Impact of Mutations on K-Ras-p120GAP Interaction

The K-Ras protein plays a key role in the signal transduction cascade. Certain mutations in K-Ras lead to a permanent “on” state which results in tumorigenesis due to failed interaction with the GTPase activating protein (GAP). In this study, we examined the mutations E31N, D33N and D38N of K-Ras coupled and decoupled to wildtype GAP-334 and mutation K935N of GAP-334 coupled and decoupled to wildtype K-Ras, to illustrate the potential mechanism by which these mutants affect the interaction between the two proteins. We identify Tyr32 in the Ras Switch I region as a critical residue that acts as a gate to the GTP binding site and which needs to be “open” during Ras coupling with GAP to allow for insertion of GAP residue Arg789. This residue plays a vital role in stabilizing the transition state during GTP hydrolysis. The different mutations studied herein caused a reduced binding affinity, and the fluctuation of the Tyr32 side chain might hinder the insertion of Arg789. This may in turn be the cause of decreased GTP hydrolysis, and permanent “on” state of K-Ras, observed for these mutants.

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Treatment time influences the effects of a low-frequency pulsed electric field on synthesis of tyrosine hydroxylase and dopamine in PC12 cells

BACKGROUND： Electromagnetic radiation can influence dopamine （DA） synthesis in brain tissues or ceils, but electromagnetic frequencies, intensities, and radiation time can produce different effects. In addition, the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial. OBJECTIVE： To determine tyrosine hydroxylase （TH） expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field （LF-PEF） stimulation, and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors. DESIGN, TIME AND SETTING： A parallel, controlled, cell experiment was performed at the Laboratory of Cell Biology, School of Life Science, East China Normal University, between January and October 2006. MATERIALS： PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Nerve growth factor was purchased from PeproTech, USA. The protein kinase A inhibitor, H-89, and mitogen-activated protein kinase kinase inhibitor, U0126, were purchased from Sigma, USA. METHODS： （1） Following routine culture in Dulbecco＇s modified eagle medium, primary PC12 cells were stimulated under LF-PEF （pulse frequency 50.Hz, pulse width 20 μs, peak field strength 1 V/m） for 5, 10, 15, 20, and 30 minutes. （2） Inhibitors （H-89 or U0126, 1 μmol/L） were added 30 minutes before LF-PEF stimulation for 10 minutes. MAIN OUTCOME MEASURES： （1） TH expression was determined by Western blot in PC12 cells at 0.5, 1,2, 3, and 4 days after LF-PEF stimulation. Similarly, DA was measured by high-performance liquid chromatography in media at 2, 3, 4, or 5 days after LF-PEE （2） TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEE RESULTS： （1） Short-term LF-PEF stimulation （5 and 10 minutes） increased TH expression and media DA levels after short-term culture （2 days） （P 〈 0.01）, but both parameters decreased with longer culture （3 4 days） （P 〈 0.01）. Long-term LF-PEF stimulation （15, 20, or 30 minutes） decreased TH and DA synthesis, followed by a rapid increase （P 〈 0.01）. （2） H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes, while the inhibition rate of U0126 was 53.2%. CONCLUSION： Short-term LF-PEF first promotes then inhibits, while long-term LF-PEF first inhibits then promotes, TH and DA synthesis. LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.

Engineering a cell-penetrating hyperstable antibody scFv(Ras)-An extraordinary approach to cancer therapeutics

In the modern pharmaceutical industry,monoclonal antibodies are often used as therapeutic agents.However,they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and maintain normal function in the reducing environment.Additionally,it can lead to cytotoxicity since it attacks cancerous cells by mimicking the human immune system.As an alternative,this study modifies the hyperstable single-chain fragment variable(scFv)antibody to eliminate cancer using its linear shape.The scFv(F8)antibody model was modified to recognize human Ras protein by altering residues in the antigen-binding site.Furthermore,a cell-penetrating peptide(CPP)was attached to the scFv(Ras)antibody model to allow entrance to the cell,creating CPP-scFv(Ras).Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),western blotting,and the binding assay were performed to prove its effectiveness.As a result,CPP-scFv(Ras)was successfully engineered and bound to the antigen,HRas(G12V).

Conservation and divergence of Grb7 family of Ras-binding domains

Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms.Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway.The Ras-effector proteins contain a Ras-associating(RA)domain through which these associate with Ras in a GTP-dependent manner.The RA domain is highly conserved among the members of the growth factor receptor-bound(Grb)7 family of proteins which includes Grb7,Grb10 and Grb14.Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel(CTRCNGA1)and inhibits the channel activity.Molecular modeling of the CTR-CNGA1 displays 50%---70%tertiary structural similarity towards Ras proteins.We named this region as Ras-like domain(RLD).The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP.It is interesting to note that Grb14 binds to GTPase-mutant Rab5,a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5.These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1.Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins,both of which might have undergone several sequence variations for functional adaptation during evolution.