Conformational studies of RGDechi peptide by natural‐abundance NMR spectroscopy

Integrins are heterodimeric cell‐surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin‐inhibitor drugs have been developed for integrin‐based medical applications, the structural determinants modulating integrin‐ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C‐terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for α_vβ3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of α_vβ₃ by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of α_vβ₃ integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance ¹⁵N and ¹³C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.     

Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.     

Metabonomics and population studies: age-related amino acids excretion and inferring networks through the study of urine samples in two Italian isolated populations

The study of two different Italian isolated populations was combined with a metabonomic approach to better understand tubular handling of amino acids. Levels of amino acids and metabolites have been analyzed by Nucleic Magnetic Resonance and expressed as ratio vs urinary creatinine concentration (mmol/mol). For most of the amino acids there is an age-related U shape pattern of excretion, with the peaks during childhood and old age, and a significant reduction in the adult age. Hierarchical cluster analysis has clearly identified three groups clustering the same amino acids: His, Thr and Ala (group one); Gly and Phe (group two) and a third larger one. Results have been further confirmed by factor and regression analysis, and used to confirm and, in some cases, infer new amino acids networks. As a matter of facts, the identification of strong evidences for clustering of urine excretion of several neutral amino acids suggests the predominant impact of relevant and common transporters     

The molecular characterization of a novel GH38 α-mannosidase from the crenarchaeon Sulfolobus solfataricus revealed its ability in de-mannosylating glycoproteins

α-Mannosidases, important enzymes in the N-glycan processing and degradation in Eukaryotes, are frequently found in the genome of Bacteria and Archaea in which their function is still largely unknown. The α-mannosidase from the hyperthermophilic Crenarchaeon Sulfolobus solfataricus has been identified and purified from cellular extracts and its gene has been cloned and expressed in Escherichia coli. The gene, belonging to retaining GH38 mannosidases of the carbohydrate active enzyme classification, is abundantly expressed in this Archaeon. The purified α-mannosidase activity depends on a single Zn²⁺ ion per subunit is inhibited by swainsonine with an IC₅₀ of 0.2 mM. The molecular characterization of the native and recombinant enzyme, named Ssα-man, showed that it is highly specific for α-mannosides and α(1,2), α(1,3), and α(1,6)-d-mannobioses. In addition, the enzyme is able to demannosylate Man₃GlcNAc₂ and Man₇GlcNAc₂ oligosaccharides commonly found in N-glycosylated proteins. More interestingly, Ssα-man removes mannose residues from the glycosidic moiety of the bovine pancreatic ribonuclease B, suggesting that it could process mannosylated proteins also in vivo. This is the first evidence that archaeal glycosidases are involved in the direct modification of glycoproteins.     

Phylogenetic divergence of fish and mammalian metallothionein: relationships with structural diversification and organismal temperature

Metallothioneins (MTs) are nonenzymatic low molecular weight proteins, that play an important role in the homeostasis and detoxification of heavy metals in a large variety of organisms. These proteins are endowed with striking features, including an unusual amino acid composition characterized by the presence of 20 cysteines out of a total of 60 residues and absence of secondary structure elements. It is generally accepted that MTs underwent few modifications during evolution because of these structural and functional constraints. Such a conclusion is founded on the studies carried out mostly on MTs of mammalian origin. For such a reason, we have decided to compare the MTs of homeothermic and poikilothermic organisms, such as mammals and fish, with the specific aim to put in relation phylogenetic divergence and structural/functional adaptation to temperature. We have included in our analysis also Antarctic Notothenioids, a fish group characterized by genetic isolation and cold-adaptation to a particular harsh environment. We have determined the average hydropathic index of ancestral MT sequences and used them to infer the temperatures of the environment housing the hypothetical ancestor organisms. Finally, we have derived phylogenetic relationships of MT molecules from the pairwise comparison of their three-dimensional structures.     

Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.     

A colorimetric assay for determination of cell viability in algal cultures

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.