Biocontrol Potential of Sodin 5, Type 1 Ribosome-Inactivating Protein from Salsola soda L. Seeds.

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as 'agretti', is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 µg/mL (1.4 µM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 µg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome-inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.

Improvement of fruit juice quality: novel endo-polygalacturonase II from Aspergillus tubingensis FAT 43 for enhanced liquefaction, clarification, and antioxidant potential.

This study focuses on the isolation, purification, and characterisation of endo-polygalacturonase II from Aspergillus tubingensis FAT43, particularly emphasising its potential applications in the fruit juice industry. A comprehensive screening test revealed the temporal dynamics of endo-polygalacturonase production during a 96-hour fermentation process. The purification process, involving ammonium sulfate and ethanol precipitation followed by ion-exchange chromatography, resulted in a 3.3-fold purification of PG II with a yield of 16% and a specific activity of 6001.67 U mg-1. Molecular analysis confirmed the identity of PG II, its gene (pgaII), and a high degree of sequence identity with Aspergillus tubingensis in the SWISS-PROT database. The optimal pH for PG II activity was 3.5-4.5, with robust stability across a broad pH spectrum (3-7). The enzyme exhibited optimal temperature activity at 45 °C, with a retention of 90% activity at 50 °C. The calculated activation energy for PG II was 62.1 kJ mol-1, indicating good stability. Inactivation kinetics revealed a half-life of 13.7 h at 40 °C, 5.4 h at 50 °C, and 0.85 h at 60 °C, with an activation energy of denaturation of 32.8 kJ mol-1. Compared to literature-reported PGs, PG II from A. tubingensis FAT43 demonstrated superior thermal stability. Hydrolysis experiments on different pectins revealed the highest specificity for non-methylated substrates (polygalacturonic acid). In fruit juice processing, PG II significantly increased juice yield and clarity, with the highest impact observed in strawberry juice. Antioxidant activity assays indicated enhanced antioxidant potential in enzyme-treated juices, especially strawberry, quince, and apple juices. The study highlights PG II's potential as an industrially valuable enzyme for fruit juice processing, offering improved thermostability and versatility across various fruit types.

Proteomics of the haemolymph of the terrestrial crustacean Porcellio scaber reveals components of its innate immunity under baseline conditions.

The terrestrial crustacean Porcellio scaber is an established test organism in environmental research. We analysed the haemolymph proteome of P. scaber using a classical proteomic approach based on one-dimensional gel electrophoresis and tandem mass spectrometry. Using a publicly available protein database and our P. scaber transcriptome data, we have identified 76 proteins involved in cytoskeleton formation, protein degradation, vesicular transport, genetic information processing, detoxification, carbohydrate and lipid metabolism reflecting haemocyte metabolic activity, active intracellular transport, and intercellular communication. Compared with the data reported for other crustaceans, 28 of these P. scaber proteins have been linked to its immunity, among them hemocyanin, α-2-macroglobulin, phenoloxidase 3, superoxide dismutase, glutathione S-transferase, haemolymph clottable protein, and histones H4 and H2B. Our results thus provide a firm base for studying the innate immune response of P. scaber at the level of the haemolymph proteome. This knowledge is of particular importance in ecotoxicity studies with various environmental stressors where understanding physiological changes is important to reveal possible modes of action.

Serine pseudoproteases in physiology and disease.

Serine proteases (SPs) constitute a very important family of enzymes, both physiologically and pathologically. The effects produced by these proteins have been explained by their proteolytic activity. However, the discovery of pharmacologically active SP molecules that show no enzymatic activity, as the so-called pseudo SPs or SP homologs (SPHs), has exposed a profoundly neglected possibility of nonenzymatic functions of these SP molecules. In this review, the most thoroughly described SPHs are presented. The main physiological domains in which SPHs operate appear to be in reproduction, embryonic development, immune response, host defense, and hemostasis. Hitherto unexplained actions of SPs should therefore be considered also as the result of the ligand-like attributes of SPs. The gain of a novel function by an SPH is a consequence of specific amino acid replacements that have resulted in a novel interaction interface or a 'catalytic trap'. Unraveling the SP/SPH interactome will provide a description of previously unknown physiological functions of SPs/SPHs, aiding the creation of innovative medical approaches.

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, ß-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat's brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom ß-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.

The Relevance of Physico-Chemical Properties and Protein Corona for Evaluation of Nanoparticles Immunotoxicity-In Vitro Correlation Analysis on THP-1 Macrophages.

Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO2, silver, and TiO2) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO2 types and SiO2 exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1ß cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1ß secretion. The induction of IL-1ß secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.

Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein.

Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.

Insect Protein-Based Diet as Potential Risk of Allergy in Dogs.

Before insects can be used widely as an alternative source of dietary protein, their allerginicity should be investigated. Therefore, the aim of our study was to assess the potential adverse reactions of the immune system of dogs against Tenebrio molitor proteins. Dogs sensitised to storage mites T. putrescentiae and A. siro were included. Clinically healthy and clinically allergic dogs were compared. Proteins were extracted from mealworm larvae and their digestibility determined by in vitro incubation with digestive proteases. Mealworm protein extracts and digests were analysed by SDS-PAGE. Canine sera tested for the presence of mite-specific IgEs were used for subsequent Western blotting. LC-MS/MS analysis was used to identify mealworm proteins and their allergenic potential was predicted with the AllermatchTM tool. The binding of canine sera IgEs to mealworm proteins was confirmed; however, the differences between the two groups of dogs were not significant. Moreover, no clear correlation was found between sensitisation to storage mites and clinical status of the dogs. Altogether, 17 different proteins were identified, including tropomyosin, α-amylase, and Tm-E1a cuticular protein that are known cross-reacting IgE-binding allergens. Our results suggest that dogs allergic to mites may clinically express also the cross-reactivity with mealworm proteins.

The first Kunitz-type proteins from a viperid venom that potentiate neuromuscular transmission.

Kunitz-type proteins that interfere with neuronal transmission have been thus far exclusively detected in venoms of elapid snakes. Here, we report for the first time that such proteins are also present in the venom of a viperid snake. From the venom of the nose-horned viper (Vipera ammodytes ammodytes; Vaa), we isolated Kunitz-type chymotrypsin inhibitors (VaaChi) and demonstrated that these molecules also significantly increase the amplitudes of an indirectly evoked simple muscle contraction of the mouse hemidiaphragm, the end-plate potential and the miniature end-plate potential. By facilitating neuromuscular transmission, these proteins resemble structurally homologous dendrotoxins from mamba (Dendroaspis spp.) venoms, which are blockers of voltage-dependent K+ channels at the presynaptic site of the neuromuscular junction. What is the mechanism behind facilitation of neuromuscular transmission by VaaChi has not been established yet, however, blocking of K+ channels does not seem to be the most probable option.

The Procoagulant Snake Venom Serine Protease Potentially Having a Dual, Blood Coagulation Factor V and X-Activating Activity.

A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell's viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

Biological Activities and Proteomic Profile of the Venom of Vipera ursinii ssp., a very Rare Karst Viper from Croatia.

The karst viper (Vipera ursinii ssp.) favours high-mountain dry grasslands in southern and south-eastern Croatia. It is medically less important than other Vipera species, because of its remote habitat and the very small amount of venom that it injects by its relatively short fangs. The scientific literature on Vipera ursinii deals mostly with the morphology, ecology and distribution range of this snake, due to the species' conservation issues, while the toxinological aspects of its venom have not so far been investigated. Here we report on the composition and biological activity of the Vipera ursinii ssp. venom. Using a proteomics approach, we have identified 25 proteins in the venom that belong to seven protein families: snake venom metalloproteinase, serine protease, secreted phospholipase A2, cysteine-rich secretory protein, snake C-type lectin-like protein, serine protease inhibitor and nerve growth factor. The Vipera ursinii ssp. venom was found to be distinctively insecticidal. Its lethal toxicity towards crickets was more than five times greater than that of Vipera ammodytes ammodytes venom, while the opposite held in mice. Interestingly, the mode of dying after injecting a mouse with Vipera ursinii ssp. venom may suggest the presence of a neurotoxic component. Neurotoxic effects of European vipers have so far been ascribed exclusively to ammodytoxins and ammodytoxin-like basic secreted phospholipases A2. Structural and immunological analyses of the Vipera ursinii ssp. venom, however, confirmed that ammodytoxin-like proteins are not present in this venom.

Vitellogenin in the European cave salamander, Proteus anguinus: Its characterization and dynamics in a captive female as a basis for non-destructive sex identification.

Vitellogenin (Vtg) is a precursor protein of egg yolk proteins in oviparous and ovoviviparous vertebrates. Except in a case of exposure to estrogenic endocrine disruptors, Vtg is a female-specific protein and could be used as a molecular marker for sex identification. This would be especially useful in the case of the endangered European cave salamander Proteus anguinus in which sexes are indistinguishable according to external morphology, which hinders the establishment of a successful captive breeding program. Here we describe the identification, partial characterization, and purification of Vtg from P. anguinus. Vtg was identified in the plasma of a vitellogenic proteus female with visible oocytes. The identification of this protein was accomplished by mass spectrometry analysis. Two-dimensional gel electrophoresis revealed proteus Vtg as a mix of 190 kDa isoforms with isoelectric points in the pH range 5.3-6.0. Vtg was purified from proteus blood by gel filtration followed by anion-exchange chromatography. Using specific staining of SDS-PAGE gels, the Vtg was found to be phosphorylated and lipidated. Unlike the case in some other aquatic vertebrates, in P. anguinus, Vtg was not present in detectable amounts in cutaneous mucus. Degradation of oocytes in the captive vitellogenic female was accompanied by simultaneous decrease of Vtg concentration. Over a period of 10 months, the concentration of Vtg dropped from maximal to sub-detectable. Our results show that Vtg is a promising molecular marker for sex identification and ovary maturation in P. anguinus, which could contribute to the development of a viable program for captive reproduction of this unique species.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy.

Components of the intrinsic blood coagulation pathway, among them factor VIIIa (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. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (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 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it 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 three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157-loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Ammodytin L is the main cardiotoxic component of the Vipera ammodytes ammodytes venom.

Venom of the nose-horned viper (V. a. ammodytes) as also venoms of some related European viperids can induce also cardiotoxic effects in mammals. In this work we demonstrated that the protein in the V. a. ammodytes venom acting on heart is a myotoxic secreted phospholipase A2 analogue ammodytin L (AtnL). In the isolated perfused rat heart AtnL induced significant and irreversible cardiotoxicity characterized by atrioventricular (AV) blockade. This venom protein induced appearance of high levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and troponin I in the sinus effluent of the isolated heart, indicative for myocardial damage, which is obviously the primary cause of its cardiotoxic action. Gel filtration chromatography subfractions C1 and C2 of the V. a. ammodytes venom harboured most of the venom cardiotoxicity. As we showed, just these two subfractions contained also AtnL. Subfraction C1 in the final CF concentration 11.3 µg/mL (containing 3.1 µg/mL AtnL) induced a complete cardiac arrest while subfraction C2 in the final CF concentration 6.0 µg/mL (containing 0.8 µg/mL AtnL) and the pure AtnL (1.0 µg/mL) did not. Contrary to AtnL, subfraction C1 at 11.3 µg/mL was not able to induce the AV blockade. This exposed the only other cardiotoxic subfractions-specific venom protein, a cysteine-rich secretory protein (CRISP), as an additional venom component potentially involved in modulation of the heart activity. Cardiotoxicity reported in some cases of the adder (V. berus) venom and the asp viper (V. aspis) venom poisonings may be assigned to AtnL in these venoms.

Disintegrins from the Venom of Vipera ammodytes ammodytes Efficiently Inhibit Migration of Breast Cancer Cells.

Integrins are plasma membrane proteins, whose dysfunction frequently results in cancer pathology, and therefore they represent important targets of anti-tumour therapy. Snake venoms are a rich source of disintegrins (Dis), proteins that specifically bind integrins and thus interfere with their functions. In an attempt to discover new molecules for treatment of breast cancer, the major type of cancer in women, we isolated a dimeric Dis (Vaa-Dis) from the venom of the nosehorned viper. By cell viability testing we demonstrated that 50 nM and higher concentrations of Vaa-Dis were toxic to highly invasive human breast adenocarcinoma cell line MDA-MB-231. Wound-healing assay revealed that already at one order of magnitude lower concentrations Vaa-Dis efficiently inhibited MDA-MB-231 cell migration. This exposed a promising anti-metastatic potential of Vaa-Dis and a good perspective of these natural snake venom proteins for further research and development towards the application in breast cancer treatment.

Formation and morphogenesis of a cuttlebone's aragonite biomineral structures for the common cuttlefish (Sepia officinalis) on the nanoscale: Revisited.

This study describes and examines the structural and morphological properties of the hierarchically organized, aragonite cuttlebone forms for the common cuttlefish (Sepia officinalis, L.), including its main structural parts, the dorsal shield, and the chambers. Specifically, it complements the mechanism for the self-organized formation of aragonite, identifies the presence, and determines the role of soluble organic matrix (SOM) proteins in the morphogenesis of the cuttlebone's biomineral structures on the nanoscale. The structure and morphology of the cuttlebone were examined using X-ray diffraction (XRD) and field-emission scanning electron microscopy (FESEM), and their thermal properties by thermo-gravimetric analysis (TGA) and differential thermal analysis (DTA). Proteins from the SOM were investigated using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), nano liquid chromatography tandem mass spectrometry (nano-LC ESI-MS) and Edman degradation. The results showed that the cuttlebone exhibited several diverse biomineral structures characterized by complex morphologies. Their formation is governed by the organic matrix, particularly proteins, which at the earliest stage of development provide templates for the initial extracellular nucleation of the aragonite nanocrystals. This is followed by a bottom-up morphogenesis, based on the nanoscale oriented aggregation and coalescence of primarily formed aragonite nanograins, which results in the hierarchically organized, nanostructured, aragonite forms. The molecular masses of the most pronounced SOM proteins from the dorsal shield were about 10, 15, 40 and 60kDa, while from the chambers they were 10, 20, 25, 30 and 45kDa. Peptide fragments corresponding to Sep7, Sep8, chitin synthase 1, ficoline-2, polyubiquitin and the ubiquitin carboxyl-terminal hydrolase 32-like protein were detected in the SOM, with these proteins having functional properties related to the biomineralization processes. In general, there are mostly acidic proteins present in alternatively glycosylated forms, which are common attributes of biomineralization-related proteins.

Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona.

Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored-the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1µm, and larger vesicles of around 10-µm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.