Bacterial expression, NMR, and electrophysiology analysis of chimeric short/long-chain alpha-neurotoxins acting on neuronal nicotinic receptors

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.     

Subcellular proteomics of cell differentiation: quantitative analysis of the plasma membrane proteome of Caco-2 cells

Human colorectal carcinoma (Caco-2) cells undergo in culture spontaneous enterocytic differentiation, characterized by polarization and appearance of the functional apical brush border membrane. To provide insights into the biology of differentiation, we have performed a comparative proteomic analysis of the plasma membranes from proliferating cells (PCs) and the apical membranes from differentiated cells (DCs). Proteins were resolved by SDS-PAGE, in-gel digested and analyzed by RP-LC and MS/MS. Alternatively, proteins were digested in solution, and tryptic peptides were labeled with isotopic tags and analyzed by 2-D LC followed by MS/MS. Among the 1125 proteins identified in both proteomes, 76 were found to be significantly increased in the membranes of DCs and 61 were increased in PCs. Majority of the proteins increased in the apical membranes were metabolic enzymes, proteins involved in the maintenance of cellular structure, transmembrane transporters, and proteins regulating vesicular transport. In contrast, majority of the proteins increased in the membranes of PCs were involved in gene expression, protein synthesis, and folding. Both groups contained many novel proteins with yet to be identified functions, which could provide potential new markers of the intestinal cells or of colorectal cancer.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Evidences against vesicle-dependent trafficking and involvement of extracellular proteasomes into cell-to-cell communications

The ubiquitin proteasome system is involved in the regulation of most basic intracellular processes, and deregulation of this system can results in certain kinds of human diseases. Proteolytic core this system, the 20S proteasome, has been found in physiological fluids of both healthy humans and patients suffering from a variety of inflammatory, autoimmune, and neoplastic diseases. The concentration of these extracellular proteasomes has been found to correlate with the diseased state, being of a prognostic significance. The transport mechanisms and functions of these proteasomes, however, are largely unclear. Previous studies revealed that the transport of extracellular proteasomes may occur via microvesicles and exosomes, which led to the hypothesis that extracellular proteasomes are implicated in cell-to-cell communication process. Here we show that microvesicles and exosomes, two major known types of intercellular vehicles, contain no detectable proteasomes. Moreover, neither affinity purified nor naturally released into conditioned medium by donor cells 20S proteasomes could penetrate recipient HeLa cells. Taken together, these results suggest that extracellular proteasomes are unlikely to be involved in the cell-to-cell communication and that their release by cells serve other biological purposes.     

Effect of bedaquiline on the functions of rat liver mitochondria

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V₃ and V_DNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V₂. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H₂O₂ production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca²⁺-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca²⁺/P_i-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca²⁺ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K⁺ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K⁺ buffer and DNP-induced K⁺ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.     

Hair Mineral and Trace Element Content in Children with Down’s Syndrome

Biological Trace Element Research - Chronic exposure to lead causes disruption to energy production mechanisms and tissue damage, in particular through its binding to thiol groups and competition...     

Prebiotic Syntheses Under Shock in the Water – Formamide – Potassium Bicarbonate – Sodium Hydroxide System

Origins of Life and Evolution of Biospheres - Syntheses under shock in nitrogen bubbled samples of the water – formamide – bicarbonate – sodium hydroxide system at...