S1 family of serine peptidases is the largest family of peptidases. They are specifically inhibited by the Kunitz/BPTI inhibitors. Kunitz domain is characterized by the compact 3D structure with the most important inhibitory loops for the inhibition of S1 peptidases. In the present study we analysed the action of site-specific positive selection and its impact on the structurally and functionally important parts of the snake venom Kunitz/BPTI family of proteins. By using numerous models we demonstrated the presence of large numbers of site-specific positively selected sites that can reach between 30-50% of the Kunitz domain. The mapping of the positively selected sites on the 3D model of Kunitz/BPTI inhibitors has shown that these sites are located in the structurally most important part of the molecule, in the inhibitory loops 1 and 2, but also in the Kunitz scaffold. Amino acid replacements are localized exclusively on the surface, and the vast majority of replacements are causing the change of the charge. The consequence of these replacements is the change in the electrostatic potential on the surface of the Kunitz/BPTI proteins that may play an important role in the precise targeting of these inhibitors into the active site of S1 family of serine peptidases. The presence of the multigene families of Kunitz/BPTIs in venomous snakes can be explained by the target-oriented arms race, since the number of S1 peptidases in vertebrate preys can reach up hundreds of representatives. Since Kunitz/BPTIs are broad spectrum inhibitors, they can be functionally diversified to target numerous and diverse S1 peptidases in their prey. Comparison of the Kunitz/BPTI inhibitors from venomous snakes with ticks and vampire bats has demonstrated that they experienced strong and widespread action of site-specific positive selection only in the venomous snakes.
COBISS.SI-ID: 29935911
Trop2 is a transmembrane signaling glycoprotein upregulated in stem and carcinoma cells. Proliferation-enhancing signaling involves regulated intramembrane proteolytic release of a short cytoplasmic fragment, which is later engaged in a cytosolic signaling complex. We propose that Trop2 function is modulated by phosphorylation of a specific serine residue within this cytosolic region (Ser303), and by proximity effects exerted on the cytosolic tail by Trop2 dimerization. Structural characterization of both the transmembrane (Trop2TM) and cytosolic regions (Trop2IC) support this hypothesis, and shows that the central region of Trop2IC forms an α-helix. Comparison of NMR structures of non-phosphorylated and phosphorylated forms suggest that phosphorylation of Trop2IC triggers salt bridge reshuffling, resulting in significant conformational changes including ordering of the C-terminal tail. In addition, we demonstrate that the cytosolic regions of two Trop2 subunits can be brought into close proximity via transmembrane part dimerization. Finally, we show that Ser303-phosphorylation significantly affects the structure and accessibility of functionally important regions of the cytosolic tail. These observed structural features of Trop2 at the membrane-cytosol interface could be important for regulation of Trop2 signaling activity.
COBISS.SI-ID: 1536305091
In the field of communication between cellular organelles, which is currently one of the focal in cell biology, we demonstrated a new way of connection between mitochondria and peroxisomes that relies the ERMES complex and the Pex11 protein. Our work received a high attention from researcher in the field and opened a way also to some other until now non-conventional views about organization of eukaryotic cells.
COBISS.SI-ID: 28442663
Vesiculation of cellular membranes is a constitutive process, although it can be induced by external physico-chemical parameters. In this study we, explored the blebbing and vesiculation of Madin-Darby canine kidney (MDCK) cells induced by fluorescently-tagged aegerolysin, OlyA-mCherry. The paper shows that vesiculation of MDCK cells induced by OlyA-mCherry is not decreased by low temperatures and does not depend on increased [Ca2+]i. Our proteomic analysis revealed that these shed EVs contained 71 proteins. These proteins were mostly of cytosolic and nuclear origin, and only a few were assigned as membrane-associated or typical membrane raft residents. Furthermore, the cellular and EVs lipidome were characterized using liquid chromatography-mass spectrometry and high-resolution nuclear magnetic resonance, which included analysis of the phospholipids and ceramide-based glycosphingolipids. We identified 218 different lipid species in these cells, and 84 different lipid species in EVs. We concluded that shed EVs are potentially interesting model for biophysical research as well as tool for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.
COBISS.SI-ID: 3971151
Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning - induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation.
COBISS.SI-ID: 4166522