Chiara Giangrande

Characterization of laccase isoforms produced by Pleurotus ostreatus in solid state fermentation of sugarcane bagasse

Laccases are oxidative enzymes linked to biological degradation of lignin. The aim of this work was to evaluate the effect of inducers and different concentrations of nitrogen on production level of total laccase activity and pattern of laccase isoforms, produced in solid state fermentation of sugarcane bagasse by a selected strain of Pleurotus ostreatus. The addition of yeast extract 5 g/L, copper sulfate 150 μM and ferulic acid 2 mM provided highest enzymatic activity (167 U/g) and zymograms indicated the presence of six laccase isoforms (POXA1b, POXA3, POXC and three other isoforms). Results of protein identification by mass spectrometry confirmed the presence of POXC and POXA3 as the main isoenzymes, and also identified a glyoxal oxidase and three galactose oxidases. The fact that the isoenzyme POXA1b was not identified in the analyzed samples can be possibly explained by its sensitivity to protease degradation.

Bacteriophage-Resistant Staphylococcus aureus Mutant Confers Broad Immunity against Staphylococcal Infection in Mice

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the MSa phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-α, IFN-γ and Il-1β genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 µl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 µg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost.

BackgroundThe use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes.ResultsDifferent bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis–Menten kinetics with a KM of 9.13 mg/ml and a vmax of 3469 μM min-1. The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C.ConclusionsIn this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion.

Technical advances in proteomics mass spectrometry: identification of post-translational modifications

Abstract The importance of post-translational modifications (PTMs) of proteins has become evident in the proteomic era as it plays a critical role in modulating cellular function, and can vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are substoichiometric, transient and reversible. Moreover, the amount of post-translationally modified proteins is generally very small when compared to their unmodified counterparts. Existing methodologies for identification of PTMs essentially relies on enrichment procedure to selectively increase the amount of modified peptides. These procedures need to be integrated with sophisticated mass spectrometric methods to enable the identifications of PTMs. Although the strategies developed so far are not optimal, a number of examples will be given where the combination of innovative separation methods along with advanced mass spectrometric analyses provide positive results. These experiences are leading the way for the next generation of proteomic approaches for identification of a wide range of PTMs. Clin Chem Lab Med 2009;47:647–65.

Innate immunity probed by lipopolysaccharides affinity strategy and proteomics

Lipopolysaccharides (LPSs) are ubiquitous and vital components of the cell surface of Gram-negative bacteria that have been shown to play a relevant role in the induction of the immune-system response. In animal and plant cells, innate immune defenses toward microorganisms are triggered by the perception of pathogen associated molecular patterns. These are conserved and generally indispensable microbial structures such as LPSs that are fundamental in the Gram-negative immunity recognition. This paper reports the development of an integrated strategy based on lipopolysaccharide affinity methodology that represents a new starting point to elucidate the molecular mechanisms elicited by bacterial LPS and involved in the different steps of innate immunity response. Biotin-tagged LPS was immobilized on streptavidin column and used as a bait in an affinity capture procedure to identify protein partners from human serum specifically interacting with this effector. The complex proteins/lipopolysaccharide was isolated and the protein partners were fractionated by gel electrophoresis and identified by mass spectrometry. This procedure proved to be very effective in specifically binding proteins functionally correlated with the biological role of LPS. Proteins specifically bound to LPS essentially gathered within two functional groups, regulation of the complement system (factor H, C4b, C4BP, and alpha 2 macroglobulin) and inhibition of LPS-induced inflammation (HRG and Apolipoproteins). The reported strategy might have important applications in the elucidation of biological mechanisms involved in the LPSs-mediated molecular recognition and anti-infection responses.

Glycoproteome Study in Myocardial Lesions Serum by Integrated Mass Spectrometry Approach: Preliminary Insights

Bottom up proteomics requires efficient and selective pre-fractionation procedures to simplify the analysis of the enormous number of peptides resulting from the hydrolysis of a cellular extract enabling the detection, identification and the structural characterization of the post-translational modifications. Glycosylation, a well-known post-translational modification, plays a key role in the enormous complexity, and heterogeneity of the human blood serum proteome. Thereby, characterization of glycosylation from serum is a challenging task, even for the existing sophisticated analytical methodologies. Here we report a glycoproteomics study on the identification of even low abundant glycoproteins, including the localization of N-glycosylation sites and the glycan profiling in human sera from healthy and myocarditis affected donors. The strategy is simply based on proteolytic digestion of total serum proteins followed by a single enrichment step of glycopeptides on ConA lectin affinity chromatography. Glycopeptides were then deglycosylated by PNGaseF treatment and nano-liquid chromatography-electrospray ionization tandem mass spectrometry analyses of the free peptides provided the basis for both identification of the individual proteins and elucidation of their modification sites. Moreover, glycan profilings could be obtained by matrix-assisted laser desorption/ionization mass spectrometry analysis of the released oligosaccharides. Our data led to the identification of 68 different glycosylation sites within 49 different proteins. Moreover, the analyses carried out on glycans represent the first picture of a glycosylation pattern in myocardial lesions. As a whole, several differences in the glycosylation patterns from different sera were observed, thus indicating glycan profiling as a possible tool to discriminate among different diseases.

Proteomic analysis of cells exposed to prefibrillar aggregates of HypF-N.

Several human diseases are associated with the deposition of stable ordered protein aggregates known as amyloid fibrils. In addition, a large wealth of data shows that proteins not involved in amyloidoses, are able to form, in vitro, amyloid-like prefibrillar and fibrillar assemblies indistinguishable from those grown from proteins associated with disease. Previous studies showed that early prefibrillar aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) are cytotoxic, inducing early mitochondria membrane depolarization, activation of caspase 9 and eventually cell death. To gain knowledge on the molecular basis of HypF-N aggregate cytotoxicity, we performed a differential proteomic analysis of NIH-3T3 cells exposed to HypF-N prefibrillar aggregates in comparison with control cells. Two-dimensional gel electrophoresis followed by protein identification by MALDI-TOF MS, allowed us to identify 21 proteins differentially expressed. The changes of the expression level of proteins involved in stress response (Hsp60 and 78 kDa glucose-regulated protein) and in signal transduction (Focal adhesion kinase1) appear particularly interesting as possible determinants of the cell fate. The levels of some of the differently expressed proteins were modified also in similar studies carried out on cells exposed to Abeta or alpha-synuclein aggregates, supporting the existence of shared features of amyloid cytotoxicity.

The multifunctional polydnavirus TnBVANK1 protein: impact on host apoptotic pathway

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.

Apolipoprotein A-I amyloidogenic variant L174S, expressed and isolated from stably transfected mammalian cells, is associated with fatty acids.

Sixteen variants of apolipoprotein A-I (ApoA-I) are associated with hereditary systemic amyloidoses, characterized by amyloid deposition in peripheral organs of patients. As these are heterozygous for the amyloidogenic variants, their isolation from plasma is impracticable and recombinant expression systems are needed. Here we report the expression of recombinant ApoA-I amyloidogenic variant Leu174 with Ser (L174S) in stably transfected Chinese hamster ovary-K1 cells. ApoA-I variant L174S was found to be efficiently secreted in the culture medium, from which it was isolated following a one-step purification procedure. Mass spectrometry analyses allowed the qualitative and quantitative definition of the amyloidogenic variant lipid content, which was found to consist of two saturated and two monounsaturated fatty acids. Interestingly, the same lipid species were found to be associated with the wild-type ApoA-I, expressed and isolated using the same cell system, with lower values of the lipid to protein molar ratios with respect to the amyloidogenic variant. A possible role of fatty acids in trafficking and secretion of apolipoproteins may be hypothesized.

A Proteomic Approach to Investigate Myocarditis

Myocarditis is an inflammatory disease of the cardiac muscle which might be related to viral (mainly parvovirus B19 and many others), protozoan (Borrellia burgdoferi, Trypanosoma cruzi, Toxoplasma gondi), bacterial (Brucella, Corynebacterium diphtheriae, Gonococcus, Haemophilus influenzae, Actinomyces, Tropheryma whipplei, Vibrio cholerae, Borrelia burgdorferi, Leptospira, Rickettsia), fungal (Aspergillus) and other non viral pathogens (Rezkalla SH et al., 2010, Blauwet LA et al., 2010, Cihakova D et al., 2010) infections; It has been reported, however, that this kind of inflammation might be caused by an hypersensitivity response to drugs (Kuhl U et al., 2009). The final effect in each case is represented by myocardial infiltration of immunocompetent cells following any kind of cardiac injury. Myocarditis presents with many symptoms, from chest pain that spontaneously resolves without treatment to cardiogenic shock and sudden death (Kuhl U et al., 2010, Taylor CL et al 2010). The major long-term consequence is dilated cardiomyopathy with chronic heart failure(Lv H et al. 2011, Stensaeth KH et al 2011). Nowadays, diagnostic tools available for this disease are mainly related to general investigations (such as electrocardiography) and analysis of the most abundant serum proteins, whose alteration is related to cardiac pathology, even if it’s not specifically connected to myocarditis itself. Here we propose preliminary speculations on the serum proteins profiling (both in the expression level and in the characterization of post translational modifications) and on free peptides identification in myocardytis affected patients (compared to healthy individual), that could be helpful in finding specific markers for this pathology. As many other inflammatory events this disease involves in fact different kinds of biological macromolecules, here we focus in particular on proteins. More than 50% of total protein content in a cell is post translationally modified. The pattern of Post Translational Modifications (PTMs) on proteins constitute a molecular code that dictates protein conformation, cellular location, macromolecular interactions and activities, depending on cell type, tissue and environmental conditions (Diernfellner AC et al., 2011, Savidge TC,