Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

BACKGROUND: Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca(2+) concentration, which is a fundamental prerequisite for any Ca(2+)-based signalling system, is accomplished by complex mechanisms including Ca(2+) binding proteins acting as Ca(2+) buffers. In this work we investigated the occurrence of Ca(2+) binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus. RESULTS: A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca(2+)-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca(2+)-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca(2+) binding ability of the M. loti protein was demonstrated by (45)Ca(2+)-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca(2+) adducts. CONCLUSIONS: The present data indicate that ferredoxin II is a major Ca(2+) binding protein in M. loti that may participate in Ca(2+) homeostasis and suggest an evolutionarily ancient origin for protein-based Ca(2+) regulatory systems. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3 epsilon

RSL3, a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating GPx4. Here we report the purification of the protein indispensable for GPx4 inactivation by RSL3. MS analysis reveals 14-3-3 isoforms as candidates and recombinant human 14-3-3epsilon confirms the identification. The function of 14-3-3\uf065 is redox-regulated. Moreover, overexpression and silencing of the gene coding for 14-3-3\uf065 consistently control the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein, operating in cell signalling, further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives

Insight into the mechanism of ferroptosis inhibition by ferrostatin-1

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides

Setting up of an innovative procedure for redox proteomics: and its application for definition of the redox status of cells with high metastatic potential

BACKGROUND: The cysteine (Cys) proteome includes 214.000 Cys with thiol and other forms. Of these, only a relatively small subset functions in cell signalling. Redox-active Cys are more susceptible to oxidation, and their oxidized form is more susceptible to reduction. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes that are collectively known as redox proteomics. Thus, it is of interest to be able to identify both the proteins and the cysteine residues affected, and to quantify the extent of the modification involved.The quantification of differences between two or more physiological states of a biological system is among the most challenging technical tasks in proteomics: liquid chromatography coupled to mass spectrometry (LC-MS) based quantification methods have gained increasing robustness and reliability over the past five years. Many authors still share a view of redox signalling in which the fate of the cell is dependent mainly on the intensity and duration of pro-oxidant stimulus: here we sustain the involvement of an equilibrium encompassing the action of both nucleophiles and electrophiles at the same time. AIM: The dual aim of my PhD work has been both to develop suitable methodology to identify and quantify redox-active proteins in complex samples and to apply it to the study of a cellular model of breast cancer (MCF10A) engineered to reproduce malignancy. METHODS: In order to pursue this aim, I took advantage of an approach integrating differential chemical sample labelling (non-isotopic) with Cys reactive probes (NEM, IAM, HPDP) and chromatographic purification of redox-sensitive proteins, with subsequent LC-MS/MS analysis and computational data handling for OpenMS-based label-free quantification. All the steps of this methodology have been developed and validated in close collaboration with experts from both the biochemistry and bioinformatics field. RESULTS: We obtained an efficient cost-effective and isotopes-free methodology to characterize the redoxome in complex protein samples. Application of our quantification protocol to benchmark dataset leads to 100% correct estimates of under/over expression of the protein moiety. Application of the methodology to the breast cancer cellular model lead to identification of more than 300 proteins and allowed us to group-up unchanged and differentially oxidized redox-sensitive proteins in the more malignant cells in respect to their less aggressive counterpart. CONCLUSION: Despite the commonly accepted association between cancer and higher oxidative-stress, this study links higher breast cancer cells malignancy to a finely tuned dynamic equilibrium in which selected protein targets are oxidized in the context of a more reduced cell environment. Preliminary results point at the enzyme G6PDH as a crucial regulator of this redox process.STATO DELL’ARTE: Il proteoma include 214.000 cisteine in forma di gruppi tiolici liberi od altra forma. Di queste, solamente un insieme relativamente ristretto ha un ruolo nella mediazione di segnali cellulari. Tali cisteine, attive dal punto di vista dell’ossido-riduzione, sono più sensibili all’ossidazione e la loro forma ossidata è più facilmente riducibile. Sono dunque necessarie specifiche tecniche di proteomica, globalmente indicate con il termine proteomica delle ossido-riduzioni, per identificare tali modifiche e studiarne la regolazione in diversi processi cellulari. Risulta quindi determinante la capacità di identificare sia le proteine che i residui coinvolti e di quantificarne il grado di modificazione. E proprio la quantificazione delle differenze tra due o più stati di un sistema biologico, si colloca tra gli obiettivi tecnicamente più sfidanti della proteomica: nel corso degli ultimi cinque anni, tecniche basate sulla spettrometria di massa associata a cromatografia in fase liquida hanno progressivamente guadagnato affidabilità e robustezza. Molti autori condividono tuttora una visione delle ossido-riduzioni nella mediazione del segnale in cui il destino cellulare dipende principalmente dall’intensità e dalla durata degli stimoli ossidanti: nel presente lavoro si vuole invece sostenere il coinvolgimento di un equilibrio che includa l’azione concomitante sia di specie nucleofile sia di specie elettrofile. OBIETTIVO: Il duplice obiettivo del mio lavoro di Dottorato è stato sia lo sviluppo di una metodologia idonea all’identificazione e quantificazione di proteine, attive dal punto di vista delle ossido-riduzioni, in campioni complessi, sia l’applicazione di tale metodologia allo studio di un sistema cellulare ingegnerizzato di carcinoma mammario (MCF10A) caratterizzato da diversi gradi di malignità. METODI: Al fine di perseguire tale obiettivo ho tratto vantaggio da un approccio che integra la marcatura chimica differenziale (non-isotopica) per mezzo di sonde reattive con i residui di cisteina (NEM, IAM, HPDP) e la purificazione cromatografica delle proteine attive dal punto di vista ossido-riduttivo, alla successiva analisi LC-MS/MS ed elaborazione informatizzata dei dati mediante OpenMS per una quantificazione label-free. Tutti i passaggi di tale metodologia sono quindi stati messi a punto e validati in stretta collaborazione con esperti biochimici e bioinformatici. RISULTATI: E’ stato sviluppato un metodo efficiente ed economico, non basato sull’utilizzo di marcatori isotopici, per la caratterizzazione delle proteine attive dal punto di vista ossido-riduttivo in campioni proteici complessi. L’applicazione del protocollo di quantificazione ad un campione test ha dato il 100% di stime corrette di sovra/sotto-espressione della miscela proteica. L’applicazione del metodo allo studio del modello cellulare di carcinoma mammario ha portato all’identificazione di più di 300 proteine ed ha permesso il raggruppamento di quelle sensibili dal punto di vista ossido-riduttivo in gruppi non differenziali e sovra- o sotto-ossidate nelle cellule più maligne rispetto alla loro controparte meno aggressiva. CONCLUSIONI: Nonostante sia comunemente riconosciuta l’associazione tra fenomeni neoplastici ed uno stress ossidativo, questo studio collega la maggiore malignità di un modello cellulare di carcinoma mammario ad un complesso equilibrio ossido-riduttivo. In questo contesto, specifici bersagli proteici sono ossidati mentre viene mantenuto un ambiente cellulare complessivamente ridotto. Risultati preliminari evidenziano poi l’enzima G6PDH come possibile elemento chiave nella regolazione di tale equilibrio

Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications

Modulation of neurogenic inflammation in osteoarthrosis patients undergoing a combined treatment of mud packs, thermal baths, and acetaminophen: a preliminary study

Osteoarthritis (OA) is the most common joint disease. Certain neuropeptides contribute to both the continuance of symptoms and cartilage damage resulting from disease. Symptomatic drugs are frequentely used for pain control, but dissatisfaction with such an intervention has led many patients to seek for other treatments, one of which is thermal heat from mud packs. The aims of the present study were to investigate a possible interaction between termal treatment and the main neuropeptides involved in OA pathogenesis and to identify any detrimental effect of heat application on OA joints

Comparison between fractioning technologies for differential protein analyses

A good fractioning method enhances the number of proteins identified by mass spectrometric analysis and improves their relative quantification. The choice of the fractioning method is therefore critical. In this study we report the results of two different separation protocols used for analysis of differential protein expression in prostate cancer cell lines PC3 and LNCaP

Setting up of an innovative procedure for redox proteomics and its application for definition of the redox status of a cellular model.

Human proteome contains 214.000 cysteine residues. In the subset of protein not-free thiols, these are alkylated or, most frequently, oxidized. While disulfides, the largely most abundant oxidized form, have a pivotal role in driving protein folding, recently, their reversible formation also came to the stage as the final outcome of cell signaling pathways under the control of the nucleophilic/electrophilic tone. Specific high throughput procedures, collectively known as redox proteomics, have been developed to pinpoint these functional redox transition. Here we present an approach for pairwise comparison, integrating differential labeling with cys reactive probes and chromatographic isolation of redox sensitive proteins