The binding of talin-F0 domain to ras-related protein 1b (Rap1b) plays an important role in the formation of thrombosis. However, since talin is a force-sensitive protein, it remains unclear whether and how force regulates the talin-F0/Rap1b interaction. To explore the effect of force on the binding affinity and the dynamics mechanisms of talin-F0/Rap1b, molecular dynamics simulation was used to observe and compare the changes in functional and conformational information of the complex under different forces. Our results showed that when the complex was subjected to tensile forces, there were at least two dissociation pathways with significantly different mechanical strengths. The key event determining the mechanical strength difference between the two pathways was whether the β4 sheet of the F0 domain was pulled away from the original β1-β4 parallel structure. As the force increased, the talin-F0/Rap1b interaction first strengthened and then weakened, exhibiting the signature of a transition from catch bonds to slip bonds. The mechanical load of 20 pN increased the interaction index of two residue pairs, ASP54-ARG41 and GLN18-THR65, which resulted in a significant increase in the affinity of the complex. This study predicts the regulatory mechanism of the talin-F0/Rap1b interaction by forces in the intracellular environment and provides novel ideas for the treatment of related diseases and drug development.
Objective Series of compl icated molecule signal pathway are involved in the bone regeneration. To explore the possibil ity of nuclear factore kappa B (NF-κB) which is taken as the “key activation” during the fracture healing and provide the laboratory evidence for the gene therapy of nonunion or delayed union of fractures. Methods Thirtythree adult male Wistar rats (weighing 180-220 g) were selected and divided randomly into 4 groups: group A (the control group, n=3), the rigth lower segments of radius were injected with normal sal ine 0.3 mL for 7 days, once per day; group B (Bay 11-7082 injection group, n=6), the middle and distal radius were injected with normal sal ine containing 50 μmol/L NF- κB inhibitor Bay 11-7082 0.3 mL for 7 days, once per day; group C (fracture group, n=12), the right middle and distal radius were cut by a sharp scissors to form per fracture model; and group D (Bay 11-7082 treatment group, n=12), based on group C, 0.3 mL of 50 μmol/L Bay 11-7082 were injected into the fracture site for 7 days, once per day. The callus tissues were harvested at 3, 7, 14, and 28 days after fracture for Western blot analysis, alkal ine phosphatase (ALP) activity assessment, prostaglandins E2 (PGE2) production assay, and histological observation. Results The rats of all groups were survivaltill the experiment completion. At 3 and 7 days after injection, there was no significant difference in the ALP activity and PGE2 production between group B and group A (P gt; 0.05); but group C was significantly higher than group A (P lt; 0.01) and group D was significantly lower than group A (P lt; 0.01). The expressions of NF-κB p65, bone morphogenetic protein 7 (BMP-7), and inhibitor of DNA binding 2 (Id2) were observed at fracture sites of 4 groups. There was no significant difference in the expressions of NF-κB p65, BMP-7, and Id2 between group B and group A (P gt; 0.05); the expressions of NF-κB p65 and BMP-7 were significantly higher and the expression of Id2 was significantly lower in group C than group A (P lt; 0.01); and the expressions of NF-κB p65 and BMP-7 were significantly lower and the expression of Id2 was significantly higher in group D than group A (P lt; 0.01). The histological observation showed that a lot of osseous callus formed in group C at 14 and 28 days, but osseous callus just began to form in group D at 28 days. Conclusion NF-κB p65 can facil itate early fracture heal ing of rat radius by elevating the PGE2 production and regulating BMP-7 and Id2 expression.