Identification of New Proteins and Potential Mitochondrial F1F0-ATPase Inhibitor Factor 1-Associated Mechanisms in Arabidopsis thaliana Using iTRAQ-Based Quantitative Proteomic Analysis

The mitochondrial synthesis of ATP makes a vital contribution to the growth and development of biological organisms, in which the enzyme mitochondrial F1F0-ATP synthase plays a pivotal role, in that it can either synthesize or hydrolyze cellular ATP. The finding of our previous study revealed that mitochondrial F1F0-ATPase inhibitor factor 1 (IF1) in Arabidopsis thaliana has a conserved function as an endogenous inhibitor affecting cellular energy status and plays an important role in plant growth and reproduction, particularly in fertility. In this study, to gain an insight into IF1-related traits, we performed isobaric tags for relative and absolute quantitation labeling analysis. In total, 67 of 4778 identified proteins were identified as differentially expressed proteins (DEPs; 59 up-regulated and 8 down-regulated) between wild-type and if1 mutant Arabidopsis thaliana seedlings. Gene ontology enrichment analysis revealed that these DEPs were the most significantly enriched in pathways such as “long-day photoperiodism, flowering,” “positive regulation of protein import into chloroplast stroma,” and “pollen sperm cell differentiation,” which are closely associated with reproductive development. Moreover, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that photosynthesis was the pathway most significantly enriched with DEPs. Collectively, our results revealed a global shift in protein abundance patterns corresponding to AtIF1 mutation, entailing changes in the abundance of multiple key proteins and metabolic processes, which will provide a valuable proteomic foundation for future studies.

Montivipera bornmuelleri Venom: Inhibitory Effect on Staphylococcus epidermidis and Escherichia coli F1F0-ATPases and Cytotoxicity on HCT116 Cancer Cell Lines

In this work, we pursued the biological characterization of the venom of Montivipera bornmuelleri, a viper from the Lebanese mountains. In relation to its antibacterial potential, the inhibitory effect of this venom on the F1F0-ATPase enzymes of Gram-positive Staphylocoocus epidermidis and Gram-negative Escherichia coli bacteria was examined. In order to determine the degree of cytotoxicity of the venom on the HCT116 human colon cancer cell lines, the biological MTT proliferation and cell viability test were implemented. After validation of the enzymatic F1F0-ATPase model by the spectrophotometric method, using quercetin as the reference ligand, results revealed that M. bornmuelleri venom is able to inhibit the activity of the enzyme of these two bacteria with a concentration of the order of 100–150 µg/mL. In addition, a venom concentration of 10 µg/mL was sufficient to kill the totality of HCT116 cell lines cultivated in vitro. These data show that M. bornmuelleri venom is a mixture of diverse molecules presenting activities of interest, and is a potential source to explore in order to discover new drug candidates.

Montivipera bornmuelleri Venom: Inhibitory Effect on Staphylococcus epidermidis and Escherichia coli F1F0-ATPases and Cytotoxicity on HCT116 Cancer Cell Lines

In this work, we pursued the biological characterization of the venom of Montivipera born-muelleri, a viper from the Lebanese mountains. In relativity to its antibacterial potential, the in-hibitory effect of this venom on the F1F0-ATPase enzymes of Gram-positive Staphylocoocus epider-midis and Gram-negative Escherichia coli bacteria was examined. In order to determine the de-gree of cytotoxicity of the venom on the HCT116 human colon cancer cell lines, the biological MTT proliferation and cell viability test were implemented. After validation of the enzymatic F1F0-ATPase model by the spectrophotometric method, using quercetin as the reference ligand, re-sults revealed that M. bornmuelleri venom is able to inhibit the activity of the enzyme of these two bacteria with a concentration of the order of 100-150 µg/mL. In addition, a venom concentration of 10 µg/mL was sufficient to kill the totality of HCT116 cell lines cultivated in vitro. These data show that M. bornmuelleri venom is a mixture of diverse molecules presenting activities of interest and is a potential source to explore in order to discover new drug candidates.

Potential Inhibitory Effect of Apis mellifera’s Venom and of Its Two Main Components—Melittin and PLA2—on Escherichia coli F1F0-ATPase

Bacterial resistance has become a worrying problem for human health, especially since certain bacterial strains of Escherichia coli (E. coli) can cause very serious infections. Thus, the search for novel natural inhibitors with new bacterial targets would be crucial to overcome resistance to antibiotics. Here, we evaluate the inhibitory effects of Apis mellifera bee venom (BV-Am) and of its two main components -melittin and phospholipase A2 (PLA2)- on E. coli F1F0-ATPase enzyme, a crucial molecular target for the survival of these bacteria. Thus, we optimized a spectrophotometric method to evaluate the enzymatic activity by quantifying the released phosphate from ATP hydrolysis catalyzed by E. coli F1F0-ATPase. The protocol developed for inhibition assays of this enzyme was validated by two reference inhibitors, thymoquinone (IC50 = 57.5 μM) and quercetin (IC50 = 30 μM). Results showed that BV-Am has a dose-dependent inhibitory effect on E. coli F1F0-ATPase with 50% inhibition at 18.43 ± 0.92 μg/mL. Melittin inhibits this enzyme with IC50 = 9.03 ± 0.27 µM, emphasizing a more inhibitory effect than the two previous reference inhibitors adopted. Likewise, PLA2 inhibits E. coli F1F0-ATPase with a dose-dependent effect (50% inhibition at 2.11 ± 0.11 μg/mL) and its combination with melittin enhanced the inhibition extent of this enzyme. Crude venom and mainly melittin and PLA2, inhibit E. coli F1F0-ATPase and could be considered as important candidates for combating resistant bacteria.