No abstract

Nerve injury leads to the release of a number of cytokines which have been shown to play an important role in cellular activation after peripheral nerve injury. The members of the signal transducer and activator of transcription (STAT) gene family are the main mediators in the signal transduction pathway of cytokines. After phosphorylation, STAT proteins are transported into the nucleus and exhibit transcriptional activity. Following axotomy in rat regenerating facial and hypoglossal neurons, a transient increase of mRNA for JAK2, JAK3, STAT1, STAT3 and STAT5 was detected using in situ hybridization and semi-quantitative polymerase chain reaction (PCR). Of the investigated STAT molecules, only STAT3 protein was significantly increased. In addition, activation of STAT3 by phosphorylation on position Tyr705 and enhanced nuclear translocation was found within 3 h in neurons and after 1 day in astrocytes. Unexpectedly, STAT3 tyrosine phosphorylation was obvious for more than 3 months. In contrast, none of these changes was found in response to axotomy of non-regenerating Clarke's nucleus neurons, although all the investigated models express c-Jun and growth-associated protein-43 (GAP-43) in response to axonal injury. Increased expression of Janus kinase (JAK) and STAT molecules after peripheral nerve transection suggests changes in the responsiveness of the neurons to signalling molecules. STAT3 as a transcription factor, which is expressed early and is activated persistently until the time of reinnervation, might be involved in the switch from the physiological gene expression to an 'alternative program' activated only after peripheral nerve injury.

BackgroundThe AutoDock family of software has been widely used in protein-ligand docking research. This study compares AutoDock 4 and AutoDock Vina in the context of virtual screening by using these programs to select compounds active against HIV protease.Methodology/Principal FindingsBoth programs were used to rank the members of two chemical libraries, each containing experimentally verified binders to HIV protease. In the case of the NCI Diversity Set II, both AutoDock 4 and Vina were able to select active compounds significantly better than random (AUC = 0.69 and 0.68, respectively; p<0.001). The binding energy predictions were highly correlated in this case, with r = 0.63 and ι = 0.82. For a set of larger, more flexible compounds from the Directory of Universal Decoys, the binding energy predictions were not correlated, and only Vina was able to rank compounds significantly better than random.Conclusions/SignificanceIn ranking smaller molecules with few rotatable bonds, AutoDock 4 and Vina were equally capable, though both exhibited a size-related bias in scoring. However, as Vina executes more quickly and is able to more accurately rank larger molecules, researchers should look to it first when undertaking a virtual screen.

Eukaryotes and archaea use a protease called the proteasome that has an integral role in maintaining cellular function through the selective degradation of proteins. Proteolysis occurs in a barrel-shaped 20S core particle, which in Thermoplasma acidophilum is built from four stacked homoheptameric rings of subunits, α and β, arranged α(7)β(7)β(7)α(7) (ref. 5). These rings form three interconnected cavities, including a pair of antechambers (formed by α(7)β(7)) through which substrates are passed before degradation and a catalytic chamber (β(7)β(7)) where the peptide-bond hydrolysis reaction occurs. Although it is clear that substrates must be unfolded to enter through narrow, gated passageways (13 Å in diameter) located on the α-rings, the structural and dynamical properties of substrates inside the proteasome antechamber remain unclear. Confinement in the antechamber might be expected to promote folding and thus impede proteolysis. Here we investigate the folding, stability and dynamics of three small protein substrates in the antechamber by methyl transverse-relaxation-optimized NMR spectroscopy. We show that these substrates interact actively with the antechamber walls and have drastically altered kinetic and equilibrium properties that maintain them in unstructured states so as to be accessible for hydrolysis.

The accessory HIV-1 Nef protein is essential for viral replication, high virus load, and progression to AIDS. These functions are mediated by the alteration of signaling and trafficking pathways and require the membrane association of Nef by its N-terminal myristoylation. However, a large portion of Nef is also found in the cytosol, in line with the observation that myristoylation is only a weak lipidation anchor for membrane attachment. We performed biochemical studies to analyze the implications of myristoylation on the conformation of Nef in aqueous solution. To establish an in vivo myristoylation assay, we first optimized the codon usage of Nef for Escherichia coli expression, which resulted in a 15-fold higher protein yield. Myristoylation was achieved by coexpression with the N-myristoyltransferase and confirmed by mass spectrometry. The myristoylated protein was soluble, and proton NMR spectra confirmed proper folding. Size exclusion chromatography revealed that myristoylated Nef appeared of smaller size than the unmodified form but not as small as an N-terminally truncated from of Nef that omits the anchor domain. Western blot stainings and limited proteolysis of both forms showed different recognition profiles and degradation pattern. Analytical ultracentrifugation revealed that myristoylated Nef prevails in a monomeric state while the unmodified form exists in an oligomeric equilibrium of monomer, dimer, and trimer associations. Finally, fluorescence correlation spectroscopy using multiphoton excitation revealed a shorter diffusion time for the lipidated protein compared to the unmodified form. Taken together, our data indicated myristoylation-dependent conformational changes in Nef, suggesting a rather compact and monomeric form for the lipidated protein in solution.