with a currently unknown functional relevance. In particular, it is unknown whether the motif is relevant for disulfide formation and to what extent it would affect the profilins structural, functional and immunological properties. Here we present crystal structures of Bet v 2 in the reduced and the oxidized state, i.e., without and with a disulfide bridge. Despite overall structural similarity, the two structures distinctly differ at their termini which are stabilized to each other in the oxidized, i.e., disulfide-linked state. These structural differences translate into differences in their proteolytic resistance. Whereas the oxidized Bet v 2 is rather resistant towards the endolysosomal protease cathepsin S, it is rapidly degraded in the reduced form. By contrast, both Bet v 2 forms exhibit similar immunological properties as evidenced by their binding to IgE antibodies from birch pollen allergic patients and by their ability to trigger histamine release in a humanized rat basophilic leukemia cells (RBL) assay, independent of the presence or absence of the disulfide bridge. Taken together our findings suggest that the oxidized Bet v 2 conformation should be the relevant species, with a much longer retention time to trigger immune responses.(VLID)235100
Distinct Roles of Catalytic Cysteine and Histidine in the Protease and Ligase Mechanisms of Human Legumain As Revealed by DFT-Based QM/MM Simulations
The cysteine protease enzyme legumain hydrolyzes peptide bonds
with high specificity after asparagine and under more acidic conditions
after aspartic acid [Baker, E. N. J. Mol. Biol. 1980, 141, 441−484; Baker, E. N.; J. Mol. Biol. 1977, 111, 207–210; Drenth, J.; Biochemistry 1976, 15, 3731–3738; Menard, R.; J. Cell. Biochem. 1994, 137; Polgar, L. Eur. J. Biochem. 1978, 88, 513–521; Storer, A. C.; Methods Enzymol. 1994, 244, 486–500. Remarkably,
legumain additionally exhibits ligase activity that prevails at pH
> 5.5. The atomic reaction mechanisms including their pH dependence
are only partly understood. Here we present a density functional theory
(DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of
the detailed reaction mechanism of both activities for human legumain
in solution. Contrasting the situation in other papain-like proteases,
our calculations reveal that the active site Cys189 must be present
in the protonated state for a productive nucleophilic attack and simultaneous
rupture of the scissile peptide bond, consistent with the experimental
pH profile of legumain-catalyzed cleavages. The resulting thioester
intermediate (INT1) is converted by water attack on the thioester
into a second intermediate, a diol (INT2), which is released by proton
abstraction by Cys189. Surprisingly, we found that ligation is not
the exact reverse of the proteolysis but can proceed via two distinct
routes. Whereas the transpeptidation route involves aminolysis of
the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted
ligation route was found. Given legumain’s important roles
in immunity, cancer, and neurodegenerative diseases, our findings
open up possibilities for targeted drug design in these fields
Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity
Background: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen–derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. Methods: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs. Results: We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. Conclusion: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes