Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth

Reactive oxygen species are well known for their damaging effects due to oxidation of lipids, proteins and DNA that ultimately result in cell death. Accumulating evidence indicates that reactive oxygen species also have important signaling functions in cell proliferation, differentiation, cell motility and apoptosis. Here, we tested the hypothesis whether reactive oxygen species play a physiological role in regulating F-actin structure and dynamics in neuronal growth cones. Lowering cytoplasmic levels of reactive oxygen species with a free radical scavenger, N -tert-butyl-α-phenylnitrone, or by inhibiting specific sources of reactive oxygen species, such as NADPH oxidases or lipoxygenases, reduced the F-actin content in the peripheral domain of growth cones. Fluorescent speckle microscopy revealed that these treatments caused actin assembly inhibition, reduced retrograde actin flow and increased contractility of actin structures in the transition zone referred to as arcs, possibly by activating the Rho pathway. Reduced levels of reactive oxygen species ultimately resulted in disassembly of the actin cytoskeleton. When neurons were cultured overnight in conditions of reduced free radicals, growth cone formation and neurite outgrowth were severely impaired. Therefore, we conclude that physiological levels of reactive oxygen species are critical for maintaining a dynamic F-actin cytoskeleton and controlling neurite outgrowth.     

Degenerative muscle fiber accelerates adipogenesis of intramuscular cells via RhoA signaling pathway

In some pathological conditions such as Duchenne muscular dystrophy, it has been known that a fatty infiltration in skeletal muscle is often observed and that is also one of primary factors to induce marked decline of muscular strength. However, the mechanism of fatty infiltration, cellular origin of accumulated adipocytes and its significance are not fully understood. The fact that persistent degenerative muscle fibers are present on dystrophic muscle leads us to hypothesize that muscle fiber condition affects fatty infiltration in skeletal muscle. We employed a single fiber culture system to determine whether fiber condition affects an appearance of adipocytes on the fibers. Artificially hyper-contracted muscle fibers (HCF), generated from isolated intact fibers (IF) of rat extensor digitrum longus muscle, were maintained as non-adherent cultures for 5–7 days. Interestingly, there appeared to be considerable numbers of mature adipocytes on HCF, whereas no adipocytes were seen on IF, indicating that cells on HCF spontaneously differentiated into mature adipocytes. Activation of RhoA signaling by the addition of thrombin decreased the number of adipocytes on HCF in a dose-dependent manner, whereas the number of MyoD-positive myoblasts increased. In contrast, Y-27632, a specific inhibitor of Rho kinases (ROCK), induced adipogenic differentiation of cells derived from IF. In addition, administration of Y-27632 into mouse regenerating muscle resulted in fat accumulation in the muscle. Taken together, the present studies clearly demonstrated that muscle fiber condition affects fat accumulation in skeletal muscle and that is possibly mediated by the RhoA signaling pathway.     

High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK

The bisubstrate fluorescent probe ARC-583 (Adc-Ahx-(d-Arg)₆-d-Lys(5-TAMRA)-NH₂) and its application for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of protein kinases PKA (cyclic AMP-dependent protein kinase) and ROCK (rho kinase) in fluorescence polarization-based assay are described. High affinity of the probe (K_D = 0.48 nM toward PKA) enables its application for the characterization of inhibitors with nanomolar and micromolar potency and determination of the active concentration of the kinase in individual experiments as well as in the high-throughput screening format. The probe can be used for the assessment of protein-protein interactions (e.g., between regulatory and catalytic subunits of PKA) and as a cyclic AMP biosensor.     

Interaction of an artificial antimicrobial peptide with lipid membranes

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.     

E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides

HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA - the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.     

Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-δ1

Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-δ1, and increases its enzymatic activity. Since PLC-δ1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-δ1. Therefore, we created a GST-PLC-δ1 construct consisting of the first 294 amino acids of PLC-δ1 (GST-PLC-δ1_1-294). In vitro binding experiments confirmed that PLC-δ1_1-294 was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-δ1, demonstrating that PLC-δ1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-δ1 were able to inhibit RalA binding to PLC-δ1_1-294. This study demonstrates that there are two binding sites for RalA in PLC-δ1 and provides further insight into the role of Ral GTPase in the regulation of PLC-δ1 function.     

The Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson’s disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.