Intermolecular interactions of the malate synthase of Paracoccidioides spp
Copyright policy )Intermolecular interactions of the malate synthase of Paracoccidioides spp
Abstract Background The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell.
- UNIVERSIDADE DE SAO PAULO Brazil
- Universidade Federal de Goiás Brazil
- Universidade Estadual Paulista Brazil
- Sao Paulo State University Brazil
- Universidade Estadual Paulista - UNESP Brazil
Centrifugation, protein binding, yeast, confocal microscopy, Mass Spectrometry, malate synthase, computer program, protein purification, Protein Interaction Mapping, lung alveolus cell, mass spectrometry, antibody production, Microscopy, Confocal, Paracoccidioides spp, heterologous expression, protein function, protein protein interaction, two hybrid system, Far Western blotting, signal transduction, Research Article, Microbiology (medical), 570, amino acid analysis, Protein-protein interactions, ligand binding, 612, macrophage, Saccharomyces cerevisiae, Molecular Dynamics Simulation, Blotting, Far-Western, in vivo study, Two-Hybrid System Techniques, protein secretion, controlled study, protein interaction, immunofluorescence, antibody specificity, genetic transcription, Fungi, Malate Synthase, Paracoccidioides, molecular docking, sequence homology, Malate synthase, molecular dynamics, amino acid sequence, Microscopy, Fluorescence, protein analysis, molecular interaction, mycelium, computer model, Murinae, molecular model, protein determination
Centrifugation, protein binding, yeast, confocal microscopy, Mass Spectrometry, malate synthase, computer program, protein purification, Protein Interaction Mapping, lung alveolus cell, mass spectrometry, antibody production, Microscopy, Confocal, Paracoccidioides spp, heterologous expression, protein function, protein protein interaction, two hybrid system, Far Western blotting, signal transduction, Research Article, Microbiology (medical), 570, amino acid analysis, Protein-protein interactions, ligand binding, 612, macrophage, Saccharomyces cerevisiae, Molecular Dynamics Simulation, Blotting, Far-Western, in vivo study, Two-Hybrid System Techniques, protein secretion, controlled study, protein interaction, immunofluorescence, antibody specificity, genetic transcription, Fungi, Malate Synthase, Paracoccidioides, molecular docking, sequence homology, Malate synthase, molecular dynamics, amino acid sequence, Microscopy, Fluorescence, protein analysis, molecular interaction, mycelium, computer model, Murinae, molecular model, protein determination
citations This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).12 popularity This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.Average influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).Average impulse This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.Top 10%
Citations provided by BIP!Popularity provided by BIP!