Current Protein and Peptide Science - Volume 26, Issue 5, 2025

Crotalus Neutralizing Factor (CNF) is a γ-type Phospholipase A2 (PLA2) inhibitor present in the blood of Crotalus durissus terrificus snake. Particularly, CNF inhibits the toxic action of Crotoxin (CTX), which is a major neurotoxin found in C. d. terrificus venom. CTX induces also myotoxic action and demonstrates high selectivity for skeletal muscle fibers. Consequently, CTX can diffuse beyond the site of infection, which can potentially evoke rhabdomyolysis. The present study has evaluated the effects of CTX on myoblasts and myotubes of muscle cells C2C12 in vitro and the effect of CNF on CTX-induced damage.

Cytotoxicity assays were performed by measuring the mitochondrial enzyme dehydrogenase levels. Furthermore, creatine kinase and lactate dehydrogenase levels were used as indicators of muscle damage.

Crotoxin has been found to have cytotoxic effects on C2C12 myoblast cells, while CNF has not shown toxic effects on these cells. Furthermore, the findings have shown CNF (50 µg/mL) to abolish CTX toxicity in myoblasts. The myotubes, differentiated cells, showed no change in mitochondrial respiration when exposed to CNF or CTX, showing greater resistance to the toxic actions of crotoxin.

The data have confirmed the potential of CNF as an anti-myotoxic agent to prevent CTX-damaged myoblasts and increase resistance to the toxic effects of crotoxin on differentiated cells.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with a high recurrence rate. A new therapeutic intervention is urgently needed to combat this lethal subtype. The identification of biomarkers is also crucial for improving outcomes in TNBC.

The cell cytotoxicity of ML364 (2-(4-Methylphenylsulfonamido)-N-(4-phenylthiazol-2-yl)-4-(trifluoromethyl)benzamide) was measured at different concentrations in TNBC-treated and untreated cells. The 2DE and LC-MS/MS analysis were used for protein identification of differentially expressed proteins. Furthermore, the quantitation of gene expression was demonstrated using RT-qPCR. TIMER, HPA, and UALCAN databases were utilized for further analysis.

Differentially expressed proteins and genes after ML364 treatment in TNBC were found to be linked with the USP2 (ubiquitin specific peptidase 2)-mediated pathway. Our results demonstrate that differentially identified proteins, including PPA1, TRIM68, and FBXO46, could be a potential prognostic biomarker for TNBC. Further analysis through the UALCAN and HPA databases shows the high expression of these proteins in primary breast tumors, which is in contrast to normal. The induction of ML364 significantly reduced the expression of PPA1, TRIM68, and FBXO46 proteins and induced cell cytotoxicity in TNBC cells.

This study provides an understanding of the USP2-mediated signaling pathway in TNBC, emphasizing the role of USP2 and its substrates with apoptotic genes. Our results offer insight into the USP2-mediated cellular mechanism after ML364 treatment in TNBC that could be a potential therapeutic candidate.

Actinomycetes, Gram-positive bacteria, are recognized for producing bioactive metabolites. Lonar Soda Lake, an alkaline ecosystem, hosts diverse actinomycetes with possible anticancer activities.

To assess the cytotoxic potential of fermentation metabolites from actinomycetes isolated from Lonar Soda Lake against HeLa cancer cells employing in-vitro and in-silico methods.

Evaluate the cytotoxicity of fermentation metabolites from Lonar Lake actinomycetes on HeLa cells. Execute molecular docking to forecast metabolite connections with cancer-related proteins.

The actinomycetes were isolated from the sediment sample of Lonar Lake using a selective medium and recognized by gene sequencing. Cytotoxicity on HeLa cells was assessed using the MTT assay, in consort with oxidative stress and apoptotic markers (GSH, MDA, TNF-α, and caspase 3). Molecular docking and molecular dynamics studies evaluated metabolite binding to cancer-related proteins (Bcl-2, TNF-α, caspase 3).

Fermentation metabolites of three Lonar Lake Sediment isolates (LLSD), LLSD-5, LLSD-7, and LLSD-9 showing promising cytotoxic activity against HeLa cell lines by MTT assay, also significantly modulate the oxidative stress parameters (GSH, MDA), and cell apoptotic marker (TNF-α, caspase 3). IC50 values were 34.17 µM (LLSD-5), 53.85 µM (LLSD-7), and 69.54 µM (LLSD-9). Furthermore, molecular docking displayed robust binding affinities to cancer-related proteins, uncovering the possible mechanism of action.

The fermentation metabolites actinomycete isolates from Lonar Lake exhibit significant cytotoxic activity against HeLa cancer cell lines. Both in-vitro and in-silico analyses support the potential of these metabolites as anticancer agents.

The rising prevalence of Mycobacterium tuberculosis (M.tb) strains resistant to aminoglycosides (amikacin and kanamycin) challenges effective TB control and treatment. Understanding the mechanisms behind this resistance is crucial since aminoglycosides are a mainstay of TB therapy.

The study aimed to analyze the cell wall proteins overexpressed in aminoglycosides-resistant isolates of Mycobacterium tuberculosis using proteomics approaches.

We used two-dimensional electrophoresis and mass spectrometry to compare the cell wall proteomes of aminoglycosides-resistant and susceptible clinical isolates. The overexpressed protein spots were excised and identified using liquid chromatography-mass spectrometry (LC/MS). The identified proteins were subsequently analyzed for molecular docking, pupylation site identification, and STRING analysis.

We found a total of nine significantly upregulated proteins in aminoglycosides-resistant isolates. Three of these proteins were the same (isoform), resulting in the identification of seven unique proteins. Specifically, Rv3841 and Rv1308 belonged to intermediary metabolism and respiration; Rv2115c to the cell wall and cell processes; Rv2501c, Rv2247 and Rv0295c to lipid metabolism; and Rv2416c to virulence, detoxification/adaptation. Notably, variations in these proteins support cell wall integrity, aiding mycobacteria's establishment and proliferation. Molecular docking study revealed that both drugs bind strongly to the proteins' active site regions. Additionally, the GPS-PUP algorithm successfully identified possible pupylation sites within these proteins, except Rv0295c. Based on interactome analysis using the STRING 12.0 database, we have identified potential interactive partners suggesting their role in aminoglycosides resistance.

Overexpressed proteins not only act to counteract or regulate drug effects but also have a role in protein dynamics that allow for resistance. Some of these identified proteins may serve as innovative drug targets and biomarkers for the early detection of drug-specific resistance in M.tb. Further research is needed to elucidate the mechanisms by which these potential protein targets contribute to resistance in AK and KM M.tb isolates.