Characterization and kinetics studies of water buffalo (Bubalus bubalis) myoglobin

The colour of buffalo (Bubalus bubalis L.) meat is darker than bovine meat. Since meat colour depends on the concentration of myoglobin (Mb) and its oxidation state, we have determined the main structural and functional properties of buffalo Mb. Buffalo Mb was purified from longissimus dorsi muscles and its molecular mass determined by ESI Q-TOF mass spectrometry. The molecular mass 17,034.50 was 86.20 Da higher than the bovine Mb. This was confirmed by analysing its primary structure, using a combined approach based on Edman degradation and MALDI-TOF mass spectrometry. Comparing the amino acid sequences of both Mbs, we found three amino acid differences out of 153 amino acid residues. One is a conservative substitution (Dbov141Ebuf), and the other two (Abov19Tbuf and Abov117Dbuf) are nonconservative. These amino acid substitutions are unlikely to cause structural changes because they are located far from the heme binding pocket, as revealed by the 3D structure of buffalo Mb elaborated by homology modelling. Stability analyses show no difference with the bovine Mb for helix E and only minor differences in the stability values for helices A and G. Moreover, autoxidation rates of purified buffalo and bovine myoglobins at 37 °C, pH 7.2, were almost identical, 0.052 ± 0.001 h- 1 and 0.054 ± 0.002 h- 1, respectively, as were their oxygen-binding Kd values, 3.7 ± 0.1 μM and 3.5 ± 0.1 μM, respectively. The percent of MetMb values were almost identical. The results presented here suggest that the darker buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393 ± 0.005 g/100 g of tissue) and consequently MetMb, which are almost twice as high as bovine meat (Mb: 0.209 ± 0.003 g/100 g of tissue). © 2006 Elsevier Inc. All rights reserved

Inducible antiviral activity and rapid production of the Ribosome-Inactivating Protein I from Phytolacca heterotepala in tobacco

We studied the in vitro and in planta antiviral activity of the PhRIP I, a type 1 Ribosome-Inactivating Protein originally purified from leaves of the Phytolacca heterotepala. This protein inhibited protein translation in a cell-free assay and limited the local lesion formation from PVX infection on tobacco leaves. We used a transient expression system based on leaf infiltration with recombinant Agrobacteria to show that tobacco can produce a correctly processed PhRIP I enzyme that retains its antiviral activity. Hence, it is possible to rapidly yield in plants a type 1 RIP by means of this transient expression system. To analyse the possible increase of virus resistance in plants, Nicotiana tabacum lines that were transformed with the PhRIP I coding sequence under the control of the wound-inducible PGIP promoter were challenged by PVX. A significantly lower number of viral lesions compared to untransformed plants was observed only after the induction of the transgene, indicating that the controlled gene expression of an antiviral protein can increase virus resistance

Pioppino mushroom in southern Italy: an undervalued source of nutrients and bioactive compounds

BACKGROUNDAgrocybe aegerita (V. Brig.) Singer, commonly known as Pioppino, is a popular edible mushroom, known in the Campania Region (Italy). Despite its habitual consumption, little nutritional and biochemical information is available. Thus, nutritional values, anti-radical properties and chemical composition of the wild Pioppino were compared to those of the cultivated Agaricus bisporus (J.E. Lange) Imbach (known as Champignon), equally analysed.RESULTSMacronutrient components (proteins, carbohydrates and lipids), free and protein amino acids and fatty acid content of poplar mushroom were achieved. Total phenol content of a defatted Pioppino alcoholic extract (PM) was determined, whereas DPPH and ABTS methods were applied to determine the radical scavenging capabilities of the extract. Ferricyanide and ORAC-fluorescein methods were also performed. Finally, LC-HRMS was used to identify and quantify the main metabolites in the extract. PM was mainly constituted of disaccharides, hexitol derivatives and malic acid. Coumaric acid isomers and C6C1 compounds were also detected.CONCLUSIONAll data revealed that wild Pioppino is an excellent functional food, by far exceeding that of the Champignon. Therefore, these data are useful to promote the consumption of this mushroom encouraging thus its biological cultivation, due to wild availability is strongly compromised by the extensive use of fungicides. (c) 2017 Society of Chemical Industr

Binding of a type 1 RIP and of its chimeric variant to phospholipid bilayers: evidence for a link between cytotoxicity and protein/membrane interactions

Ribosome-inactivating proteins (RIPs) are enzymes, almost all identified in plants, able to kill cells by depurination of rRNAs. Recently, in order to improve resistance to proteolysis of a type 1 RIP (PD-L4), we produced a recombinant chimera combining it with a wheat protease inhibitor (WSCI). Resulting chimeric construct, named PD-L4UWSCI, in addition to present the functions of the two domains, shows also an enhanced cytotoxic action on murine cancer cells when compared to PD-L4. Since different ways of interaction of proteins with membranes imply different resulting effects on cells, in this study we investigate conformational stability of PD-L4 and PD-L4UWSCI and their interaction with membrane models (liposomes). Circular dichroism analysis and differential scanning calorimetry measurements indicate that PD-L4 and PD-L4UWSCI present high and similar conformational stability, whereas analysis of their binding to liposomes, obtained by isothermal titration calorimetry and differential scanning calorimetry, clearly indicate that chimera is able to interact with biomembranes more effectively. Overall, our data point out that WSCI domain, probably because of its flexibility in solution, enhances the chimeric protein interaction with membrane lipid surfaces without however destabilizing the overall protein structure. Analysis of interactions between RIPs or RIP based conjugates and lipid surfaces could provide novel insights in the search of more effective selective membrane therapeutics

Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture

Structural and functional characterization of the cytotoxic protein ledodin, an atypical ribosome‐inactivating protein from shiitake mushroom (Lentinula edodes)

Producción CientíficaWe have purified ledodin, a cytotoxic 22-kDa protein from shiitake mushroom (Lentinula edodes) consisting of a 197 amino acid chain. Ledodin possessed N-glycosylase activity on the sarcin-ricin loop of mammalian 28S rRNA and inhibited protein synthesis. However, it was not active against insect, fungal and bacterial ribosomes. In vitro and in silico studies suggested that ledodin exhibits a catalytic mechanism like that of DNA glycosylases and plant ribosome-inactivating proteins. However, the sequence and structure of ledodin was not related to any protein of known function, although ledodin-homologous sequences were found in the genome of several species of fungi, some edible, belonging to different orders of the class Agaricomycetes. Therefore, ledodin could be the first of a new family of enzymes widely distributed among this class of basidiomycetes. The interest of these proteins lies both, in the fact that they can be a toxic agent of some edible mushrooms and in their application in medicine and biotechnology.Junta de Castilla y León (Consejería de Sanidad - grants BIO39/VA39/14 and BIO/VA17/15)Junta de Castilla y León (Consejería de Educación - grant VA033G19)NUTRABEST PON I&C 2014–2020 Prog.n. F/200050/01-03/X4

Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane- proximal external region (MPER) of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization: first, they bind and pre-concentrate at the viral membrane through their long, hydrophobic CDRH3 loops and second, they form a high-affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol- conjugated antibodies bind to lipid-raft domains on the membrane and because of this enrichment, they show increased antiviral potency. In particular we find that cholesterol conjugation: (i) rescues the antiviral activity of CDRH3- mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane binding HIV antibody D5 10-100 fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears a general strategy to boost potency of antiviral antibodies and, since membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies

Free amino acid profile of Bubalus bubalis L. meat from the Campania region

ABSTRACT In this study, we determined the amount of carnosine and anserine in water buffalo meat without hanging treatment and the free amino acid profile by using amino acid analyser with post-column ninhydrin derivatization procedure. The main free amino acids present in samples were glutamic acid (>60 mg/100 g), followed by alanine, glycine, and arginine. Other protein amino acids were detected in minor amounts (less than 2 mg/100 g). Among the non-protein amine-containing compounds, taurine and urea were the most abundant. The analysis showed that 50% of the total free amino acids was represented by dipeptides carnosine (average ~130.3 mg/100 g) and anserine (average ~17.9 mg/100 g). Thus, this study for the first time reports the free amino acids profile of water buffalo meat and the content of carnosine and anserine, potentially involved in the darkening meat process and their ratio, that could be used to estimate the water buffalo meat portion in mixed meat products

A new age for biomedical applications of Ribosome Inactivating Proteins (RIPs): from bioconjugate to nanoconstructs.

Ribosome-inactivating proteins (RIPs) are enzymes (3.2.2.22) that possess N-glycosilase activity that irreversibly inhibits protein synthesis. RIPs have been found in plants, fungi, algae, and bacteria; their biological role is still under investigation, even if it has been recognized their role in plant defence against predators and viruses. Nevertheless, several studies on these toxins have been performed to evaluate their applicability in the biomedical field making RIPs selectively toxic towards target cells. Indeed, these molecules are extensively used to produce chimeric biomolecules, such as immunotoxins or protein/peptides conjugates. However, to date, clinical use of most of these bioconiujates has been limited by toxicity and immunogenicity. More recently, material sciences have provided a wide range of nanomaterials to be used as excellent vehicles for toxin-delivery, since they are characterized by improved stability, solubility, and in vivo pharmacokinetics. This review discusses progresses in the development of RIPs bioconjugates, with particular attention to the recent use of nanomaterials, whose appropriate design opens up a broad range of different possibilities to the use of RIPs in novel therapeutic approaches in human diseases