Publications by authors named "Francesco Bonomi"

65 Publications

Redox Titration of Flavoproteins: An Overview.

Methods Mol Biol 2021 ;2280:119-133

Section of Chemical and Biomolecular Sciences, DeFENS, University of Milan, Milan, Italy.

Redox titration of flavoproteins allows to detect and analyze (1) the determinants of the stabilization of individual redox forms of the flavin by the protein; (2) the binding of the redox-active cofactor to the protein; (3) the effects of other components of the systems (such as micro- or macromolecular interactors) on parameters 1 and 2; (4) the pattern of electron flow to and from the flavin cofactor to other redox-active chemical species, including those present in the protein itself or in its physiological partners. This overview presents and discusses the fundamentals of the methodological approaches most commonly used for these purposes, and illustrates how data may be obtained in a reliable way, and how they can be read and interpreted.
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http://dx.doi.org/10.1007/978-1-0716-1286-6_8DOI Listing
January 2021

Cefoxitin treatment of MRSA leads to a shift in the IL-12/IL-23 production pattern in dendritic cells by a mechanism involving changes in the MAPK signaling.

Mol Immunol 2021 Mar 3;134:1-12. Epub 2021 Mar 3.

Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of β-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria - as denoted by increased sensitivity to mutanolysin -caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that β-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus.
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http://dx.doi.org/10.1016/j.molimm.2021.02.025DOI Listing
March 2021

Begelomab for severe refractory dermatomyositis: A case report.

Medicine (Baltimore) 2021 Mar;100(9):e24372

Division of Immunology, Transplantation and Infectious diseases, IRCCS Ospedale San Raffaele.

Rationale: Severe refractory idiopathic inflammatory myopathy (IIM) represents a challenge for the clinician. The lack of efficacy of available tools reflects our incomplete insight into the molecular events sustaining the inflammatory tissue damage in these patients. We present the first case of refractory IIM treated with anti-dipeptidyl peptidase-4 (DPP-4)/cluster of differentiation 26 (CD26) monoclonal antibody.

Patient Concerns: A 55-year old man presented with proximal muscle weakness, diffuse erythematous skin lesions which rapidly evolved into ulcerations, dysphagia and dysphonia.

Diagnosis: Increased serum creatine kinase levels and histological findings at muscle and skin biopsies were compatible with the diagnosis of dermatomyositis (DM). Several lines of treatment failed to control the disease including steroids, mycophenolate mofetil, tacrolimus, intravenous immunoglobulins and rituximab. Despite therapy, the patient also had recurrent intestinal vasculitis causing bowel perforation. Concurrently, DPP-4/CD26 expression in the patient's skin and skeletal muscle was observed.

Interventions: The patient was treated with begelomab, a murine immunoglobulin G2b monoclonal antibody against DPP-4/CD26.

Outcomes: Dysphagia, skin lesions and intestinal vasculitis resolved and the patient experienced a significant improvement of his quality of life.

Conclusion: Blockade of DPP-4/CD26, which is expressed on T cells and mediates T cell activation and function, is safe and might be effective in patients with refractory DM.
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http://dx.doi.org/10.1097/MD.0000000000024372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939186PMC
March 2021

Bio-Functional and Structural Properties of Pasta Enriched with a Debranning Fraction from Purple Wheat.

Foods 2020 Feb 8;9(2). Epub 2020 Feb 8.

Department of Food, Environmental, and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Via G. Celoria 2, 20133 Milan, Italy.

A colored and fiber-rich fraction from the debranning of purple wheat was incorporated at 25% into semolina- and flour-based pasta produced on a pilot-plant scale, with the aim of increasing anthocyanin and total phenolic content with respect to pasta obtained from whole pigmented grains. The debranning fraction impaired the formation of disulfide-stabilized protein networks in semolina-based systems. Recovery of phenolics was impaired by the pasta making process, and cooking decreased the phenolic content in both enriched samples. Cooking-related losses in anthocyanins and total phenolics were similar, but anthocyanins in the cooked semolina-based pasta were around 20% of what was expected from the formulation. HPLC (High Performance Liquid Chromatography) profiling of phenolics was carried out on extracts from either type of enriched pasta both before and after cooking and indicate possible preferential retention of specific compounds in each type of enriched pasta. Extracts from cooked samples of either enriched pasta were tested as inhibitors of enzymes involved in glucose metabolism and uptake, as well as for their capacity of suppressing the response to inflammatory stimuli. Results of both biological tests indicate that the phenolics in extracts from both cooked pasta samples had inhibitory capacities higher than extracts of the original debranning fraction at identical concentrations of total bioactives.
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http://dx.doi.org/10.3390/foods9020163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073741PMC
February 2020

Inhibition of Pancreatic α-amylase by Resveratrol Derivatives: Biological Activity and Molecular Modelling Evidence for Cooperativity between Viniferin Enantiomers.

Molecules 2019 Sep 5;24(18). Epub 2019 Sep 5.

Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Via Celoria 2, 20133 Milano, Italy.

To improve the current understanding of the role of stilbenoids in the management of diabetes, the inhibition of the pancreatic α-amylase by resveratrol derivatives was investigated. To approach in a systematic way, the mechanistic and structural aspects of the interaction, potential bioactive agents were prepared as single molecules, that were used for the biological evaluation of the determinants of inhibitory binding. Some dimeric stilbenoids-in particular, viniferin isomers- were found to be better than the reference drug acarbose in inhibiting the pancreatic α-amylase. Racemic mixtures of viniferins were more effective inhibitors than the respective isolated pure enantiomers at an equivalent total concentration, and displayed cooperative effects not observed with the individual enantiomers. The molecular docking analysis provided a thermodynamics-based rationale for the measured inhibitory ability and for the observed synergistic effects. Indeed, the binding of additional ligands on the surface of the alpha-amylase was found to decrease the dissociation constant of inhibitors bound to the active site of the enzyme, thus providing a mechanistic rationale for the observed inhibitory synergies.
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http://dx.doi.org/10.3390/molecules24183225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766848PMC
September 2019

Effects of starch addition on the activity and specificity of food-grade lipases.

Biotechnol Appl Biochem 2019 Jul 30;66(4):607-616. Epub 2019 May 30.

Section of Chemical and Biomolecular Sciences, Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Milan, Italy.

Lipases are surface-active enzymes, acting on their substrates at the polar/nonpolar interface in emulsions. This study was aimed to test whether their activity, specificity, and the rates of formation/degradation of the various hydrolysis intermediates (i.e., mono- and diglycerides of interest as surface-active agents) could be modulated by adhesion of the triglyceride substrates as a thin layer on the surface of solids. These hypotheses were tested by using an array of food-grade lipases used in bakery, testing various types of starch as the "solid" phase. Starch-dependent increase in the hydrolysis rate was tested by pH-stat techniques on pure triglycerides and on food-grade oils in diluted emulsions. Starch-related improvements in the rate of fatty acids release were most evident at temperatures above 40 °C, and when using maize starch instead of wheat starch. Starch-dependent changes in the nature of the hydrolysis products were tested by chromatographic profiling of ethyl ether extracts from aqueous slurries containing up to 33% fat and 33% starch. Accumulation of mono- and diglycerides as hydrolysis intermediates was found to be modulated by the type of oil being used, by the reaction conditions, as well as by the enzyme nature and amount.
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http://dx.doi.org/10.1002/bab.1761DOI Listing
July 2019

Greetings from foodland: Teaching biochemistry to BS students in food-related courses in Italy.

Biochem Mol Biol Educ 2019 07 15;47(4):394-403. Epub 2019 Apr 15.

Department of Food, Environmental, and Nutritional Sciences (DeFENS), University of Milan, Milan, Italy.

Biochemistry has always been a mandatory topic within BS courses aimed at food science students at the University of Milan, namely: Food Science & Technology and Catering Sciences. Addressing biochemistry topics in this peculiar setting requires: (i) specific focus on topics that are seldom considered in courses offered in bio-medical curricula; (ii) close integration with other area disciplines, such as food biotechnology; (iii) ad hoc design of laboratory classes; and (iv) an array of elective courses covering specific aspects of biochemistry. In this context, for example, protein chemistry is presented by using food proteins of known structure and discussed in terms of structural features in the raw materials and of structural and chemical modifications occurring upon processing. Along the same lines, metabolic pathways and their regulation are presented starting from widespread metabolism-related issues and to issues related to food safety (including food allergies and intolerances). A similar "hands on" approach is used for laboratory classes, that cover about one third of total credits and are aimed at providing fundamental-type information by analyzing practical situations in the food chain. In spite of their inherent complexity and volume, biochemistry courses score very well with the students in mandatory anonymous surveys. Our approach to biochemistry courses seems to help the students in "visualizing" the practical implications of concepts acquired in other courses within their curricula. The students' appreciation is confirmed by the sizeable attendance to elective and specialized biochemical-themed courses. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):394-403, 2019.
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http://dx.doi.org/10.1002/bmb.21245DOI Listing
July 2019

Topological features of the intermolecular contacts in gluten-forming proteins: Exploring a novel methodological approach based on gold nanoparticles.

Food Res Int 2019 05 8;119:492-498. Epub 2019 Jan 8.

Section of Chemistry and Biomolecular Sciences, DeFENS, University of Milan, Italy.

This work introduces a novel methodological approach to study both the geometry of complex protein networks and the nature of the interacting proteins. This approach is based on the high reactivity of Au ions on the surface of gold nanoparticles (AuNPs) towards thiols, that allows fast formation of a covalent bond between accessible protein thiols and AuNPs. In the case of the durum wheat semolina used in the exploratory studies reported here, the nature of proteins covalently bound to AuNPs is expected to be affected by both the compactness of the protein network and by the AuNPs size. Simple centrifugation procedures allowed recovery of the protein-loaded AuNPs that remained soluble, and the protein(s) covalently bound on the surface of soluble AuNP were identified by MS analysis of their proteolytic fragments. Gluten-forming proteins were found to be bound to soluble AuNPs only when detergents or chaotropes were added to the semolina/AuNPs suspension at room temperature. AuNPs-bound proteins also included gluten-forming proteins with no reported free thiols, suggesting that they are piggybacked on other thiol-containing gluten-forming proteins via disulfide bonds already present in the otherwise untreated semolina. The potential of this approach is discussed in terms of the possibility of developing a methodology suitable for further clarification of the geometrical features of protein networks, of the nature of the involved proteins, and of the type of interaction they establish, as well as any modifications of these features upon processing.
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http://dx.doi.org/10.1016/j.foodres.2019.01.013DOI Listing
May 2019

Surface Layer of MIMLh5 Promotes Endocytosis by Dendritic Cells.

Appl Environ Microbiol 2019 05 18;85(9). Epub 2019 Apr 18.

Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production. Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.
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http://dx.doi.org/10.1128/AEM.00138-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6495763PMC
May 2019

The Bio-Functional Properties of Pigmented Cereals may Involve Synergies among Different Bioactive Species.

Plant Foods Hum Nutr 2019 Mar;74(1):128-134

Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, via Giovanni Celoria 2, 20133, Milan, Italy.

This study was aimed at characterizing the anthocyanins and phenolics profile in different varieties of pigmented corn and wheat and in some of their milling fractions. Acid/ethanol extracts were used to assess total anthocyanins, overall antioxidant activity, the overall polyphenol profile, and for evaluating the inhibition of pancreatic α-amylase and of intestinal α-glucosidase. Both enzymes were inhibited in a dose-dependent manner by all extracts, but individual extracts had specific effects on each enzyme. Anti-inflammatory response was evaluated by using acid-free extracts and Caco-2 cells transiently transfected with a luciferase reporter gene responding to cytokine stimulation. The immune response of interleukin-stimulated cells decreased significantly in a dose-dependent manner in the presence of 20-50 μM/l anthocyanins from all grains extracts, again with a different efficiency. The inhibitory ability and the anti-inflammatory capability of these extracts are in most cases higher than in similar extracts from other sources, suggesting that activities in each extract may imply specific synergies between anthocyanins and other phenolics.
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http://dx.doi.org/10.1007/s11130-019-0715-4DOI Listing
March 2019

Enriching gluten-free rice pasta with soybean and sweet potato flours.

J Food Sci Technol 2018 Jul 30;55(7):2641-2648. Epub 2018 Apr 30.

1Department of Food, Environmental and Nutritional Sciences, University of Milan, Via G. Celoria 2, 20133 Milan, Italy.

The development of innovative rice products is a way to exploiting and adding value to low-grade African rice varieties. To this purpose, rice-based pasta was enriched with flours from soybean and orange-fleshed sweet potato, that are common ingredients in the African tradition. Four different formulations based on pre-gelatinized rice flour and liquid egg albumen, and containing soybean and/or sweet potato (up to 20%) were prepared and characterized via a multidisciplinary approach. Soybean and sweet potato enrichment leads to a decrease in the pasta consistency and in significant changes in the color of the resulting samples, likely due to Maillard-type reactions. E-sensing approaches indicated that the sensory profile of the various pasta products strongly depends on the type of enrichment. Data collected after cooking suggest that both soybean and sweet potato have a role in defining the firmness and water absorption, as well as the optimum cooking time. Structural characterization of proteins in the uncooked products indicates the presence of protein aggregates stabilized by hydrophobic interactions and disulfide bonds in all samples, although structural properties of the aggregates related to specific compositional traits.
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http://dx.doi.org/10.1007/s13197-018-3185-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033823PMC
July 2018

Affinity and selectivity of plant proteins for red wine components relevant to color and aroma traits.

Food Chem 2018 Aug 16;256:235-243. Epub 2018 Feb 16.

DeFENS, University of Milan, Italy.

The effects of fining with various plant proteins were assessed on Aglianico red wine, using both the young wine and wine aged for twelve and twenty-four months, and including wine unfined or fined with gelatin as controls. Color traits and fining efficiency were considered, along with the content of various types of phenolics and of aroma-related compounds of either varietal or fermentative origin. All agents had comparable fining efficiency, although with distinct kinetics, and had similar effects on wine color. Individual plant proteins and enzymatic hydrolyzates differed in their ability to interact with some anthocyanins, with specific proanthocyanidins complexes, and with some aroma components of fermentative origin. Changes in varietal aroma components upon fining were very limited or absent. Effects of all the fining agents tested in this study on the anthocyanidin components were most noticeable in young red wine, and decreased markedly with increasing wine ageing.
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http://dx.doi.org/10.1016/j.foodchem.2018.02.085DOI Listing
August 2018

Structural consequences of the interaction of puroindolines with gluten proteins.

Food Chem 2018 Jul 31;253:255-261. Epub 2018 Jan 31.

Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, via G. Celoria 2, 20133 Milan, Italy.

The effect of puroindolines (PINs) on structural characteristics of wheat proteins was investigated in Triticum turgidum ssp. durum (cv. Svevo) and Triticum aestivum (cv. Alpowa) and in their respective derivatives in which PIN genes were expressed (Soft Svevo) or the distal end of the short arm of chromosome 5D was deleted and PINs were not expressed (Hard Alpowa). The presence of PINs decreased the amount of cold-SDS extractable proteins and the accessibility of protein thiols to specific reagents, but resulted in facilitated solvation of gluten proteins, as detected by tryptophan fluorescence measurements carried out on minimally mixed flour/water mixtures. We propose that PINs and gluten proteins are interacting in the grain or flour prior to mixing. Hydrophobic interactions between PINs and some of the gluten proteins modify the pattern of interactions among gluten proteins, thus providing an additional mechanistic rationale for the effects of PINs on kernel hardness.
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http://dx.doi.org/10.1016/j.foodchem.2018.01.146DOI Listing
July 2018

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

Molecules 2018 Jan 6;23(1). Epub 2018 Jan 6.

Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, via Orabona, 4, I-70126 Bari, Italy.

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in and purified with a yield of 25 mg·L of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (k about 2.8 min), as well as FAD pyrophosphorolysis in a strictly Mg-dependent manner. The synthesis of FAD is inhibited by HgCl₂. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.
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http://dx.doi.org/10.3390/molecules23010116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017331PMC
January 2018

Macromolecular Traits in the African Rice Oryza glaberrima and in Glaberrima/Sativa Crosses, and Their Relevance to Processing.

J Food Sci 2017 Oct 29;82(10):2298-2305. Epub 2017 Aug 29.

Dipto. di Scienze per gli Alimenti, la Nutrizione e l'Ambiente (DeFENS), Univ.à degli Studi di Milano, Italy.

Molecular properties of proteins and starch were investigated in 2 accessions of Oryza glaberrima and Oryza sativa, and in one NERICA cross between the 2 species, to assess traits that could be relevant to transformation into specific foods. Protein nature and organization in O. glaberrima were different from those in O. sativa and in NERICA. Despite the similar cysteine content in all samples, thiol accessibility in O. glaberrima proteins was higher than in NERICA or in O. sativa. Inter-protein disulphide bonds were important for the formation of protein aggregates in O. glaberrima, whereas non-covalent protein-protein interactions were relevant in NERICA and O. sativa. DSC and NMR studies indicated only minor differences in the structure of starch in these species, as also made evident by their microstructural features. Nevertheless, starch gelatinization in O. glaberrima was very different from what was observed in O. sativa and NERICA. The content of soluble species in gelatinized starch from the various species in the presence/absence of treatments with specific enzymes indicated that release of small starch breakdown products was lowest in O. glaberrima, in particular from the amylopectin component. These findings may explain the low glycemic index of O. glaberrima, and provide a rationale for extending the use of O. glaberrima in the production of specific rice-based products, thus improving the economic value and the market appeal of African crops.

Practical Application: The structural features of proteins and starch in O. glaberrima are very different from those in O. sativa and in the NERICA cross. These results appear useful as for extending the use of O. glaberrima cultivars in the design and production of specific rice-based products (for example, pasta), that might, in turn, improve the economic value and the market appeal of locally sourced raw materials, by introducing added-value products on the African market.
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http://dx.doi.org/10.1111/1750-3841.13853DOI Listing
October 2017

Serum Proteome in a Sporadic Amyotrophic Lateral Sclerosis Geographical Cluster.

Proteomics Clin Appl 2017 Dec 25;11(11-12). Epub 2017 Sep 25.

Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Milan, Italy.

This study is meant to characterize the serum proteome in a small geographical cluster of sporadic ALS subjects originating from a restricted geographical area and sharing the same environmental exposure, in a broader context of evaluating the relevance of environmental factors to disease onset, status, and progression. An Artificial Neural Network based software is used to compare the relative abundance of proteins identified as different (by means of bi-dimensional electrophoresis and mass spectrometry) in the serum proteome of patients and age-matched healthy controls. The patient's group is characterized by altered levels of acute phase reactants and of proteins involved in lipid homeostasis, along with over-representation of the APOE*4 allele. Characterization of the serum proteome in a small cluster of sporadic ALS patients, originating from a geographically restricted area with a high prevalence of the disease and evaluation of the results with software based on artificial neural networks, highlights the association of the relative abundance of some proteins (most notably, acute phase reactants and lipid homeostasis proteins) with the disease presence and status.
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http://dx.doi.org/10.1002/prca.201700043DOI Listing
December 2017

Stabilization of beta-lactoglobulin by polyols and sugars against temperature-induced denaturation involves diverse and specific structural regions of the protein.

Food Chem 2017 Nov 21;234:155-162. Epub 2017 Apr 21.

Section of Chemical and Biomolecular Sciences, DeFENS, Università degli Studi di Milano, Milano, Italy.

Temperature sensitivity of bovine milk beta-lactoglobulin (BLG) was assessed in the presence/absence of non-reducing sugars (sucrose and trehalose) and polyols (glycerol and sorbitol). None of them affected the structural features of the protein at room temperature, where the only observed effect was an increased affinity towards hydrophobic probes in the presence of all co-solutes but glycerol. Although most of the observed effects in temperature-ramp experiments are due to entropic effects (fitting within the "preferential exclusion" theory of protein stabilization), this study indicates that each co-solute exhibit different efficacy at stabilizing specific regions of BLG, suggesting that each of them acts in a specific way on the solvent/protein system. The relevance of these observations with respect to systems of practical relevance is discussed, given the widespread use of heat-polymerizing proteins - such as BLG - in many food formulations that very often include significant amounts of sugars and/or polyols.
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http://dx.doi.org/10.1016/j.foodchem.2017.04.132DOI Listing
November 2017

Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from Arabidopsis thaliana.

Biochemistry 2017 04 7;56(15):2116-2125. Epub 2017 Apr 7.

Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche , Via Bassini 15a, 20133 Milano, Italy.

The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol-disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their catalytic activity and cellular function are still poorly understood. To gain insight into the role of higher-plant class A PDIs, the biochemical properties of rAtPDI-A, the recombinant form of Arabidopsis thaliana PDI-A, have been investigated. As expressed, rAtPDI-A has only little oxidoreductase activity, but it appears to be capable of binding an iron-sulfur (Fe-S) cluster, most likely a [2Fe-2S] center, at the interface between two protein monomers. A mutational survey of all cysteine residues of rAtPDI-A indicates that only the second and third cysteines of the CXXXCKHC stretch, containing the putative catalytic site CKHC, are primarily involved in cluster coordination. A key role is also played by the lysine residue. Its substitution with glycine, which restores the canonical PDI active site CGHC, does not influence the oxidoreductase activity of the protein, which remains marginal, but strongly affects the binding of the cluster. It is therefore proposed that the unexpected ability of rAtPDI-A to accommodate an Fe-S cluster is due to its very unique CKHC motif, which is conserved in all higher-plant class A PDIs, differentiating them from all other members of the PDI family.
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http://dx.doi.org/10.1021/acs.biochem.6b01257DOI Listing
April 2017

Blood trace metals in a sporadic amyotrophic lateral sclerosis geographical cluster.

Biometals 2017 06 23;30(3):355-365. Epub 2017 Mar 23.

Department of Food, Environmental and Nutritional Sciences (DEFENS), Division of Chemical and Biomolecular Sciences, University of Milan, 20133, Milan, Italy.

Amyotrophic lateral sclerosis (ALS) is a fatal disorder with unknown etiology, in which genetic and environmental factors interplay to determine the onset and the course of the disease. Exposure to toxic metals has been proposed to be involved in the etiology of the disease either through a direct damage or by promoting oxidative stress. In this study we evaluated the concentration of a panel of metals in serum and whole blood of a small group of sporadic patients, all living in a defined geographical area, for which acid mine drainage has been reported. ALS prevalence in this area is higher than in the rest of Italy. Results were analyzed with software based on artificial neural networks. High concentrations of metals (in particular Se, Mn and Al) were associated with the disease group. Arsenic serum concentration resulted lower in ALS patients, but it positively correlated with disease duration. Comet assay was performed to evaluate endogenous DNA damage that resulted not different between patients and controls. Up to now only few studies considered geographically well-defined clusters of ALS patients. Common geographical origin among patients and controls gave us the chance to perform metallomic investigations under comparable conditions of environmental exposure. Elaboration of these data with software based on machine learning processes has the potential to be extremely useful to gain a comprehensive view of the complex interactions eventually leading to disease, even in a small number of subjects.
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http://dx.doi.org/10.1007/s10534-017-0011-4DOI Listing
June 2017

Soybean-Enriched Snacks Based on African Rice.

Foods 2016 May 20;5(2). Epub 2016 May 20.

Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Milan 20133, Italy.

Snacks were produced by extruding blends of partially-defatted soybean flour with flours from milled or parboiled African-grown rice. The interplay between composition and processing in producing snacks with a satisfactory sensory profile was addressed by e-sensing, and by molecular and rheological approaches. Soybean proteins play a main role in defining the properties of the protein network in the products. At the same content in soybean flour, use of parboiled rice flour increases the snack's hardness. Electronic nose and electronic tongue discriminated samples containing a higher amount of soybean flour from those with a lower soybean flour content.
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http://dx.doi.org/10.3390/foods5020038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302339PMC
May 2016

Structural changes in emulsion-bound bovine beta-lactoglobulin affect its proteolysis and immunoreactivity.

Biochim Biophys Acta 2016 Jul 13;1864(7):805-13. Epub 2016 Apr 13.

Section of Chemical and Biomolecular Sciences, DeFENS, University of Milan, Via G. Celoria 2, 20133 Milano, Italy.

Adsorption on the surface of sub-micrometric oil droplets resulted in significant changes in the tertiary structure of bovine beta-lactoglobulin (BLG), a whey protein broadly used as a food ingredient and a major food allergen. The adsorbed protein had increased sensitivity to trypsin, and increased immunoreactivity towards specific monoclonal antibodies. In spite of the extensive tryptic breakdown of emulsion-bound BLG, some sequence stretches in BLG became trypsin-insensitive upon absorption of the protein on the fat droplets. As a consequence - at contrast with free BLG - proteolysis of emulsion-bound BLG did not decrease the immunoreactivity of the protein, and some of the large peptides generated by trypsinolysis of emulsion-bound BLG were still recognizable by specific monoclonal antibodies. Structural changes occurring in emulsion-bound BLG and their consequences are discussed in comparison with those occurring when the tertiary structure of BLG is modified by lipophilic salts, by urea, or upon interaction with solid hydrophobic surfaces. Such a comparison highlights the relevance of situation-specific structural modifications, that in turn may affect physiologically relevant features of the protein.
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http://dx.doi.org/10.1016/j.bbapap.2016.04.007DOI Listing
July 2016

Functional implications of the interaction between HscB and IscU in the biosynthesis of FeS clusters.

J Biol Inorg Chem 2015 Sep 6;20(6):1039-48. Epub 2015 Aug 6.

Section of Chemical and Biomolecular Sciences, DeFENS, University of Milan, Via Celoria 2, 20133, Milan, Italy.

In bacteria, HscB is the cochaperone of HscA in modulating the transfer of 2Fe2S clusters from a cluster-loaded form of the scaffold protein IscU to acceptor apoproteins. HscB binding to the IscU apoform (apoIscU) reportedly impairs the structural flexibility of apoIscU, but the effects of HscB on cluster formation on IscU have never been assessed. We report that presence of HscB impaired the rate-but not the equilibrium-of the appearance of the distinctive circular dichroism signals associated with formation of a stable 2Fe-2S cluster on IscU in reconstitution experiments. This impairment: (1) was independent of the source of cluster sulfide; (2) was not observed for HscB mutants unable to bind IscU; (3) implied formation of a 1/1 HscB/IscU complex; (4) was not observed for a D39A mutant of IscU, with a much more rigid structure than wt IscU. The cluster species assembled on IscU in the presence of HscB were transferred to apoferredoxin at a slower rate than those formed in the absence of HscB, unless ATP and HscA were also present. At contrast, HscB was found to improve the "catalytic" function of IscU with respect to cluster assembly in the presence of a large apoferredoxin excess. Thus, the HscB/IscU interaction may modulate formation and transfer of FeS clusters by accelerating cluster biosynthesis when appropriate target apoproteins are abundant or by slowing it down when the rate of apoprotein synthesis is slow, and cluster-loaded IscU is more likely to play a role as a "FeS storage" protein.
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http://dx.doi.org/10.1007/s00775-015-1285-zDOI Listing
September 2015

Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis.

Front Chem 2015 22;3:30. Epub 2015 Apr 22.

Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy ; Dipartimento di Scienze della Vita, Istituto di Biomembrane e Bioenergetica, CNR Bari, Italy.

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.
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http://dx.doi.org/10.3389/fchem.2015.00030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406087PMC
May 2015

Esterases as stereoselective biocatalysts.

Biotechnol Adv 2015 Sep-Oct;33(5):547-65. Epub 2015 Feb 10.

Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, Via Mangiagalli 25, 20133 Milan, Italy.

Non-lypolitic esterases are carboxylester hydrolases with preference for the hydrolysis of water-soluble esters bearing short-chain acyl residues. The potential of esterases as enantioselective biocatalysts has enlarged in the last few years due to the progresses achieved in different areas, such as screening methodologies, overproduction of recombinant esterases, structural information useful for understanding the rational behind enantioselectivity, and efficient methods in protein engineering. Contributions of these complementary know-hows to the development of new robust enantioselective esterases are critically discussed in this review.
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http://dx.doi.org/10.1016/j.biotechadv.2015.01.006DOI Listing
March 2016

Rubredoxin refolding on nanostructured hydrophobic surfaces: evidence for a new type of biomimetic chaperones.

Proteins 2014 Nov 3;82(11):3154-62. Epub 2014 Sep 3.

Section of Chemistry and Biomolecular Sciences, DeFENS, University of Milan, Milan, Italy.

Rubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)4 site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron-uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at "catalytic" NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific "iron-uptake-competent" conformations of apoRd on the NP surface. This bio-mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor-triggered refolding, particularly when over-expressed under limited cofactor accessibility.
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http://dx.doi.org/10.1002/prot.24675DOI Listing
November 2014

Significance of redox-active cysteines in human FAD synthase isoform 2.

Biochim Biophys Acta 2014 Dec 15;1844(12):2086-95. Epub 2014 Aug 15.

Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "A. Moro", via Orabona 4, I-70126 Bari, Italy; Istituto di Biomembrane e Bioenergetica (IBBE), CNR, via Amendola 165/A, I-70126 Bari, Italy. Electronic address:

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.
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http://dx.doi.org/10.1016/j.bbapap.2014.08.005DOI Listing
December 2014

TgaA, a VirB1-like component belonging to a putative type IV secretion system of Bifidobacterium bifidum MIMBb75.

Appl Environ Microbiol 2014 Sep 20;80(17):5161-9. Epub 2014 Jun 20.

Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland.

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.
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http://dx.doi.org/10.1128/AEM.01413-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136095PMC
September 2014

Murein lytic enzyme TgaA of Bifidobacterium bifidum MIMBb75 modulates dendritic cell maturation through its cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) amidase domain.

Appl Environ Microbiol 2014 Sep 9;80(17):5170-7. Epub 2014 May 9.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.
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http://dx.doi.org/10.1128/AEM.00761-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136110PMC
September 2014

Unfolding of beta-lactoglobulin on the surface of polystyrene nanoparticles: experimental and computational approaches.

Proteins 2014 Jul 15;82(7):1272-82. Epub 2014 Jan 15.

Section of Chemistry and Biomolecular Sciences, DeFENS, University of Milan, Via Celoria 2, 20133, Milan, Italy.

Structural changes ensuing from the non-covalent absorption of bovine beta-lactoglobulin (BLG) on the surface of polystyrene nanoparticles were investigated by using spectroscopic approaches, by assessing the reactivity of specific residues, and by limited proteolysis/mass spectrometry. Also, the immunoreactivity of absorbed and free BLG was compared. All these approaches indicated substantial rearrangements of the protein structure in the absorbed state, in spite of the reported structural rigidity of BLG. Changes made evident by experimental measurements were confirmed by computational approaches. These indicate that adsorption-related changes are most marked in the area between the main C-terminal alpha helix and the beta-barrel, and lead to full exposure of the thiol on Cys121 , consistent with experimental measurements. In the computational model of bound BLG, both Trp61 and Trp19 also move away from their neighboring quenchers and become solvent-exposed, as indicated by fluorescence measurement. Upon binding, the beta-barrel also loosens, with a substantial increase in immunoreactivity and with noticeable changes in the trypsinolytic pattern. The possible general significance of the structural changes reported here for non-covalently adsorbed BLG is discussed with respect to recognition events involving surface-bound proteins, as are aspects related to the carrier function(s) of BLG, and to its use as a common ingredient in many food systems.
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http://dx.doi.org/10.1002/prot.24493DOI Listing
July 2014

Binding of curcumin to milk proteins increases after static high pressure treatment of skim milk.

J Dairy Res 2013 May 3;80(2):152-8. Epub 2013 Apr 3.

Department of Food Science, University of Guelph, Guelph, Ontario N1H 2W1, Canada.

Curcumin is a bioactive polyphenolic compound extracted from turmeric with known anti-inflammatory properties, and its hydrophobic nature restricts its solubility and its bioaccessibility. Solubility may be improved upon binding of curcumin to native or treatment-modified casein micelles. The present work demonstrated that high hydrostatic pressure treatment of skim milk increases the binding of curcumin to caseins. The association of curcumin to casein micelles was assessed using fluorescence spectroscopy, either directly or by tryptophan quenching. The amount of curcumin associated with the milk proteins increased in pressure-treated milk, and a further improvement in the amount of bound curcumin was observed upon pressure treatment of a milk/curcumin mixture. However, in this case, some of the curcumin dissociated during storage, contrarily to what was observed for untreated milk. From a molecular standpoint, the data presented here indicate that structural modifications induced by high-pressure treatment and known to affect the structure of milk proteins result in a rearrangement of the amino acid residues in close proximity to the protein-associated curcumin.
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http://dx.doi.org/10.1017/S0022029913000125DOI Listing
May 2013