Phylogeographic structure in the chromosomally polymorphic rodent Cricetulus barabensis sensu lato (Mammalia,Cricetidae)

Striped hamsters (Cricetulus barabensis sensu lato) represent a complex of chromosomally distinct allopatric lineages/taxa of either species or subspecies rank. They are widely distributed across the steppes of eastern and central Palearctic. Phylogenetic analysis of cytochrome b gene sequences based on 496 specimens from 112 localities revealed five well‐supported lineages divergent at 2%–4%. Two of them correspond to “griseus” (2n = 22) and “pseudogriseus” (2n = 24) karyomorphs and are placed as sister taxa. The “barabensis” (2n = 20) karyomorph is represented by three other branches and appears non‐monophyletic. All mtDNA lineages are distributed allopatrically or parapatrically; no indications of gene flow between populations of different chromosomal races were found. The results of the molecular clock analysis suggest that the main lineages diverged in the late Middle Pleistocene. The inferred evolutionary scenario implies that the common ancestor of the recent lineages belonged to the 2n = 20 karyomorph and originated in the eastern part of the contemporary range.     

Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss Physcomitrella patens

Plant secretome comprises dozens of secreted proteins. However, little is known about the composition of the whole secreted peptide pools and the proteases responsible for the generation of the peptide pools. The majority of studies focus on target detection and characterization of specific plant peptide hormones. In this study, we performed a comprehensive analysis of the whole extracellular peptidome, using moss Physcomitrella patens as a model. Hundreds of modified and unmodified endogenous peptides that originated from functional and nonfunctional protein precursors were identified. The plant proteases responsible for shaping the pool of endogenous peptides were predicted. Salicylic acid (SA) influenced peptide production in the secretome. The proteasome activity was altered upon SA treatment, thereby influencing the composition of the peptide pools. These results shed more light on the role of proteases and posttranslational modification in the “active management” of the extracellular peptide pool in response to stress conditions. It also identifies a list of potential peptide hormones in the moss secretome for further analysis.     

Genotoxic activity of 1,2,3‐triazolyl modified furocoumarins and 2,3‐dihydrofurocoumarins

The furocoumarin backbone is a promising platform for chemical modifications aimed at creating new pharmaceutical agents. However, the high level of biological activity of furocoumarins is associated with a number of negative effects. For example, some of the naturally occurring ones and their derivatives can show genotoxic and mutagenic properties as a result of their forming crosslinks with DNA molecules. Therefore, a particularly important area for the chemical modification of natural furocoumarins is to reduce the negative aspects of their bioactivity. By studying a group of 21 compounds—1,2,3‐triazolyl modified derivatives of furocoumarin and peucedanin—using the SOS chromotest, the Ames test, and DNA‐comet assays, we revealed modifications that can neutralize the structure's genotoxic properties. Theoretical aspects of the interaction of the compound library were studied using molecular modeling and this identified the leading role of the polyaromatic molecular core that takes part in stacking‐interactions with the pi‐systems of the nitrogenous bases of DNA.     

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.