Deciphering of interactions between platinated DNA and HMGB1 by hydrogen/deuterium exchange mass spectrometry

A high mobility group box 1 (HMGB1) protein has been reported to recognize both 1,2-intrastrand crosslinked DNA by cisplatin (1,2-cis-Pt-DNA) and monofunctional platinated DNA using trans-[PtCl2(NH3)(thiazole)] (1-trans-PtTz-DNA). However, the molecular basis of recognition between the trans-PtTz-DNA and HMGB1 remains unclear. In the present work, we described a hydrogen/deuterium exchange mass spectrometry (HDX-MS) method in combination with docking simulation to decipher the interactions of platinated DNA with domain A of HMGB1. The global deuterium uptake results indicated that 1-trans-PtTz-DNA bound to HMGB1a slightly tighter than the 1,2-cis-Pt-DNA. The local deuterium uptake at the peptide level revealed that the helices I and II, and loop 1 of HMGB1a were involved in the interactions with both platinated DNA adducts. However, docking simulation disclosed different H-bonding networks and distinct DNA-backbone orientations in the two Pt-DNA-HMGB1a complexes. Moreover, the Phe37 residue of HMGB1a was shown to play a key role in the recognition between HMGB1a and the platinated DNAs. In the cis-Pt-DNA-HMGB1a complex, the phenyl ring of Phe37 intercalates into a hydrophobic notch created by the two platinated guanines, while in the trans-PtTz-DNA-HMGB1a complex the phenyl ring appears to intercalate into a hydrophobic crevice formed by the platinated guanine and the opposite adenine in the complementary strand, forming a penta-layer π–π stacking associated with the adjacent thymine and the thiazole ligand. This work demonstrates that HDX-MS associated with docking simulation is a powerful tool to elucidate the interactions between platinated DNAs and proteins

Responses of rumen microorganisms and metabolites to different roughage of domesticated Tibetan sheep

Tibetan sheep can utilize high fiber feeds well. However, the mechanisms of rumen microbiota and metabolites in response to different roughage in a housed environment are still unclear. We fed Tibetan sheep with three different roughage diets: 50% whole corn silage (TS), 50% wheatgrass group (TW), and 25% each of whole corn silage and wheatgrass (TM). Subsequently, meat traits, rumen contents 16S rRNA and metabolomics were studied. The results showed that feeding wheat straw to Tibetan sheep significantly increased the abundance of bacteria such as Ruminococcus and Succiniclasticum in the rumen. These microorganisms significantly increased metabolites such as beta-alanyl-L-lysine, butanoic acid and prostaglandin E2. Eventually, production performance, such as carcass weight and intramuscular fat and meat quality characteristics, such as color and tenderness were improved by altering the rumen’s amino acid, lipid and carbohydrate metabolism. This study demonstrated that including 25% wheatgrass and 25% whole corn silage in the diet improved the performance of Tibetan sheep, revealing the effect of the diet on the performance of Tibetan sheep through rumen microorganisms and metabolites

2D, 3D-QSAR study and docking of vascular endothelial growth factor receptor 3 (VEGFR3) inhibitors for potential treatment of retinoblastoma

Background: Retinoblastoma is currently the most common malignant tumor seen in newborns and children’s eyes worldwide, posing a life-threatening hazard. Chemotherapy is an integral part of retinoblastoma treatment. However, the chemotherapeutic agents used in clinics often lead to drug resistance. Thus there is a need to investigate new chemotherapy-targeted agents. VEGFR3 inhibitors are anti-tumour-growth and could be used to develop novel retinoblastoma-targeted agents.Objective: To predict drug activity, discover influencing factors and design new drugs by building 2D, 3D-QSAR models.Method: First, linear and non-linear QSAR models were built using heuristic methods and gene expression programming (GEP). The comparative molecular similarity indices analysis (COMISA) was then used to construct 3D-QSAR models through the SYBYL software. New drugs were designed by changing drug activity factors in both models, and molecular docking experiments were performed.Result: The best linear model created using HM had an R2, S2, and R2cv of 0.82, 0.02, and 0.77, respectively. For the training and test sets, the best non-linear model created using GEP had correlation coefficients of 0.83 and 0.72 with mean errors of 0.02 and 0.04. The 3D model designed using SYBYL passed external validation due to its high Q2 (0.503), R2 (0.805), and F-value (76.52), as well as its low standard error of SEE value (0.172). This demonstrates the model’s reliability and excellent predictive ability. Based on the molecular descriptors of the 2D model and the contour plots of the 3D model, we designed 100 new compounds using the best active compound 14 as a template. We performed activity prediction and molecular docking experiments on them, in which compound 14.d performed best regarding combined drug activity and docking ability.Conclusion: The non-linear model created using GEP was more stable and had a more substantial predictive power than the linear model built using the heuristic technique (HM). The compound 14.d designed in this experiment has the potential for anti-retinoblastoma treatment, which provides new design ideas and directions for retinoblastoma-targeted drugs

Mutual synergistic protein folding in split intein

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with the concomitant ligation of the flanking polypeptides. Split inteins are expressed in two separated halves, and the recognition and association of two halves are the first crucial step for initiating trans-splicing. In the present study, we carried out the structural and thermodynamic analysis on the interaction of two halves of DnaE split intein from Synechocystis sp. PCC6803. Both isolated halves (IN and IC) are disordered and undergo conformational transition from disorder to order upon association. ITC (isothermal titration calorimetry) reveals that the highly favourable enthalpy change drives the association of the two halves, overcoming the unfavourable entropy change. The high flexibility of two fragments and the marked thermodynamic preference provide a robust association for the formation of the well-folded IN/IC complex, which is the basis for reconstituting the trans-splicing activity of DnaE split intein

Cellular uptake of nickel by NikR is regulated by phase separation

Summary: Bacterial cells were long thought to be “bags of enzymes” with minimal internal structures. In recent years, membrane-less organelles formed by liquid-liquid phase separation (LLPS) of proteins or nucleic acids have been found to be involved in many important biological processes, although most of them were studied on eukaryotic cells. Here, we report that NikR, a bacterial nickel-responsive regulatory protein, exhibits LLPS both in solution and inside cells. Analyses of cellular nickel uptake and cell growth of E. coli confirm that LLPS enhances the regulatory function of NikR, while disruption of LLPS in cells promotes the expression of nickel transporter (nik) genes, which are negatively regulated by NikR. Mechanistic study shows that Ni(II) ions induces the accumulation of nik promoter DNA into the condensates formed by NikR. This result suggests that the formation of membrane-less compartments can be a regulatory mechanism of metal transporter proteins in bacterial cells

Tetrathiomolybdate inhibits the reaction of cisplatin with human copper chaperone Atox1

Cisplatin is a widely used anticancer drug in clinic, and ammonium tetrathiomolybdate ([(NH 4 ) 2 MoS 4 ], TM) is a copper chelator used in clinic for the treatment of Wilson's disease. Recently, TM has been found to enhance the therapeutic effect of cisplatin; however, the origin of this effect is not clear. Here we found that TM can inhibit the reaction of cisplatin with Cu-Atox1 and prevent the protein unfolding and aggregation induced by cisplatin. Although Ag(i) binds to Atox1 in a way similar to Cu(i)-Atox1, TM does not prevent the reaction of Ag-Atox1 with cisplatin. This result indicates that the formation of a Mo-centered trimeric protein cluster in the TM-Cu-Atox1 system plays a role in the inhibitory effect. This work provides new insights into the mechanism by which TM enhances the cytotoxic efficacy of cisplatin and helps to circumvent cisplatin resistance of tumor cells