The establishment of plant-fungus mutualistic interaction requires bidirectional molecular crosstalk. Therefore, the analysis of the interacting organisms secretomes would help to understand how such relationships are established. Here, a gel-free shotgun proteomics approach was used to identify the secreted proteins of the plant Arabidopsis thaliana and the mutualistic fungus Trichoderma atroviride during their interaction. A total of 126 proteins of Arabidopsis and 1027 of T. atroviride were identified. Among them, 118 and 780 were differentially modulated, respectively. Bioinformatic analysis unveiled that both organisms' secretomes were enriched with enzymes. In T. atroviride, glycosidases, aspartic endopeptidases, and dehydrogenases increased in response to Arabidopsis. Additionally, amidases, protein-serine/threonine kinases, and hydro-lyases showed decreased levels. Furthermore, peroxidases, cysteine endopeptidases, and enzymes related to the catabolism of secondary metabolites increased in the plant secretome. In contrast, pathogenesis-related proteins and protease inhibitors decreased in response to the fungus. Notably, the glutamate:glyoxylate aminotransferase GGAT1 was secreted by Arabidopsis during its interaction with T. atroviride. Our study showed that GGAT1 is partially required for plant growth stimulation and on the induction of the plant systemic resistance by T. atroviride. Additionally, GGAT1 seems to participate in the negative regulation of the plant systemic resistance against B. cinerea through a mechanism involving H2O2 production.

Metalloproteinases are zinc-dependent endopeptidases that function as primary effectors of tissue remodeling, cell-signaling, and many other roles. Their regulation is ferociously complex, and is exquisitely sensitive to their molecular milieu, making in vivo studies challenging. Phenanthroline (PhN) is an inexpensive, broad-spectrum inhibitor of metalloproteinases that functions by chelating the catalytic zinc ion, however its use in vivo has been limited due to suspected off-target effects. PhN is very similar in structure to phenanthrene (PhE), a well-studied poly aromatic hydrocarbon (PAH) known to cause toxicity in aquatic animals by activating the aryl hydrocarbon receptor (AhR). We show that zebrafish are more sensitive to PhN than PhE, and that PhN causes a superset of the effects caused by PhE. Morpholino knock-down of the AhR rescues the effects of PhN that are shared with PhE, suggesting these are due to PAH toxicity. The effects of PhN that are not shared with PhE (specifically disruption of neural crest development and angiogenesis) involve processes known to depend on metalloproteinase activity. Furthermore these PhN-specific effects are not rescued by AhR knock-down, suggesting that these are bona fide effects of metalloproteinase inhibition, and that PhN can be used as a broad spectrum metalloproteinase inhibitor for studies with zebrafish in vivo.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which play a pivotal role in invasion, migration, and angiogenesis of glioma. Therefore, controlling MMPs is potentially an important therapeutic strategy for glioma. In the present study, we found that exogenous cell-permeable short-chain C2 ceramide inhibits phorbol myristate acetate (PMA)-induced MMP-1, -3, and -9 gene expressions in U87MG and U373MG human astroglioma cells. In addition, C2 ceramide inhibited the protein secretion and enzymatic activities of MMP-1, -3, and -9. The Matrigel invasion assay and wound healing assay showed that C2 ceramide suppresses the in vitro invasion and migration of glioma cells, which appears to be involved in strong inhibition of MMPs by C2 ceramide. Subsequent mechanistic studies revealed that C2 ceramide inhibits PMA-induced mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor (NF)-κB/activator protein (AP)-1 DNA binding activities. Furthermore, C2 ceramide significantly inhibited PMA-induced reactive oxygen species (ROS) production and NADPH oxidase 4 (NOX4) expression, and inhibition of ROS by diphenylene iodonium (DPI, NADPH oxidase inhibitor) mimicked the effects of C2 ceramide on MMP expression and NF-κB/AP-1 via inhibition of p38 MAPK. The results suggest C2 ceramide inhibits MMP expression and glioma invasion, at least partly, by modulating ROS-p38 MAPK signaling axis and other MAPK signaling pathways.

Matrix metalloproteinases (MMPs) are endopeptidases that degrade components of the extracellular matrix, but also modulate inflammation. During bacterial infections, MMPs are important in the recruitment and migration of inflammatory cells. Besides facilitating cell migration by degrading extracellular matrix components, they potentiate the action of several inflammatory molecules, including cytokines, chemokines, and antimicrobial peptides. Staphylococcus aureus secretes an arsenal of immune evasion molecules that interfere with immune cell functioning and hamper proper immune responses. An earlier study identified staphylococcal superantigen-like protein 5 (SSL5) as an MMP9 inhibitor. Since multiple MMPs are involved in neutrophil recruitment, we set up an in-depth search for additional MMP inhibitors by testing a panel of over 70 secreted staphylococcal proteins on the inhibition of the two main neutrophil MMPs: MMP8 (neutrophil collagenase) and MMP9 (neutrophil gelatinase B). We identified SSL1 and SSL5 as potent inhibitors of both neutrophil MMPs and show that they are actually broad range MMP inhibitors. SSL1 and SSL5 prevent MMP-induced cleavage and potentiation of IL-8 and inhibit the migration of neutrophils through collagen. Thus, through MMP-inhibition, SSL1 and SSL5 interfere with neutrophil activation, chemotaxis, and migration, all vital neutrophil functions in bacterial clearance. Studies on MMP-SSL interactions can have therapeutic potential and SSL based derivatives might prove useful in treatment of cancer and destructive inflammatory diseases.

Matrix metalloproteinases (MMPs) are calcium-dependent zinc-containing endopeptidases involved in multiple cellular processes. Among the MMP isoforms, MMP-9 regulates cancer invasion, rheumatoid arthritis, and osteoarthritis by degrading extracellular matrix proteins present in the tumor microenvironment and cartilage and promoting angiogenesis. Here, we identified two potent natural product inhibitors of the non-catalytic hemopexin domain of MMP-9 using a novel quantum mechanical fragment molecular orbital (FMO)-based virtual screening workflow. The workflow integrates qualitative pharmacophore modeling, quantitative binding affinity prediction, and a raw material search of natural product inhibitors with the BMDMS-NP library. In binding affinity prediction, we made a scoring function with the FMO method and applied the function to two protein targets (acetylcholinesterase and fibroblast growth factor 1 receptor) from DUD-E benchmark sets. In the two targets, the FMO method outperformed the Glide docking score and MM/PBSA methods. By applying this workflow to MMP-9, we proposed two potent natural product inhibitors (laetanine 9 and genkwanin 10) that interact with hotspot residues of the hemopexin domain of MMP-9. Laetanine 9 and genkwanin 10 bind to MMP-9 with a dissociation constant (KD) of 21.6 and 0.614 μM, respectively. Overall, we present laetanine 9 and genkwanin 10 for MMP-9 and demonstrate that the novel FMO-based workflow with a quantum mechanical approach is promising to discover potent natural product inhibitors of MMP-9, satisfying the pharmacophore model and good binding affinity.