Gianluca Paredi

Muscle to meat molecular events and technological transformations: The proteomics insight ☆

Cellular death is characterized by a complex pattern of molecular events that depend on cell type. Specifically, muscle cells first undergo rigor mortis due to ATP depletion, and later, on the time scale of days, muscle fiber degradation due to proteolytic enzyme activity. In the present review, we will refer to proteomic investigations on the post-mortem evolution of the protein patterns of animal muscle cells. These studies, carried out with the application of either bottom-up or top-down methods, are relevant for understanding the biochemical reactions that i) convert muscle to meat, ii) are associated with meat aging and iii) impact on meat tenderness, a feature of significant commercial value. We also report on the proteomic investigations that have been made to analyze the transformation of meat in industrial processes. These studies are primarily aimed at identifying protein patterns and/or individual proteins diagnostic of the quality of the final product.

Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria.

We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis.

Molecular insights into dimerization inhibition of c-Maf transcription factor

The Maf protein family belongs to the activator protein 1 (AP-1) superfamily of transcription factors that bind specific DNA target sequences through a basic region and exploit a leucine zipper (LZ) motif for protein-protein interactions leading to homo- or hetero-dimerization. Mafs unique DNA-binding domain contains a highly conserved extended homology region (EHR) that allows to recognize longer DNA sequences than other basic leucine zipper (bZIP) transcription factors. Inspired by the fact that overexpression of Mafs is observed in about 50% of cases of multiple myeloma, a hematological malignant disorder, we undertook a peptide inhibitor approach. The LZ domain of c-Maf, one of large Mafs, was produced by solid phase peptide synthesis. We characterized its secondary structure and dimerization properties, and found that dimerization and folding events are strictly coupled. Moreover, potential peptidic c-Maf dimerization inhibitors were computationally designed and synthesized. These compounds were demonstrated by circular dichroism (CD) spectroscopy and MALDI-TOF mass spectrometry to bind to c-Maf LZ monomers, to drive folding of their partially disordered structure and to efficiently compete with dimerization, suggesting a way for interfering with the function of c-Maf and, more generally, of intrinsically disordered proteins, till now considered undruggable targets.

Insight of Saffron Proteome by Gel-Electrophoresis

Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds.

Gel-Based and Gel-Free Analytical Methods for the Detection of HMW-GS and LMW-GS in Wheat Flour

Durum wheat (Triticum turgidum L.) flour is instrumental for the production of pasta worldwide. The quality of this food rests on flour processing and on its protein content and composition. Gluten proteins as high and low-molecular weight glutenins (GS) are important to predict the flour technological property in pasta making. Different methods were compared to separate, identify and quantify GS in flours from two wheat cultivars. Sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) gave in a fast way information about the GS assets. Two-dimensional gel electrophoresis (2D-GE) allowed for the highest resolution in detecting and quantifying single GS, subsequently identified by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS). Reversed-phase high-performance liquid chromatography (RP-HPLC) is a non-gel alternative system for separation and quantification of single GS that when combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) gave information about their exact masses. This method gives also quantitative indications of each individual GS. Different GS patterns and contents were detected in the flour of the two cultivars, underlining the importance of these analytical methods before determining the best flour processing procedure in pasta making. The different methods were evaluated with a modular approach consisting of a grid of different parameters and a non-linear score within each module.

Structural insight into the interaction of O-acetylserine sulfhydrylase with competitive, peptidic inhibitors by saturation transfer difference-NMR.

O‐acetylserine sulfhydrylase (OASS), the enzyme catalysing the last step of cysteine biosynthesis in bacteria, is involved in antibiotic resistance and biofilm formation. Since mammals lack OASS, it is a potential target for antimicrobials. However, a limited number of inhibitors has been developed and crystallized in complex with OASS. STD‐NMR was applied to study the interaction of the inhibitory pentapeptide MNYDI with the CysK and CysM OASS isozymes from Salmonella Typhimurium. Results are in excellent agreement with docking and SAR studies and confirm the important role played by the C‐terminal Ile5 and the arylic moiety at P3 in dictating affinity.

Proteomics of Parma Dry-Cured Ham: Analysis of Salting Exudates

The production of Parma dry-cured ham involves the steps of salting, drying, and ripening. Although sea salt is the only preserving agent, there are strategies being developed with the goal of reducing salt content in order to decrease its negative impact on consumer health. A 24 h pressure treatment was applied before salting to reduce thickness and inequalities in shape. To evaluate the potential impact of the pressure step on the process outcome, differential proteomic analyses by complementary 2D-PAGE and LC-MS/MS were carried out on exudates collected at day 1, 5, and 18 of the salting phase for hams treated or untreated with pressure. Specific proteins were found differentially abundant in exudates from pressed vs unpressed hams and as a function of time. These changes include glycolytic enzymes and several myofibrillar proteins. These findings indicate that pressure causes a faster loosening of the myofibrillar structure with the release of specific groups of proteins.

Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

In bacteria and plants, serine acetyltransferase (CysE) and O‐acetylserine sulfhydrylase‐A sulfhydrylase (CysK) collaborate to synthesize l‐Cys from l‐Ser. CysE and CysK bind one another with high affinity to form the cysteine synthase complex (CSC). We demonstrate that bacterial CysE is activated when bound to CysK. CysE activation results from the release of substrate inhibition, with the Ki for l‐Ser increasing from 4 mm for free CysE to 16 mm for the CSC. Feedback inhibition of CysE by l‐Cys is also relieved in the bacterial CSC. These findings suggest that the CysE active site is allosterically altered by CysK to alleviate substrate and feedback inhibition in the context of the CSC.

Molecular basis for covalent inhibition of glyceraldehyde-3-phosphate dehydrogenase by a 2-phenoxy-1,4-naphthoquinone small molecule

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2‐phenoxy‐1,4‐naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2‐phenoxy‐1,4‐naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2‐phenoxy‐1,4‐naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non‐covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2‐phenoxy‐1,4‐naphthoquinone displays selectivity for GAPDHs over two other cysteine‐dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.

High- and low-affinity PEGylated hemoglobin-based oxygen carriers: Differential oxidative stress in a Guinea pig transfusion model

Abstract Hemoglobin‐based oxygen carriers (HBOCs) are an investigational replacement for blood transfusions and are known to cause oxidative damage to tissues. To investigate the correlation between their oxygen binding properties and these detrimental effects, we investigated two PEGylated HBOCs endowed with different oxygen binding properties ‐ but otherwise chemically identical ‐ in a Guinea pig transfusion model. Plasma samples were analyzed for biochemical markers of inflammation, tissue damage and organ dysfunction; proteins and lipids of heart and kidney extracts were analyzed for markers of oxidative damage. Overall, both HBOCs produced higher oxidative stress in comparison to an auto‐transfusion control group. Particularly, tissue 4‐hydroxynonenal adducts, tissue malondialdehyde adducts and plasma 8‐oxo‐2’‐deoxyguanosine exhibited significantly higher levels in comparison with the control group. For malondialdehyde adducts, a higher level in the renal tissue was observed for animals treated with the high‐affinity HBOC, hinting at a correlation between the HBOCs oxygen binding properties and the oxidative stress they produce. Moreover, we found that the high‐affinity HBOC produced greater tissue oxygenation in comparison with the low affinity one, possibly correlating with the higher oxidative stress it induced. Graphical abstract Figure. No Caption available. HighlightsTwo hemoglobin‐based oxygen carriers led to oxidative stress in a transfusion model.Both products caused an increase in markers of heart damage and kidney dysfunction.Tissue and plasma markers of oxidative stress were validated for the model.The highest‐affinity oxygen carrier induced higher oxidative stress.