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Interaction between alpha-lactalbumin and lipids: conformational features and effects on protein aggregation
No full textThe topic of this PhD project concerns aspects of the general problem of the protein folding and unfolding, in line with the research conducted in the laboratory of Protein Chemistry at CRIBI, where the activities were mostly performed. The mechanism of acquisition of the three-dimensional structure of proteins (folding) is an important biological event. It is generally a multi-stage process involving the formation of intermediates, which are partly folded states having some structural features of the native protein, but not the final side chains interactions that allow the protein to exert its specific function. The failure in achieving the correct folding (misfolding) may cause protein aggregation. In fact, partly folded proteins can easily self-assembly in regular insoluble aggregates (amyloid fibrils), which are associated with serious diseases, the so called amyloidosis (Chiti & Dobson, 2006).
In particular, my PhD research was focused on ?-lactalbumin (?-LA), a milk metalloprotein, widely used as a model to study protein folding. A new wave of interest in this protein appeared in the last decade after the discovery of a variant of the protein that, besides its physiological role in lactation, is able to induce apoptosis of tumor cells (Svensson et al., 1999). The cytotoxic activity resides in the formation of a complex with a milk fatty acid, oleic acid (OA), named HAMLET (Human Alpha-lactalbumin Made LEthal to Tumour cells) (Svensson et al., 2000). The cell events caused by HAMLET were extensively studied (Kohler et al., 2001; Duringer et al., 2003). On the contrary, the mechanism and the nature of the interaction between ?-LA and OA and the physico-chemical properties of the complex are not completely understood yet. The preparation of the complex is a controversial aspect. Indeed, despite the effective association of OA with the protein in solution (Polverino de Laureto et al., 2002), the resulting ?-LA/OA complex was believed to possess lower antitumor activity than HAMLET, obtained using a chromatographic procedure (Svensson et al., 2000). Other debated matters focus on the protein/fatty acid stoichiometry and the monomeric/oligomeric state of ?-LA in the complex. Moreover, a systematic investigation of the effect of the protein on the phase behavior of OA is still lacking.
The aim of this PhD Thesis was the investigation of the OA binding propensity and cytotoxic activity of three proteolytic derivatives of bovine ?-LA, obtained by limited proteolysis with pepsin at acidic pH. The use of proteolytic dissection of a protein has been widely employed to study the folding of several proteins (Wetlaufer, 1981; Gegg et al., 1997; Llinás & Marqusee, 1998). The characterization of protein fragments containing specific structural elements of the intact protein (?-helix, ?-sheet) and able of autonomous folding was successfully used to unravel folding features of ?-LA (Polverino de Laureto et al., 1999; 2001). Indeed, fragments corresponding to structural domains in multidomain proteins were shown in some cases to acquire in solution a native-like conformation (Fontana et al., 2004).
The ?-LA fragments investigated are: species 1–40/53–123, lacking the ?-subdomain of the native protein; 1–40/104–123, given by the N-terminal fragment 1–40 covalently linked by two disulfide bridges to the C-terminal fragment 104–123 and containing three of the four ?-LA helices; 53–103, containing the C-helix and the calcium binding loop of intact ?-LA (Polverino de Laureto et al., 1999; 2001). The conformational differences between the three fragments were used as the rationale for a study of their efficacy to bind OA, thus giving an insight into the mechanism of this binding. This study has also some physiological relevance: pepsin is an enzyme of the stomach and therefore these species might be generated in vivo, under the same conditions of low pH in which the complex HAMLET was hypothesized to form, thus contributing to the apoptotic activity of the complex formed by the intact protein.
The complexes between the three fragments and OA were prepared first by the chromatographic procedure described for HAMLET, then by directly mixing in solution the two components. The conformational properties of the complexes were characterized by circular dichroism, showing that the complexes prepared by both procedures display similar conformations and acquire ?-helix. The effect of calcium on the conformation of the complexes was then investigated by circular dichroism. Fluorescence spectroscopy was used to study the involvement of Trp residues in the interaction with OA. Moreover, the ability of the complexes to induce apoptosis-like cell death was evaluated, in line with the cytotoxic activity displayed by HAMLET. In order to verify the physical state of OA involved in the interaction with ?-LA, the OA aggregation behavior was investigated using different techniques, such as transmission electron microscopy (TEM), titration with a fluorescent dye and turbidimetric analyses, and an effect of solubilization of OA was observed. A manuscript with these results will be soon submitted to an international journal and is herewith attached.
In the second part of the PhD, the research was focused on the aggregation propensity of ?-LA and its three proteolytic fragments. ?-LA is able to form amyloid fibrils in vitro, even if not disease-related. ?-LA fibrils are formed when the structure of the protein is partially destabilized, e.g. at low pH, upon reduction of three disulphide bridges at neutral pH (Goers et al., 2002), or after proteolytic cleavage (Polverino de Laureto et al., 2005). Lipids may act as an effective catalyst of fibrillogenesis, providing a generic environment where protein molecules adopt conformation and orientation promoting their assembly into fibrillar structures (Thirumalai et al., 2003; Stefani, 2004; Sparr et al., 2004; Zhao et al., 2004). In particular, since fatty acid–protein interactions are known to modulate the process of fibrillation (Kim & Takahashi 2006), aggregation processes of ?-LA and its fragments were investigated to elucidate whether and how OA might affect fibrils formation. Firstly, aggregation was followed at pH 2.0, as a comparison with the known fibrillogenic behavior of intact ?-LA. Secondly, the aggregation was investigated at pH 7.4, since in this physiological condition the conformational changes induced by OA on the fragments structure were studied and their cytotoxic activity was analyzed, in the first part of the Thesis. The formation of fibrils was followed by thioflavin T (ThT) fluorescence assay, circular dichroism and TEM. All three fragments were able to produce amyloid fibrils at pH 2.0, similarly to ?-LA. In the used range of protein concentration and protein/lipid ratio, OA seems to accelerate the rate of fibrillation for ?-LA and the three fragments at pH 2.0. At pH 7.4, ?-LA is not able to form fibrils both in the absence and in the presence of OA. Also the three fragments did not form amyloid fibrils at neutral pH, while in the presence of OA they underwent a conformational change to ?-sheet structure, were able to bind ThT and to form aggregates with the typical amyloid morphology.
During the PhD course, I also collaborated to an ongoing project of the laboratory at CRIBI, focused on the characterization of oligomeric species on the aggregation pathway of human lysozyme, which belongs to the so called “lysozyme/lactalbumin superfamily”. Soluble oligomers of lysozyme were produced at low pH and high temperature, and then analyzed by a range of techniques including binding to fluorescent probes, Fourier-transform infrared (FTIR) spectroscopy and limited proteolysis. Oligomers have solvent-exposed hydrophobic patches, and FTIR spectra are indicative of highly misfolded species. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis than both the monomeric protein and the mature fibrils, indicating their lack of organized structure. This study showed that the soluble lysozyme oligomers are structurally flexible species present at low concentration during the initial phases of aggregation. The results of this study were accepted for publication in an international journal and the ‘in press’ manuscript is attached to the Thesis.
During the third year of PhD, I spent a six months period at the University of Cambridge (UK), in the Cambridge Centre for Proteomics, under the supervision of Dr. Kathryn Lilley. The aim of this period was to learn proteomic methodologies for large scale identification of proteins, improving my background in mass spectrometry. I was involved in an ongoing project of the laboratory, focused on a parallel affinity purification method coupled to mass spectrometry for identifying proteins and their binding partners in Drosophila melanogaster embryos (Veraksa et al., 2005). Triple tagged proteins were generated (Spradling et al., 1999) and isolated from a variety of tissues in embryos. Affinity purification using two tags in parallel allowed the isolation of intact native protein complexes. The high sensitivity and high mass accuracy of the hybrid LTQ-Orbitrap instrument ensured maximal coverage of low abundance complex components, generating high confident data with low false discovery rates. The software ProteinCenterTM (Proxeon) was utilized for the visualization and the statistical comparison of the datasets. The data were compared with datasets published in public databases, which validate the data and increased the certainty of the detected interaction sets. Also novel protein interactions not previously reported were mapped. These high confidence in vivo protein datasets add high confidence data to the currently incomplete D. melanogaster proteome and interactome. Here, the results of five different size proteins from different cellular localizations are reported in detail to show the workflow and the efficiency of the methodology.
Summing up, this PhD Thesis is composed of a major part dealing with the characterization of the interaction of ?-LA and oleic acid and the effects on the protein aggregation, and a minor part dealing with the mass spectrometry analysis of Drosophila protein complexes, besides the publication on the oligomeric species in the aggregation pathway of lysozyme.L’argomento di questa Tesi di Dottorato riguarda in generale aspetti del problema del folding e unfolding proteico, in linea con la tematica di ricerca condotta nel laboratorio di Chimica delle Proteine al CRIBI, dove le attività sono state per la maggior parte svolte. Il meccanismo di acquisizione della struttura tridimensionale di una proteina (folding) è un evento biologico importante. In generale, è un processo multi-stadio che coinvolge la formazione di intermedi, che sono stati parzialmente strutturati contenenti alcune caratteristiche strutturali della proteina nativa, ma non le interazioni finali tra le catene laterali che permettono alla proteina di esercitare la sua funzione specifica. Il fallimento nell’acquisizione del corretto folding (misfolding) può causare aggregazione proteica. Infatti, proteine parzialmente strutturate possono facilmente auto-assemblarsi in aggregati regolari insolubili (fibrille amiloidi), associati a gravi malattie, le cosiddette amiloidosi (Chiti & Dobson, 2006).
In particolare, il mio progetto di Dottorato è focalizzato sull’?-lattalbumina (?-LA), una metallo-proteina del latte, ampiamente utilizzata come modello di studio del folding proteico. Nell’ultimo decennio, questa proteina ha suscitato un nuovo interesse per la scoperta di una variante che, oltre al ruolo fisiologico nella lattazione, è in grado di indurre apoptosi nelle cellule tumorali (Svensson et al., 1999). L’attività citotossica risiede nella formazione di un complesso con un acido grasso del latte, l’acido oleico (OA), denominato HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) (Svensson et al., 2000). Gli eventi cellulari causati da HAMLET sono stati estesamente studiati (Kohler et al., 2001; Duringer et al., 2003). Al contrario, il meccanismo e la natura dell’interazione tra ?-LA e OA e le proprietà fisico-chimiche del complesso non sono ancora completamente chiariti. Infatti, nonostante l’effettiva associazione dell’OA con la proteina in soluzione (Polverino de Laureto et al., 2002), si ritiene che il complesso così ottenuto possieda attività antitumorale minore dell’HAMLET, preparato secondo una procedura cromatografica (Svensson et al., 2000). Altre questioni dibattute riguardano la stechiometria proteina/acido grasso e lo stato monomerico/oligomerico dell’?-LA nel complesso. Inoltre, manca un’indagine sistematica dell’effetto della proteina sul comportamento di fase dell’OA.
Lo scopo di questa Tesi di Dottorato è lo studio della capacità di legare OA ed esprimere attività citotossica di tre derivati proteolitici di ?-LA bovina, ottenuti per proteolisi limitata con pepsina a pH acido. L’uso di un approccio di proteolytic dissection di una proteina è stato largamente impiegato per studiare il folding di molte proteine (Wetlaufer, 1981; Gegg et al., 1997; Llinás & Marqusee, 1998). La caratterizzazione di frammenti proteici, contenenti elementi strutturali specifici della proteina intera (?-elica, ?-sheet) e in grado di assumere struttura in modo autonomo, è stata usata con successo per chiarire caratteristiche strutturali di ?-LA (Polverino de Laureto et al., 1999; 2001). Infatti, frammenti corrispondenti a domini strutturali in proteine multi-dominio hanno mostrato in alcuni casi la capacità di acquisire in soluzione una conformazione simile a quella nativa (Fontana et al., 2004).
I frammenti di ?-LA studiati sono: la specie 1–40/53–123, priva di parte del dominio ? della?proteina nativa; 1–40/104–123, formato dal frammento N-terminale 1-40 legato covalentemente al frammento C-terminale 104-123 mediante due ponti disolfuro e contenente tre delle quattro ?-eliche di ?-LA; 53-103, contenente l’elica C e il sito di legame al calcio (Polverino de Laureto et al., 1999; 2001). Le differenze conformazionali dei tre frammenti sono state utilizzate come razionale per studiare la loro efficacia di legame all’OA, e per approfondire quindi il meccanismo di questo legame. Questo studio ha anche rilevanza fisiologica: la pepsina è un enzima dello stomaco e quindi queste specie potrebbero essere generate in vivo, nelle stesse condizioni di pH acido in cui si è ipotizzata la formazione del complesso HAMLET, contribuendo così all’attività apoptotica del complesso formato dalla proteina intera.
I complessi dei tre frammenti con OA sono stati preparati prima seguendo la procedura cromatografica descritta per l’HAMLET, poi per diretto miscelamento in soluzione dei due componenti. Le proprietà conformazionali dei complessi sono state caratterizzate mediante dicroismo circolare, mostrando che i complessi preparati attraverso entrambe le procedure presentano conformazioni simili e acquisizione di ?-elica. Inoltre, è stato valutato l’effetto del calcio sulla conformazione dei complessi mediante dicroismo circolare. La spettroscopia di fluorescenza è stata utilizzata per analizzare il coinvolgimento dei residui di Trp nell’interazione con OA. Inoltre, è stata studiata la capacità dei complessi di indurre morte cellulare per apoptosi, in linea con l’attività citotossica mostrata dall’HAMLET. Per analizzare lo stato fisico dell’OA coinvolto nell’interazione con l’?-LA, il comportamento di aggregazione di OA è stato studiato con diverse tecniche, quali microscopia elettronica a trasmissione (TEM), titolazione con un colorante fluorescente e analisi turbidimetriche, ed è stato osservato un effetto di solubilizzazione dell’OA. Questi risultati hanno permesso di preparare un articolo che sarà presto spedito ad una rivista internazionale e che è allegato alla Tesi.
Nella seconda parte del Dottorato, la ricerca è stata focalizzata sulla tendenza di ?-LA e dei suoi tre frammenti proteolitici ad aggregare. ?-LA è in grado di formare fibrille amiloidi in vitro, sebbene non sia associata a patologie. Fibrille di ?-LA si producono quando la struttura della proteina è parzialmente destabilizzata, ad esempio a pH acido, per riduzione di tre ponti disolfuro a pH neutro (Goers et al., 2002), o per taglio proteolitico (Polverino de Laureto et al., 2005). I lipidi possono agire come efficaci catalizzatori della fibrillogenesi, creando un ambiente in cui le molecole proteiche adottano una conformazione e un’orientazione che promuove il loro assemblaggio in strutture fibrillari (Thirumalai et al., 2003; Stefani, 2004; Sparr et al., 2004; Zhao et al., 2004). In particolare, poiché è noto che le interazioni proteina-acido grasso modulano il processo di fibrillogenesi (Kim & Takahashi 2006), i processi di aggregazione di ?-LA e dei suoi frammenti sono stati studiati per capire se e come l’OA possa influenzare la formazione di fibrille. In primo luogo, l’aggregazione è stata seguita a pH 2.0, in parallelo con il già noto comportamento fibrillogenico di ?-LA intera. In secondo luogo, l’aggregazione è stata studiata a pH 7.4, poiché in questa condizione fisiologica nella prima parte della Tesi sono stati studiati i cambiamenti conformazionali indotti dall’OA sulla struttura dei frammenti e la loro attività citotossica. La formazione di fibrille è stata seguita mediante saggi di fluorescenza con la tioflavina T (ThT), dicroismo circolare e TEM. Tutti e tre i frammenti sono in grado di produrre fibrille amiloidi a pH 2.0, analogamente all’?-LA. Ai valori di concentrazione proteica e rapporto proteina/lipide utilizzati, OA sembra accelerare la velocità di formazione di fibrille di ?-LA e dei frammenti a pH 2.0. A pH 7.4, ?-LA non forma fibrille amiloidi sia in assenza sia in presenza di OA. Anche i tre frammenti non sono stati in grado di formare fibrille a pH neutro, mentre in presenza di OA hanno mostrato un cambiamento conformazionale verso una struttura ?-sheet, hanno legato ThT e formato aggregati con la tipica morfologia amiloide.
Durante il Dottorato, ho inoltre collaborato ad un progetto in corso nel laboratorio al CRIBI, relativo alla caratterizzazione di specie oligomeriche nel processo di aggregazione del lisozima umano, che appartiene alla cosiddetta “superfamiglia lisozima/lattalbumina”. Oligomeri solubili di lisozima sono stati prodotti a pH acido e alta temperatura, e quindi analizzati con varie tecniche, quali legame a molecole fluorescenti, spettroscopia infrarosso in trasformata di Fourier (FTIR) e proteolisi limitata. Gli oligomeri presentano superfici idrofobiche esposte al solvente, e gli spettri FTIR sono indicativi di specie altamente destrutturate. Inoltre, gli aggregati oligomerici di lisozima si sono rivelati più suscettibili alla proteolisi rispetto sia alla proteina monomerica sia alle fibrille mature, indicando la mancanza di una struttura organizzata. Questo studio ha dimostrato che gli oligomeri solubili di lisozima sono specie strutturalmente flessibili presenti a bassa concentrazione durante le fasi iniziali dell’aggregazione. I risultati di questo studio sono stati accettati per la pubblicazione in una rivista internazionale e il manoscritto in press è allegato alla Tesi.
Durante il terzo anno di Dottorato, ho trascorso un periodo di sei mesi all’Università di Cambridge (UK), presso il Cambridge Centre for Proteomics, sotto la supervisione della Dr.ssa Kathryn Lilley. L’obiettivo di questo periodo è stato di apprendere metodologie di proteomica per l’identificazione di proteine su larga scala, ottimizzando le mie conoscenze di spettrometria di massa. Ho collaborato ad un progetto in corso nel laboratorio, incentrato su un metodo di purificazione di affinità in parallelo accoppiata a spettrometria di massa per identificare complessi proteici in embrioni di Drosophila melanogaster (Veraksa et al., 2005). Proteine con tre tags sono state generate (Spradling et al., 1999) e isolate da vari tessuti embrionali. La purificazione di affinità usando due tags in parallelo ha permesso di isolare complessi proteici nativi intatti. L’alta sensibilità e l’alta accuratezza di massa dello strumento ibrido LTQ-Orbitrap ha assicurato la massima copertura di componenti di complessi poco abbondanti, generando dati ad alta confidenza con basse false discovery rates. È stato utilizzato il software ProteinCenterTM (Proxeon) per la visualizzazione e l’analisi statistica dei set di dati. I risultati sono stati confrontati con dati presenti in database pubblici, confermandone la validità e aumentando l’autenticità delle interazioni individuate. Sono state inoltre mappate nuove interazioni proteiche non riportate in precedenza. Questi set di dati in vivo aggiungono alta confidenza al proteoma e ‘interattoma’ di D. melanogaster attualmente incompleto. In questa Tesi sono riportati in dettaglio i risultati di cinque proteine di diverse dimensioni e localizzazione cellulare, per presentare la procedura e l’efficienza della metodologia.
In sintesi, questa Tesi di Dottorato è composta da una parte principale che riguarda la caratterizzazione dell’interazione tra ?-LA e acido oleico e gli effetti sull’aggregazione proteica, e una parte minore relativa all’analisi di spettrometria di massa di complessi proteici in Drosophila, oltre alla pubblicazione sulle specie oligomeriche studiate nel processo di aggregazione del lisozima
Caratterizzazione di isoforme di chitinasi da succo d’uva
No full textLe chitinasi dell’uva sono considerate tra le principali responsabili della instabilità proteica nei vini bianchi. In questo lavoro le proteine del mosto (cv. Manzoni bianco) sono state frazionate utilizzando una cromatografia a scambio anionico seguita da una cromatografia a interazione idrofobica. In base all’analisi elettroforetica (SDS-PAGE) e alla
rilevazione dell’attività enzimatica su gel sono state identificate sei diverse bande proteiche con attività chitinolitica. Queste bande sono state sottoposte a spettrometria di massa (MALDI-TOF/TOF MS), che ha identificato tutte le frazioni come Chitinasi di classe IV di Vitis vinifera. Tali isoforme, che mostrano differenti caratteristiche elettroforetiche e cromatografiche, hanno probabilmente un diverso comportamento nel determinare gli intorbidamenti del vino. Quindi il loro studio è importante per meglio capire il coinvolgimento di ogni singola componente nella formazione di torbidità e per sviluppare tecniche enologiche specifiche mirate alla prevenzione della casse proteica dei vini bianchi
Analysis of Commercial Wines by LC-MS/MS Reveals the Presence of Residual Milk and Egg White Allergens
No full textFining agents of animal origin are commonly used in winemaking process to clarify and stabilize wines and to optimize their organoleptic properties thanks to the removal of phenolic compounds that cause bitterness and astringency. Considering the potential allergenicity of proteins used as fining agents, the need of label declaration for wines treated with such compounds is compelling. However, the difficulties in detecting proteins in a wine matrix by immunological assays have given rise to a search for alternative methods that can overcome the limitations of classical approaches. Mass spectrometry (MS) has recently emerged as a powerful and sensitive technique to detect residual fining proteins in wines. In this study we show that a simple and straightforward mass spectrometric approach can be used to reliably detect egg and milk allergens in commercial bottled wines. We tested 25 different wines by liquid chromatography coupled with tandem MS (LC-MS/MS) and found proteins of animal origin in 8 samples, so demonstrating that the proposed method allows to monitor the presence of potential allergenic proteins in fined commercial wine
Proteomic iTRAQ analysis of the bacterial plant symbiont Rhizobium leguminosarum induced by its host legume flavonoid signal
No full textCharacterization of chitinase isoforms from grape juice
Full text linkGrape chitinases are recognized as being mainly responsible for protein haze formation in white wines. Vitis vinifera L. cv. Manzoni Bianco grape juice proteins were fractionated
using anion exchange and hydrophobic interaction chromatographies. According to SDSPAGE and zymography, six protein bands with chitinolytic activity were subjected to
mass spectrometry (MALDI-TOF/TOF MS), which assigned all the bands to Vitis vinifera class IV chitinases. These grape chitinase isoforms showing different electrophoretic and
chromatographic behaviours are likely to be also distinct in their functionality in wine. This could be relevant to understand the involvement of single chitinase components in
wine hazing and to develop specific winemaking techniques for their removal from wine
ITRAQ-based proteomic analysis reveals several novel flavonoid-inducible genes in Rhizobium leguminosarum bv. viciae
No full textCalcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis
No full textRhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant-microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca2+ involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca2+ chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca2+ influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed
Biochemical and quantitative proteomics investigations in Arabidopsis ggt1mutant leaves reveal a role for the gamma-glutamyl cycle in plant's adaptation to environment
No full textThe existence of a gamma-glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma-glutamyl transferase (GGT)-assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild-type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low-molecular-weight range compared to WT. The combined iTRAQ and LC-MS/MS based quantitative proteomics approach identified 70 proteins (out of 1,013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH-associated genes showed that disruption of gamma-glutamyl cycle in ggt1 knockout-leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma-glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism and photosynthesis, with strong implications for plant's adaptation to environment
Circulating myeloid calcifying cells have anti-angiogenic activity via thrombospondin-1 overexpression
Full text linkMyeloid calcifying cells (MCCs) represent a subpopulation of human monocytes with procalcific potential and are characterized by coexpression of osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, an in-depth proteomic investigation of MCCs based on fluorescence-activated cell sorting, protein extraction and digestion, isobaric tag for relative and absolute quantitation labeling, fractionation, and analysis on matrix-assisted laser desorption/ionization-time of flight/time of flight and LTQ Orbitrap mass spectrometers identified and quantified more than 700 proteins and revealed pathways activated in OC+BAP+ MCCs compared with those in OC-BAP- cells. Among proteins referable to angiogenesis, the thrombospondin-1 pathway was markedly up-regulated in MCCs vs. control cells. Up-regulation of the thrombospondin-1 pathway was confirmed by a genome-wide transcriptional analysis. Using in vitro and in vivo angiogenesis assays, we found that freshly isolated MCCs and cultured MCCs display an antiangiogenic function by means of both paracrine activity (conditioned medium) and altered spatial localization in cocultures with endothelial cells. Thrombospondin-1 inhibition by antibody-mediated neutralization or gene knockdown restored the angiogenic activity of OC+BAP+ MCCs toward normal values and abolished the antiangiogenic effects of MCC conditioned medium. These data indicate that circulating MCCs exert antiangiogenic activity by virtue of their overexpression of thrombospondin-1. The study highlights the successful identification and validation of a pathogenic pathway by a gold standard proteomic/transcriptomic analysis of blood cell
Mass spectrometry detection of egg proteins in red wines treated with egg white
No full textThe need of label declaration for egg proteins is temporarily suspended when they are used as a processing
aid in winemaking, because of a lack of scientific data concerning their actual permanence as
residual proteins in fined wines.
The possibility to detect residual egg proteins in red wines treated with a commercial egg white
preparation was studied. By using an immunochemical method residual egg proteins were detected in
the experimental red wines only for doses of fining agent of 50 g/hL or higher, whereas no residual
proteins were detected by this system in a commercial red wine.
A simple method based on the recovery and identification of the wine fining proteins by liquid
chromatography coupled with tandem MS (LC-MS/MS) in a gel-free approach was developed. This
allowed the detection of egg proteins in red wines fined down to 5 g/hL of commercial egg white
preparation and also in the commercial red wine. These results indicate that the analytical approach here
suggested is superior to the immunochemical methods in detecting egg proteins in wines. Therefore
hypersensitivity reactions after consumption of wines treated with egg proteins can be a real risk for eggallergic
people
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