Thermal Stabilization of Dihydrofolate Reductase Using Monte Carlo Unfolding Simulations and Its Functional Consequences

Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r = 0.65–0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing mutations.     

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

A novel TXNIP‐based mechanism for Cx43‐mediated regulation of oxidative drug injury

Gap junctions (GJs) play an important role in the regulation of cell response to many drugs. However, little is known about their mechanisms. Using an in vitro model of cytotoxicity induced by geneticin (G418), we explored the potential signalling mechanisms involved. Incubation of cells with G418 resulted in cell death, as indicated by the change in cell morphology, loss of cell viability and activation of caspase‐3. Before the onset of cell injury, G418 induced reactive oxygen species (ROS) generation, activated oxidative sensitive kinase P38 and caused a shift of connexin 43 (Cx43) from non‐phosphorylated form to hyperphosphorylated form. These changes were largely prevented by antioxidants, suggesting an implication of oxidative stress. Downregulation of Cx43 with inhibitors or siRNA suppressed the expression of thioredoxin‐interacting protein (TXNIP), activated Akt and protected cells against the toxicity of G418. Further analysis revealed that inhibition of TXNIP with siRNA activated Akt and reproduced the protective effect of Cx43‐inhibiting agents, whereas suppression of Akt sensitized cells to the toxicity of G418. Furthermore, interference of TXNIP/Akt also affected puromycin‐ and adriamycin‐induced cell injury. Our study thus characterized TXNIP as a presently unrecognized molecule implicated in the regulatory actions of Cx43 on oxidative drug injury. Targeting Cx43/TXNIP/Akt signalling cascade might be a promising approach to modulate cell response to drugs.     

Sirt3 is essential for apelin‐induced angiogenesis in post‐myocardial infarction of diabetes

Heart failure following myocardial infarction (MI) is the leading cause of death in diabetic patients. Angiogenesis contributes to cardiac repair and functional recovery in post‐MI. Our previous study shows that apelin (APLN) increases Sirtuin 3 (Sirt3) expression and ameliorates diabetic cardiomyopathy. In this study, we further investigated the direct role of Sirt3 in APLN‐induced angiogenesis in post‐MI model of diabetes. Wild‐type (WT) and Sirt3 knockout (Sirt3KO) mice were induced into diabetes by i.p. streptozotocin (STZ). STZ mice were then subjected to MI followed by immediate intramyocardial injection with adenovirus‐apelin (Ad‐APLN). Our studies showed that Sirt3 expression was significantly reduced in the hearts of STZ mice. Ad‐APLN treatment resulted in up‐regulation of Sirt3, angiopoietins/Tie‐2 and VEGF/VEGFR2 expression together with increased myocardial vascular densities in WT‐STZ+MI mice, but these alterations were not observed in Sirt3KO‐STZ+MI mice. In vitro, overexpression of APLN increased Sirt3 expression and angiogenesis in endothelial progenitor cells (EPC) from WT mice, but not in EPC from Sirt3KO mice. APLN gene therapy increases angiogenesis and improves cardiac functional recovery in diabetic hearts via up‐regulation of Sirt3 pathway.     

Evaluation of Methyl Bromide Alternatives Efficacy against Soil-Borne Pathogens,Nematodes and Soil Microbial Community

Methyl bromide (MB) and other alternatives were evaluated for suppression of Fusarium spp., Phytophthora spp., and Meloidogyne spp. and their influence on soil microbial communities. Both Fusarium spp. and Phytophthora spp. were significantly reduced by the MB (30.74 mg kg^-1), methyl iodide (MI: 45.58 mg kg^-1), metham sodium (MS: 53.92 mg kg^-1) treatments. MS exhibited comparable effectiveness to MB in controlling Meloidogyne spp. and total nematodes, followed by MI at the tested rate. By contrast, sulfuryl fluoride (SF: 33.04 mg kg^-1) and chloroform (CF: 23.68 mg kg^-1) showed low efficacy in controlling Fusarium spp., Phytophthora spp., and Meloidogyne spp. MB, MI and MS significantly lowered the abundance of different microbial populations and microbial biomass in soil, whereas SF and CF had limited influence on them compared with the control. Diversity indices in Biolog studies decreased in response to fumigation, but no significant difference was found among treatments in PLFA studies. Principal component and cluster analyses of Biolog and PLFA data sets revealed that MB and MI treatments greatly influenced the soil microbial community functional and structural diversity compared with SF treatment. These results suggest that fumigants with high effectiveness in suppressing soil-borne disease could significantly influence soil microbial community.