In order to associate with the cytoplasmic leaflet of the plasma membrane, many cytosolic signalling proteins possess a distinct lipid binding domain as part of their overall fold. Here, a multiscale simulation approach has been used to investigate three membrane-binding proteins involved in cellular processes such as growth and proliferation. The pleckstrin homology (PH) domain from the general receptor for phosphoinositides 1 (GRP1-PH) binds phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) with high affinity and specificity. To investigate how this peripheral protein is able to locate its target lipid in the complex membrane environment, Brownian dynamics (BD) simulations were employed to explore association pathways for GRP1-PH binding to PI(3,4,5)P₃ embedded in membranes with different surface charge densities and distributions. The results indicated that non-PI(3,4,5)P₃ lipids can act as decoys to disrupt PI(3,4,5)P₃ binding, but that at approximately physiological anionic lipid concentrations steering towards PI(3,4,5)P₃ is actually enhanced. Atomistic molecular dynamics (MD) simulations revealed substantial membrane penetration of membrane-bound GRP1-PH, evident when non-equilibrium, steered MD simulations were used to forcibly dissociate the protein from the membrane surface. Atomistic and coarse grained (CG) MD simulations of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumour suppressor, which also binds PI(3,4,5)P₃, detected numerous non-specific protein-lipid contacts and anionic lipid clustering around PTEN that can be modulated by selective in silico mutagenesis. These results suggested a dual recognition model of membrane binding, with non-specific membrane interactions complementing the protein-ligand interaction. Molecular docking and MD simulations were used to characterise the lipid binding properties of kindlin-1 PH. Simulations demonstrated that a dynamic salt bridge was responsible for controlling the accessibility of the binding site. Electrostatics calculations applied to a variety of PH domains suggested that their molecular dipole moments are typically aligned with their ligand binding sites, which has implications for steering and ligand electrostatic funnelling.

Computational methods, including homology modelling, in-silico dockings, and molecular dynamics simulations have been used to study the functional dynamics and interactions of K⁺ channels. Molecular models were built of the inwardly rectifying K⁺ channel Kir2.2, the bacterial homolog K⁺ channel KirBac3.1, and the twin pore (K2P) K⁺ channels TREK-1 and TRESK. To investigate the electrostatic energy profile of K⁺ permeating through these homology models, continuum electrostatic calculations were performed. The primary mechanism of KirBac3.1 gating is believed to involve an opening at the helix bundle crossing (HBC). However, simulations of Kir channels have not yet revealed opening at the HBC. Here, in simulations of the new KirBac3.1-S129R X-ray crystal structure, in which the HBC was trapped open by the S129R mutation in the inner pore-lining helix (TM2), the HBC was found to exhibit considerable mobility. In a simulation of the new KirBac3.1-S129R-S205L double mutant structure, if the S129R and the S205L mutations were converted back to the wild-type serine, the HBC would close faster than in the simulations of the KirBac3.1-S129R single mutant structure. The double mutant structure KirBac3.1-S129R-S205L therefore likely represents a higher-energy state than the single mutant KirBac3.1-S129R structure, and these simulations indicate a staged pathway of gating in KirBac channels. Molecular modelling and MD simulations of the Kir2.2 channel structure demonstrated that the HBC would tend to open if the C-linker between the transmembrane and cytoplasmic domain was modelled helical. The electrostatic energy barrier for K⁺ permeation at the helix bundle crossing was found to be sensitive to subtle structural changes in the C-linker. Charge neutralization or charge reversal of the PIP2-binding residue R186 on the C-linker decreased the electrostatic barrier for K⁺ permeation through the HBC, suggesting an electrostatic contribution to the PIP2-dependent gating mechanism. Multi-scale simulations determined the PIP2 binding site in Kir2.2, in good agreement with crystallographic predictions. A TREK-1 homology model was built, based on the TRAAK structure. Two PIP2 binding sites were found in this TREK-1 model, at the C-terminal end, in line with existing functional data, and between transmembrane helices TM2 and TM3. The TM2-TM3 site is in reasonably good agreement with electron density attributed to an acyl tail in a recently deposited TREK-2 structure.

Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1beta-mediated inflammatory signaling. Resveratrol suppressed IL-1beta-induced activation of NF-kappaB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1beta-induced NF-kappaB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IkappaB kinase, IkappaBalpha phosphorylation, and inhibition of nuclear translocation of NF-kappaB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-kappaB activation. Inhibition of PI3K by wortmannin attenuated IL-1beta-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1beta-induced activation of NF-kappaB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-kappaB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-kappaB.