In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.     

Prion protein amino acid determinants of differential susceptibility and molecular feature of prion strains in mice and voles

The bank vole is a rodent susceptible to different prion strains from humans and various animal species. We analyzed the transmission features of different prions in a panel of seven rodent species which showed various degrees of phylogenetic affinity and specific prion protein (PrP) sequence divergences in order to investigate the basis of vole susceptibility in comparison to other rodent models. At first, we found a differential susceptibility of bank and field voles compared to C57Bl/6 and wood mice. Voles showed high susceptibility to sheep scrapie but were resistant to bovine spongiform encephalopathy, whereas C57Bl/6 and wood mice displayed opposite features. Infection with mouse-adapted scrapie 139A was faster in voles than in C57Bl/6 and wood mice. Moreover, a glycoprofile change was observed in voles, which was reverted upon back passage to mice. All strains replicated much faster in voles than in mice after adapting to the new species. PrP sequence comparison indicated a correlation between the transmission patterns and amino acids at positions 154 and 169 (Y and S in mice, N and N in voles). This correlation was confirmed when inoculating three additional rodent species: gerbils, spiny mice and oldfield mice with sheep scrapie and 139A. These rodents were chosen because oldfield mice do have the 154N and 169N substitutions, whereas gerbil and spiny mice do not have them. Our results suggest that PrP residues 154 and 169 drive the susceptibility, molecular phenotype and replication rate of prion strains in rodents. This might have implications for the assessment of host range and molecular traceability of prion strains, as well as for the development of improved animal models for prion diseases.     

DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: zeta potential measurements and electron spin resonance spectra

Non-viral vectors represent an important alternative in gene delivery. Among these vectors, cationic liposomes are widely studied, because of their ability to form stable complexes with DNA fragments (lipoplexes). In the present work, we report on the characterization by electron spin resonance (ESR) spectroscopy and zeta potential measurements of cationic liposomes and of their complexes with oligonucleotides. Liposomes were made with a zwitterionic lipid, DOPE, and a cationic lipid, either DOTAP or DC-Chol. Oligonucleotides were the 20-base single strand polyA, the 20-base single strand polyT, and the corresponding double strand dsAT. The zeta potential as a function of the oligonucleotide/lipid⁺ ratio gave an S-shaped titration curve. Well-defined surface potential changes took place upon charge compensation between the cationic lipid heads and the phosphate groups on the oligonucleotides. The inversion point depended on the specific system under study. The bilayer properties and the changes that occurred with the incorporation of DNA fragments were also monitored by ESR spectroscopy of appropriately tailored spin probes. For all the systems investigated, the ESR spectra showed that no major alteration took place after lipoplex formation and molecular packing remained substantially unchanged. Both zeta potential and ESR measurements were in favor of an external mode of packing of the lipoplexes.     

Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice

Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M(3) muscarinic receptor was implicated in the relaxing action of ACh and M(1) muscarinic receptor (M(1)AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M(1)AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M(1)AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M(1)AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M(1)AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."     

Derlin-1 facilitates the retro-translocation of cholera toxin

Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade, leading to secretory diarrhea. How CT is targeted to the site of retro-translocation in the ER membrane to initiate translocation is unclear. Using a semipermeabilized-cell retro-translocation assay, we demonstrate that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. These findings indicate that Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport.     

Molecular ecology, speciation, and evolution of the reef fish genus Anisotremus

Grunts (family Haemulidae) in the genus Anisotremus comprise 10 described species which occur predominantly on coral reefs and subtropical rocky reefs in the Neotropics of the Tropical Eastern Pacific the Caribbean and adjacent waters. In this study, the phylogenetic relationships for all described species were examined based on one mitochondrial gene (cytochrome b) and one nuclear marker (the first intron of the ribosomal protein S7). Closely related species of the genus Haemulon and the single representative of the genus Genyatremus, G. luteus were also included in the analysis, following the suggestion, based on morphological data, that they may be closely related to, or possibly placed within, Anisotremus. Two sweetlips, genus Plectorhinchus, which belong to the recognized sister subfamily Plectorhynchinae, were used as outgroups. Two species pairs in the ingroup genus Anisotremus are geminate species, which presumably diverged after the rise of the Isthmus of Panama, thus providing an internal molecular clock calibration. This allowed for a comprehensive evolutionary scenario for the genus. Our data were consistent with two independent allopatric speciation events (geminate species), and one likely sympatric speciation event. Our data were also consistent with the placement of Genyatremus and Haemulon within the genus Anisotremus, thus breaking its monophyletic status. As proposed for other coral reef species, Anisotremus seems to have colonized coral reefs from non-coral reef habitats. In addition, ancestral species seem to have been widespread, since species that are currently found in Brazilian and Pacific waters are basal on the phylogenetic tree. Finally, this study adds to the hypothesis of a secondary evolutionary center in the New World.     

Occurrence of Euetheola humilis (Burmeister) (Coleoptera: Scarabaeidae) in Eucalyptus saligna Smith (Myrtaceae), in Rio Grande do Sul State, Brazil

Euetheola humilis (Burmeister) is a scarabaeid pest of rice, sugarcane and corn cultures, that attacks the crops during both larval and adult phase. The occurrence of E. humilis was observed in a forest area with Eucalyptus saligna Smith, from Votorantim Celulose & Papel (Fazenda Aroeira), in the Municipality of Candiota, Rio Grande do Sul State. Plants approximately 1m high were damaged by E. humilis adult insects near the stem base, what unraveled its cortex and led some of them to death. The attack was verified in about 10% of the eucalypts plants, in an area of approximately 30 ha.     

Effects of Vitamin C on Fibroblasts from Sporadic Alzheimer’s Disease Patients

Several therapies for Alzheimer’s Disease (AD) are currently under investigation. Some studies have reported that concentration of vitamins in biological fluids are lower in AD patients compared to control subjects and clinical evidence has shown the therapeutic potential of vitamin C and E in delaying AD progression. However, the molecular mechanism(s) that are engaged upon their administration in the APP metabolism in vitro or in vivo still need clarifying. Here, we investigate the effects of vitamin C supplementation, at physiological concentration, in skin fibroblasts obtained from SAD and FAD patients. This study shows that SAD patients’ fibroblasts exhibited the exclusive appearance of C-terminal fragments, derived from APP processing, without giving rise to the β-amyloid peptide, other than corresponding decreased levels of lysosomal enzymes, such as β-hexosaminidase, α-mannosidase and cathepsins B, L, and D. Special issue article in honor of Anna Maria Giuffrida-Stella.