We report the proteomic characterization of the venom of the medically important North American western diamondback rattlesnake, Crotalus atrox, using two complementary approaches: snake venomics (to ...gain an insight of the overall venom proteome), and two solid-phase combinatorial peptide ligand libraries (CPLL), followed by 2D electrophoresis and mass spectrometric characterization of in-gel digested protein bands (to capture and “amplify“ low-abundance proteins). The venomics approach revealed ∼24 distinct proteins belonging to 2 major protein families (snake venom metalloproteinases, SVMP, and serine proteinases), which represent 69.5% of the total venom proteins, 4 medium abundance families (medium-size disintegrin, PLA2, cysteine-rich secretory protein, and l-amino acid oxidase) amounting to 25.8% of the venom proteins, and 3 minor protein families (vasoactive peptides, endogenous inhibitor of SVMP, and C-type lectin-like). This toxin profile potentially explains the cytotoxic, myotoxic, hemotoxic, and hemorrhagic effects evoked by C. atrox envenomation. Further, our results showing that C. atrox exhibits a similar level of venom variation as Sistrurus miliarius points to a ”diversity gain” scenario in the lineage leading to the Sistrurus catenatus taxa. On the other hand, the two combinatorial hexapeptide libraries captured distinct sets of proteins. Although the CPLL-treated samples did not retain a representative venom proteome, protein spots barely, or not at all, detectable in the whole venom were enriched in the two CPLL-treated samples. The amplified low copy number C. atrox venom proteins comprised a C-type lectin-like protein, several PLA2 molecules, PIII-SVMP isoforms, glutaminyl cyclase isoforms, and a 2-cys peroxiredoxin highly conserved across the animal kingdom. Peroxiredoxin and glutaminyl cyclase may participate, respectively, in redox processes leading to the structural/functional diversification of toxins, and in the N-terminal pyrrolidone carboxylic acid formation required in the maturation of bioactive peptides such as bradykinin-potentiating peptides and endogenous inhibitors of metalloproteases. Our findings underscore the usefulness of combinatorial peptide libraries as powerful tools for mining below the tip of the iceberg, complementing thereby the data gained using the snake venomics protocol toward a complete visualization of the venom proteome.
Nanomedicine is an interdisciplinary field of research, comprising science, engineering, and medicine. Many are the clinical applications of nanomedicine, such as molecular imaging, medical ...diagnostics, targeted therapy, and image‐guided surgery. Despite major advances during the past 20 years, many efforts must be done to understand the complex behavior of nanoparticles (NPs) under physiological conditions, the kinetic and thermodynamic principles, involved in the rational design of NP. Once administrated in physiological environment, NPs interact with biomolecules and they are surrounded by protein corona (PC) or biocorona. PC can trigger an immune response, affecting NPs toxicity and targeting capacity. This review aims to provide a detailed description of biocorona and of parameters that are able to control PC formation and composition. Indeed, the review provides an overview about the role of PC in the modulation of both cytotoxicity and immune response as well as in the control of targeting capacity.
The plasma membrane controls the selective internalization of (macro)molecules through different mechanisms, often relaying on specialized outward-facing carriers such as exofacial proteins thiols ...(EPTs). Although the interchange of critical thiols and disulphides between EPTs and exogenous cargoes is the first critical event, the identification of specific cell interactors remains to be thoroughly explored. Besides, it is likewise evident that only the relatively little suite of EPTs truly reactive can be considered theranostic targets.
We were aimed at developing a stepwise procedure for the isolation and identification of a subset of EPTs, that we named chemically reactive EPS, which are potential theranostic targets.
In the present study, EPTs that displayed permissive sulfhydryls on the surface of live cells in vitro underwent i) chemo-selective capture by means of thiolated superparamagnetic microbeads (isolation step), followed by ii) their prompt release via disulphide breakage through the addition of DTT reducing agent (elution step) and iii) analysis by means of SDS-PAGE and LCMS/ MS (identification step).
In total cell lysates, most of the proteins recovered were intracellular. Conversely, this methodology allowed a 2.6-fold enrichment in chemically reactive EPTs recovered and identified, corresponding up to 37% of the total cellular proteins. The key element of our approach was the reversible chemo-selective capture through disulphide linkages between chemically reactive EPTs and free thiols on microbeads.
We devised an enabling methodology to selectively pick up, recover and characterize chemically reactive EPTs.
The ‘enzyme prodrug therapy’ represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an ...active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric flavoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing five aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efficacy under low O
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concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O
2
concentration. In order to evaluate the nanocarriers’ biocompatibility, PEG-MWCNTs-DAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has influenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic effect controlled by the substrate addition, against different tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug’s biodistribution.
The use of combinatorial peptide ligand libraries (CPLLs), containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, or with a terminal tertiary amine, ...allowed discovering and identifying a large number of previously unreported egg yolk proteins. Whereas the most comprehensive list up to date K. Mann, M. Mann, Proteomics, 8 (2008) 178–191 tabulated about 115 unique gene products in the yolk plasma, our findings have more than doubled this value to 255 unique protein species. From the initial non-treated egg yolk it was possible to find 49 protein species; the difference was generated thanks to the use of the three combined CPLLs. The aberrant behaviour of some proteins, upon treatment via the CPLL method, such as proteins that do not interact with the library, is discussed and evaluated. Simplified elution protocols from the CPLL beads are taken into consideration, of which direct elution in a single step via sodium dodecyl sulphate desorption seems to be quite promising. Alternative methods are suggested. The list of egg yolk components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.
Treatment of 50μM RBBR with 60μg of N. gerenzanensis peroxidase preparation: the dye decolorizing activity was clearly observed.
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•Actinomycetes represent an attractive source of ...ligninolytic enzymes.•43 actinomycetes were screened for laccase and peroxidase activities.•The novel species N. gerenzanensis produces a valuable bacterial peroxidase activity.•The dye-decolorizing activity paves the way for an industrial use of this peroxidase.
Degradation of lignin constitutes a key step in processing biomass to become useful monomers but it remains challenging. Compared to fungi, bacteria are much less characterized with respect to their lignin metabolism, although it is reported that many soil bacteria, especially actinomycetes, attack and solubilize lignin. In this work, we screened 43 filamentous actinomycetes by assaying their activity on chemically different substrates including a soluble and semi-degraded lignin derivative (known as alkali lignin or Kraft lignin), and we discovered a novel and valuable peroxidase activity produced by the recently classified actinomycete Nonomuraea gerenzanensis. Compared to known fungal manganese and versatile peroxidases, the stability of N. gerenzanensis peroxidase activity at alkaline pHs and its thermostability are significantly higher. From a kinetic point of view, N. gerenzanensis peroxidase activity shows a Km for H2O2 similar to that of Phanerochaete chrysosporium and Bjerkandera enzymes and a lower affinity for Mn2+, whereas it differs from the six Pleurotus ostreatus manganese peroxidase isoenzymes described in the literature. Additionally, N. gerenzanensis peroxidase shows a remarkable dye-decolorizing activity that expands its substrate range and paves the way for an industrial use of this enzyme. These results confirm that by exploring new bacterial diversity, we may be able to discover and exploit alternative biological tools putatively involved in lignin modification and degradation.
Combinatorial peptide ligand libraries have recently allowed considerable advances in the mapping of chicken egg yolk and white proteomics. Data from literature have been regrouped and elaborated for ...network and pathway analyses in order to convey a unified view of these proteomes. Redundant proteins were excluded, while isoforms of the same proteins were maintained to reach a total of 260 distinct gene products for egg yolk and 148 for egg white having a match in the database. From these analyses, a role for proteins involved in cell development, proliferation and migration, cell-to-cell interaction and hematological system development emerged. Although it might turn out that, notwithstanding the extensive mapping, the currently available datasets might be still incomplete, a valuable insight could still be obtained about specific proteins playing a crucial role in antimicrobial responses, mainly histones, lysozyme and vitamin-binding proteins. In particular, SERPINB3 (ovalbumin Y, or Squamous Cell Carcinoma Antigen, SCCA1) was individuated in 8 out of 10 top score pathways in egg yolk and in 6 out 10 in egg white. SERPINB3 is a member of the ov-serpin family, participating in coagulation and inflammation responses. However, it is yet to be assessed how these observations could correlate with previous analyses about the role of egg yolk derived proteins in counteracting blood coagulation.
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The proteome of Hevea brasiliensis latex has been explored in depth via combinatorial peptide ligand libraries. A total of 300 unique gene products have been identified in this latex, whose proteome ...has been largely unknown up to the present. In search for unknown allergens, control latex and eluates from the ligand libraries have been fractionated by two-dimensional mapping, blotted and confronted with sera of 18 patients. In addition to the already known and named Hevea major allergens, we have unambiguously detected several others like, for instance: heat shock protein (81kDa), proteasome subunit (30kDa), protease inhibitor (8kDa), hevamine A (43kDa) and glyceraldehyde-3-phosphate dehydrogenase (37kDa). Gene Ontology analysis of analyzed fractions has shown that major functions are substantially unchanged after sample treatment, while novel biological functions appeared that were undetectable in the crude sample.
The capture of the Hevea latex proteome via two combinatorial peptide ligand libraries has permitted the discovery of a total of 300 unique gene products as shown in the Venn diagram. Display omitted
The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related ...disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.
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