Differential roles of ERK1/2 and JNK in retinal development and degeneration

Programmed cell death is well established as a key factor in the development of the vertebrate nervous system of which the retina is a unique sensory component. However, it is of utmost importance for the survival of post-mitotic tissues such as the retina that the execution of the cell death program is kept under stringent control once development is complete. This is exemplified by the many retinal dystrophies where aberrant apoptosis results in loss of distinct cell layers in the mature retina and often culminates in blindness. In this study, we report that the extracellular signal-regulated kinase (ERK1/2) pathway plays a key role in the regulation of apoptosis during retinal development. We show that as the retina matures, the emphasis shifts towards survival and ERK1/2 is activated resulting in phosphorylation of the potent BH3-only protein Bim(EL) and a dramatic decline in Bim(EL) expression via proteasomal degradation. We find that activation of ERK1/2 also occurs in response to injury in retinal explants. However, this is a transient response and appears to be overcome by Jun N-terminal kinase activation resulting in induction of Bim(EL) mRNA and photoreceptor apoptosis. Our findings provide new insights into the intracellular pathways responsible for regulating apoptosis during neuronal development and degeneration.     

Molecular mechanisms of viral and host cell substrate recognition by hepatitis C virus NS3/4A protease

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.     

Plasma antioxidant enzymes and clastogenic factors as possible biomarkers of colorectal cancer risk

Oxidative damage plays an important role in the pathogenesis of colorectal (CR) cancer. This study investigated the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione-S-transferase (GST) in plasma of 82 participants of a screening program for CR cancer prevention (30 females and 52 males; age 50-70 years). All subjects resulted positive to fecal occult blood test and were subsequently classified, according to the colonoscopy and histological findings, in patients with CR cancer, patients with colorectal polyps or controls. Furthermore, the activity of clastogenic factors (CFs) in plasma from study population was measured as the ability of inducing micronuclei (MN) in vitro in peripheral of a healthy donor. CAT and GR activities were significantly lower in CR cancer patients compared to controls (P<0.05) and polyps groups (P<0.05). SOD activity was significantly higher in patients with CR cancer than in polyp (P<0.05) and control (P<0.05) groups. GST activity was not significantly different in plasma of the three groups. An increase of CFs induction was observed in plasma of CR cancer patients (MN: 8.89±3.42) with respect to control (MN: 6.37±0.96 P<0.05). These results can contribute to define plasma biomarkers associated to oxidative stress damage that could predictive of CR cancer risk.     

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.     

Human mutation within Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) causes basal insulin hypersecretion

PAS kinase (PASK) is a glucose-regulated protein kinase involved in the control of pancreatic islet hormone release and insulin sensitivity. We aimed here to identify mutations in the PASK gene that may be associated with young-onset diabetes in humans. We screened 18 diabetic probands with unelucidated maturity-onset diabetes of the young (MODY). We identified two rare nonsynonymous mutations in the PASK gene (p.L1051V and p.G1117E), each of which was found in a single MODY family. Wild type or mutant PASKs were expressed in HEK 293 cells. Kinase activity of the affinity-purified proteins was assayed as autophosphorylation at amino acid Thr307 or against an Ugp1p-derived peptide. Whereas the PASK p.G1117E mutant displayed a ～25% increase with respect to wild type PASK in the extent of autophosphorylation, and a ～2-fold increase in kinase activity toward exogenous substrates, the activity of the p.L1051V mutant was unchanged. Amino acid Gly1117 is located in an α helical region opposing the active site of PASK and may elicit either: (a) a conformational change that increases catalytic efficiency or (b) a diminished inhibitory interaction with the PAS domain. Mouse islets were therefore infected with adenoviruses expressing wild type or mutant PASK and the regulation of insulin secretion was examined. PASK p.G1117E-infected islets displayed a 4-fold decrease in glucose-stimulated (16.7 versus 3 mM) insulin secretion, chiefly reflecting a 4.5-fold increase in insulin release at low glucose. In summary, we have characterized a rare mutation (p.G1117E) in the PASK gene from a young-onset diabetes family, which modulates glucose-stimulated insulin secretion.     

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.     

New insights on the involvement of Nerve Growth Factor in allergic inflammation and fibrosis

Nerve Growth Factor (NGF), that was originally discovered for its properties of stimulating growth and differentiation of neurons, is now also considered responsible for several activities in the immune system and beyond. Mast cells and eosinophils, key cells of allergic inflammation, are a source of NGF and are influenced by it. These observations have prompted studies on NGF in allergy and tissue repair. Recent evidences link NGF and these two processes. While NGF is clearly a new tool in the management of untreatable ulcers, its role in allergic inflammation, although appearing to be pro-inflammatory, is still not clearly defined.