Analyzing the mechanism by which oyster peptides target IL-2 in melanoma cell apoptosis based on RNA-seq and m6A-seq.

Melanoma is a kind of skin cancer with high malignancy and strong proliferation and invasion abilities. Chemotherapy drugs in the clinic have the disadvantages of high price and high toxicity. Peptides are natural active ingredients that have many functions and are safe and effective. Previous studies have shown that oysters are rich in protein and have antitumor effects. In this study, a high-throughput strategy combined with MALDI TOF/TOF-MS and molecular docking was developed to screen peptides with antitumor functions from oyster hydrolysate. Three dominant peptides were predicted to have similar functions to IL-2 via molecular docking. Then, the activity of the peptides was confirmed in B16 cells, and we found that the three peptides increased the apoptosis of B16 cells. Furthermore, via RNA-seq and m6A-seq of B16 cells treated with the peptides, we found that ILADSAPR downregulates the expression of Pcna, Tlr4, and Ncbp2 and upregulates the expression of Bax, Bad, Pak4, Rasa2, Cct6, and Gbp2. ILADSAPR inhibited B16 cell proliferation and promoted cell apoptosis by regulating the expression of these genes. In addition, the result of metabolic pathway analysis also proved this point. This study provides a preliminary reference for antitumor research on oyster peptides.

In vitro synergistic action of TAT-RasGAP317-326 peptide with antibiotics against Gram-negative pathogens.

OBJECTIVES: Multidrug-resistant (MDR) bacteria are a continuously increasing threat for medicine, causing infections recalcitrant to antibiotics. Antimicrobial peptides (AMPs) were identified as alternatives to antibiotics, being naturally occurring short peptides and part of the innate immune system of a vast majority of organisms. However, the clinical application of AMPs is limited by suboptimal pharmacokinetic properties and relatively high toxicity. Combinatorial treatments using AMPs and classical antibiotics may decrease the concentrations of AMPs required for bacterial eradication, thus lowering the side effects of these peptides. METHODS: Here, we investigate the in vitro efficiency of combinations of the recently described antimicrobial peptide TAT-RasGAP317-326 with a panel of commonly used antimicrobial agents against three Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii, using checkerboard and time-kill assays. RESULTS: We identified synergistic combinations towards all three bacteria and demonstrated that these combinations had an increased bactericidal effect compared to individual drugs. Moreover, combinations were also effective against clinical isolates of A. baumannii. Finally, combination of TAT-RasGAP317-326 and meropenem had a promising antibiofilm effect towards A. baumannii. CONCLUSIONS: Taken together, our results indicate that combinations of TAT-RasGAP317-326 with commonly used antimicrobial agents may lead to the development of new treatment protocols against infections caused by MDR bacteria.

Ras signals principally via Erk in G1 but cooperates with PI3K/Akt for Cyclin D induction and S-phase entry.

Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.

TAT-RasGAP317-326 requires p53 and PUMA to sensitize tumor cells to genotoxins.

Although chemotherapy has revolutionized cancer treatment, the associated side effects induced by lack of specificity to tumor cells remain a challenging problem. We have previously shown that TAT-RasGAP(317-326),a cell-permeable peptide derived from RasGAP, specifically sensitizes cancer cells to the action of genotoxins. The underlying mechanisms of this sensitization were not defined however. Here, we report that TAT-RasGAP(317-326) requires p53, but not the Ras effectors Akt and extracellular signal-regulated kinase, to mediate its tumor sensitization abilities. The TAT-RasGAP(317-326) peptide, although not modulating the transcriptional activity of p53 or its phosphorylation and acetylation status, nevertheless requires a functional p53 cellular status to increase the sensitivity of tumor cells to genotoxins. Genes regulated by p53 encode proapoptotic proteins, such as PUMA, and cell cycle control proteins, such as p21. The ability of TAT-RasGAP(317-326) to sensitize cancer cells was found to require PUMA but not p21. TAT-RasGAP(317-326) did not affect PUMA levels, however, but increased genotoxin-induced mitochondrial depolarization and caspase-3 activation. These results indicate that TAT-RasGAP(317-326) sensitizes tumor cells by activating signals that intersect with the p53 pathway downstream of, or at the level of, proapoptotic p53 target gene products to increase the activation of the mitochondrial death pathway.

Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking.

Rho GTPases, which control polarized cell growth through cytoskeletal reorganization, have recently been implicated in the control of endo- and exocytosis. We now report that both Rho1p and Cdc42p have a direct role in mediating the docking stage of homotypic vacuole fusion. Vacuoles prepared from strains with temperature-sensitive alleles of either Rho1p or Cdc42p are thermolabile for fusion. RhoGDI (Rdi1p), which extracts Rho1p and Cdc42p from the vacuole membrane, blocks vacuole fusion. The Rho GTPases can not fulfill their function as long as priming and Ypt7p-dependent tethering are inhibited. However, reactions that are reversibly blocked after docking by the calcium chelator BAPTA have passed the point of sensitivity to Rdi1p. Extraction and removal of Ypt7p, Rho1p and Cdc42p from docked vacuoles (by Gdi1p, Gyp7p and Rdi1p) does not impede subsequent membrane fusion, which is still sensitive to GTPgammaS. Thus, multiple GTPases act in a defined sequence to regulate the docking steps of vacuole fusion.