Components of the intrinsic blood coagulation pathway, among them FVIIIa, have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. In this work we described a unique glycoprotein from the nose-horned viper venom, VaaSPH-1, structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a threedimensional model of VaaSPH-1 bound to FVIIIa and proposed the most appropriate structural elements of VaaSPH-1 to constitute small FVIIIa-binding molecules, potential new generation of anticoagulants.
COBISS.SI-ID: 31735847
The ß-neurotoxic secreted phospholipases A2 (sPLA2s) block neuro-muscular transmission by poisoning nerve terminals. Damage inflicted by such sPLA2s (ß-ntx) on neuronal mitochondria is characteristic, very similar to that induced by structurally homologous endogenous group IIA sPLA2 when its activity is elevated, as, for example, in the early phase of Alzheimer’s disease. Using ammodytoxin (Atx), the ß-ntx from the venom of the nose-horned viper (Vipera a. ammodytes), we detected the sPLA2 receptor R25 in neuronal mitochondria. Here we describe its purification from mitochondria by a new Atx-affinity-based chromatographic procedure and its structural characterization. R25 is the subunit II of cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. We confirmed CCOX as the first intracellular membrane receptor for sPLA2 by alternative Atx-affinity-labellings of purified CCOX and by demonstrating the encounter of Atx and CCOX in PC12 cells. This discovery suggests the explanation of the mechanism by which ß-ntx hinders production of ATP in poisoned nerve endings. It also provides a new insight into the potential function and dysfunction of endogenous GIIA sPLA2 in mitochondria.
COBISS.SI-ID: 32050215
The pulmonary delivery of nanoparticles is a promising approach in nanomedicine. For the efficient and safe use of inhalable nanoparticles, understanding of nanoparticle interference with lung surfactant metabolism is needed. Our results on A549 human lung cancer cells, a model of alveolar type II cells, demonstrate that non-cytotoxic concentrations of silica-coated superparamagnetic iron oxide nanoparticles (SiO2-SPIONs) interfere with surfactant metabolism and lamellar bodies (LB) biogenesis, leading to disturbed ability to reduce hypophase surface tension. To ensure the safe use of nanoparticles for pulmonary delivery, we propose that potential nanoparticle interference with LB biogenesis is obligatorily taken into account.
COBISS.SI-ID: 30433319
RecA protein is a hallmark of the bacterial response to insults inflicted on DNA. It catalyses the strand exchange step of homologous recombination and stimulates self-inactivation of the LexA transcriptional repressor. Importantly, by these activities, RecA contributes to the antibiotic resistance of bacteria. An original way to decrease the acquisition of antibiotic resistance would be to block RecA association with LexA. To engineer inhibitors of LexA–RecA complex formation, we have mapped the interaction area between LexA and active RecA–ssDNA filament (RecA*) and generated a three–dimensional model of the complex. The model revealed that one subunit of the LexA dimer wedges into a deep helical groove of RecA*, forming multiple interaction sites along seven consecutive RecA protomers. Based on the model, we predicted that LexA in its DNA–binding conformation also forms a complex with RecA* and that the operator DNA sterically precludes interaction with RecA*, which guides the induction of SOS gene expression. Moreover, the model shows that besides the catalytic C-terminal domain of LexA, its N-terminal DNA–binding domain also interacts with RecA*. Because all the model based predictions have been confirmed experimentally, the presented model offers a validated insight into the critical step of the bacterial DNA damage response.
COBISS.SI-ID: 26953767
For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in venoms of mollusks. In order to shed more light on cone snail venom HMM components, the proteins of dissected and injected venom of a fishhunting cone snail, Conus consors, were extensively assessed. HMM venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the venom duct and assigned a biological function, thus pointing to their potential role in venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups of HMM proteins passing the venom selection process – echotoxins and hyaluronidases – were clearly present in the injected venom. They are suggested to contribute to the envenomation process. This newly devised integrated HMM proteomic analysis is a big step towards identification of the protein arsenal used in a cone snail venom apparatus for venom production, maturation and function.
COBISS.SI-ID: 26042407
EpCAM (epithelial cell adhesion molecule; a modular transmembrane glycoprotein) has been known for several decades as an important tumor marker, and in the recent years several therapeutic aproaches, based on EpCAM targeted drug delivery, were developed, however their efficacy was rather low due to lack of knowledge on EpCAM structure and function. In the paper we for the first time described the crystal structure of extracellular part of EpCAM (solved in our group), showed that the molecule forms a dimer on cell surface, explored dimerization of the transmembrane part by molecular dynamics, and presented a model of intercellular tetrameric unit. Results are an excellent base for enhancement of existing and development of new therapeutic approaches.
COBISS.SI-ID: 1764911
Viruses that infect bacteria (phages) represent the most abundant biological entities on the planet, having an enormous impact on microbial communities and bacterial evolution. For the successful development of phage progeny they have evolved diverse and sophisticated mechanisms to take over essential bacterial processes. To date such host takeover mechanisms have only been thoroughly studied in detail for a handful of phages. The phage GIL01 infects the insect pathogen Bacillus thuringiensis. We resolved that a small GIL01-encoded protein, gp7, modulates host LexA transcription repressor to establish GIL01 dormant state inside bacterium. Here we show that the second small phage protein, gp6, is the genetic switch, the inducer of the GIL01 resurrection leading to page particle formation and initiation of the host cell lysis. Surprisingly, gp6 evolved from the host LexA, thus we provide a rare example of how two related proteins, with opposing functions, control the phage life cycle switch. Here resolved GIL01 molecular mechanisms inspired us to set forth research to develop novel antibacterial compounds based on GIL01 small proteins.
COBISS.SI-ID: 4764239
We traced the genesis and regulatory wiring of the Metaviridae-derived domesticated genes (DGs) through phylogenomic analysis, using whole-genome information from more than 90 chordate genomes. Mammalian retroelement-derived DGs (RDDGs) have been shown to originate in several steps by independent domestication events and to diversify later by gene duplications. Analysis of syntenic loci has shown that diverse RDDGs and their chromosomal positions were fully established in the ancestor of placental mammals. By analysis of active Metaviridae lineages in amniotes, we have demonstrated that RDDGs originated from retroelement remains. During the domestication process, de novo acquisition of regulatory regions is shown to be a prerequisite for the survival of the DGs. The origin and evolution of de novo acquired promoters and untranslated regions in diverse mammalian RDDGs have been explained by comparative analysis of orthologous gene loci. The origin of placental mammal-specific innovations and adaptations, such as placenta and newly evolved brain functions, was most probably connected to the regulatory wiring of DGs and their rapid fixation in the ancestor of placental mammals.
COBISS.SI-ID: 26492711
In this breakthrough paper, we describe the ability of lipid droplets to promote breast cancer cell survival and a provide evidence of a novel “metabolic” action of secreted phospholipases A2 (sPLA2s) in cancer. Previous reports have revealed various and often contrasting effects of sPLA2s on cancer cells, with many unsuccessful attempts to describe their action by activation of signalling pathways and arachidonic acid (AA)-derived lipid mediators. The ability of sPLA2 to release various fatty acids in addition to AA was not in the forefront of research and their activity was never associated with lipid droplets or energy metabolism. We show in this paper that sPLA2-released fatty acids from breast cancer cell membranes induce lipid droplet biogenesis and enable cell survival during nutrient stress. The paper elegantly connects the major points of the mechanism, describing the global changes in lipid metabolism that enable cancer cell survival, including activation of mitochondrial fatty acid oxidation, suppression of de novo lipogenesis and activation of AMP-activated protein kinase (AMPK). The ability of sPLA2-induced lipid droplets to protect cancer cells from stress was associated with lipid droplet breakdown and suppressed by inhibitors of fatty acid oxidation, thus implicating lipolysis and mitochondrial oxidation in the protection of breast cancer cells from nutrient stress.
COBISS.SI-ID: 27087655
Recently, Pleurotus aegerolysins OlyA, PlyA2 and erylysin A (EryA) were demonstrated to preferentially bind to artificial lipid membranes containing 50 mol% ceramide phosphoethanolamine (CPE), the main sphingolipid in invertebrate cell membranes. In this study, we demonstrate that OlyA6, PlyA2 and EryA bind to insect cells and to artificial lipid membranes with physiologically relevant CPE concentrations. Moreover, these aegerolysins permeabilize these membranes when combined with PlyB. These aegerolysin/PlyB complexes show selective toxicity toward western corn rootworm larvae and adults and Colorado potato beetle larvae. These data strongly suggest that these aegerolysin/PlyB complexes recognize CPE as their receptor molecule in the insect midgut. This mode of binding is different from those described for similar aegerolysin-based bacterial complexes, or other Bacillus thuringiensis Cry toxins, which have protein receptors. Targeting of Pleurotus aegerolysins to CPE and formation of transmembrane pores in concert with PlyB suggest the use of aegerolysin/PlyB complexes as novel biopesticides for the control of western corn rootworm and Colorado potato beetle.
COBISS.SI-ID: 5013839