Carlo Barnaba | Michigan State University (original) (raw)
Papers by Carlo Barnaba
Food and Chemical Toxicology
Autophagy is a catabolic pathway required for the clearance and recycling of cytoplasmic material... more Autophagy is a catabolic pathway required for the clearance and recycling of cytoplasmic materials. Upregulation and dysfunction of autophagy contributes to the pathology of cancer and neurogenerative diseases, respectively. To define the molecular mechanisms that control autophagic flux it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of 9 cell lines expressing HaloTagged autophagy factors from their endogenous loci, we systematically analyze the abundance, single-molecule dynamics, and autophagosome association kinetics of a wide variety of autophagy proteins involved in the initiation and maturation of the autophagosome. Our results reveal that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles and tethering of these ATG9 vesicles to donor membranes by ATG2 is a key step in phagophore maturation. In addition, we demonstrate that the overall lifetime of an autophagosome is...
Up to the 60% of the Western diet is characterized by consumption of high amounts of Ultra-proces... more Up to the 60% of the Western diet is characterized by consumption of high amounts of Ultra-processed foods (UPFs). From a nutritional standpoint, the high consumption of UPFs, including Fast-foods and Ready-to-Eat (RTE), emerges as a critical topic for public health linking nutritional quality and food safety. In the present work, we provide a systematic database of fatty acids composition of the most consumed UPFs in the US mid-western region. Saturate (SFA) and monounsaturated (MUFA) fatty acids were predominant in both FF and RTE, while health-beneficial polyunsaturated (PUFA) fatty acids were abundant only in seafood meals. Nutritional and non-nutritional attributes were correlated, finding positive correlation between caloric and sodium content. The significance of this study relies on providing new quantitative data for the most consumed UPFs, with the opportunity to define priority interventions for more advanced precision nutrition, especially for vulnerable populations.
Advances in Experimental Medicine and Biology, 2019
Flavonoids are plant secondary metabolites that act as protectants against harmful effects of UV-... more Flavonoids are plant secondary metabolites that act as protectants against harmful effects of UV-B radiation inasmuch as biotic stress, conferring at the same time pigmentation of fruits and leaves [67]. The term "flavonoid" refers to phenolics having a basic skeleton of diphenylpropane (C6-C3-C6), which consists of two aromatic rings linked through three carbons that usually form an oxygenated heterocycle [25, 52]. Flavonoids are broken down into several different sub-categories based on their chemical structure. The main subclasses commonly found in food items are: flavonols, flavones, flavanones, flavan-3-ols, proanthocyanidins, and anthocyanins [44, 67]. Figure 19.1 depicts the major classification of flavonoids according to their chemical structure. Their occurrence in food matrices has been extensively reviewed [39, 44], and has been subject of extensive research in the last decades. Table 19.1 contains a few examples of compounds from each of the subcategory, with the fruit (berry) in which they are commonly found. The monomeric unit of flavonoids can dimerize and polymerize to form other important high molecular weight molecules; this is the case of proanthocyanidins, that are polymers of flavan-3-ols or flavanols. Not only do these compounds act as plant protectants, but they can also be very beneficial to human health. Cohorts studies performed in the early '90 have shown that dietary consumption of flavonoids was inversely associated with morbidity and mortality from coronary heart disease [31, 32]. These findings have opened an intensive field of research on the effects of flavonoids and flavonoids-rich food extracts in cardiovascular diseases (CVD) progression, particularly in the modulating CVD-associated oxidative stress and inflammation. In this short review, we will summarize the current findings in flavonoids beneficial effects in preventing CVD through inhibition of initial stages of CVD progression. Given the magnitude of scientific literature in the field, we will focus on two strictly mechanistic aspects: inhibition of chemical-induced LDL oxidation, and the effect of flavonoids in the monocyte/macrophages activation pathways.
medRxiv, 2020
Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutrition... more Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutritional source during the first six months of life. IF is available in a variety of commercial presentations, although from a manufacturing standpoint, they can be categorized in powder- (PIF) or liquid- (LIF) based formulations. Herein, thirty commercial IFs were analyzed in their oxidative and non-oxidative lipidomics profiles. Results show that LIFs have a characteristic lipidomic fingerprint, enriched in an oxidated form of cholesterol, and a lower load of phytosterols. We identified 7-ketocholesterol - a major end-product of cholesterol oxidation - as a potential biomarker of IF manufacturing. Our data allowed re-classification of IF based on their metabolomic fingerprint, resulting in three groups assigned with low-to-high oxidative status. Finally, we modeled the dietary intake for cholesterol, sterols, and 7-ketocholesterol in the first year of life. The database provided in this stud...
Journal of Clinical Lipidology, 2020
Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. Ho... more Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in animal models. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique in determining that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic stimulation activation at a single partic...
Biophysical Journal, 2018
HIV enters human T cells through the fusion of viral and host-cell membranes. This fusion process... more HIV enters human T cells through the fusion of viral and host-cell membranes. This fusion process is mediated by a surface protein, gp41, and the platform provided by the cholesterol-rich viral membrane. The membrane-bound region of gp41 plays critical roles in this fusion process and is a major target of anti-gp41 antibodies and vaccine design. Here, EPR and spin-labeling techniques were used to dissect the interactions between the viral membrane and the membrane-bound region of gp41, including the membrane proximal ectodomain region (MPER) and the transmembrane domain (TM). The analyses revealed a helix-hinge-helix structure of the MPER sitting on the membrane headgroup region in a manner impacted by para-hinge and other residues. The MPER exerts its fusogenic activity by perturbing membrane properties and by inducing significant lipid order and membrane permeability changes. The MPER-induced membrane property changes are modulated by cholesterol content and the TM. In addition, anti-gp41 neutralizing antibodies disrupt MPER-lipid interactions by altering MPER conformation on the membrane and by abolishing the membrane-perturbing activity of the MPER. These findings suggest that the membrane-bound region of gp41 facilitates HIV infection through its interaction with the cholesterol-rich viral membrane, and this interaction is disrupted by anti-gp41 neutralizing antibodies.
Chemical communications (Cambridge, England), Jan 14, 2018
Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the s... more Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.
Chemphyschem : a European journal of chemical physics and physical chemistry, Oct 19, 2018
Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, and re... more Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, and requires enzymatic partners as well as environmental factors that tune its complex dynamic activity. P450 and its reducing counterparts are membrane-bound proteins which are believed to dynamically interact to form functional complexes. Increasing experimental evidence signifies the role (s) of protein-lipid interactions in P450's catalytic function and efficiency. The challenges posed by the membrane have severely limited high-resolution understanding of the molecular interfaces of these interactions. Nevertheless, recent NMR studies have provided piercing insights into the dynamic structural interactions that enable the function of P450. In this review, we will discuss different biomimetic approaches relevant to unveil molecular interplays at the membrane, focusing on our recent work on lipid-nanodiscs. We also highlight the need to expand the use of nanodiscs, and the power of a com...
Food and Chemical Toxicology, 2018
Food consumption can lead to the accumulation of certain chemical compounds able to exert toxic a... more Food consumption can lead to the accumulation of certain chemical compounds able to exert toxic activities against humans. Of mayor interests are those molecules generated during food processing and handling, since their occurrence and distribution depend of many intrinsic and extrinsic factors. Cholesterol-a lipid constituent of mammalian cells-is the precursor of several toxic molecules known as cholesterol oxidation products (COPs). In the last decades, it has been demonstrated that food processing can dramatically trigger COPs accumulation in meats, eggs, dairy products, fish and poultry. On the other hand, countless scientific evidences have pointed out the highly toxic and pathogenic activities of COPs, from cancer stimulation to neurodegenerative disorders, via molecular mechanisms that are largely unexplored. The aim of this review is to merge the evidence on COPs accumulation in foods and their toxic activities through dietary intake, as from in vivo and in vitro studies. We consider that is imperative to systematically monitor the formation of COPs and bridge these quantitative efforts with risk exposure assessment on sensitive populations.
Angewandte Chemie (International ed. in English), Jan 19, 2018
Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, th... more Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.
Biophysical Journal, 2017
The degree of lipid saturation is a key feature of membranes that determines lipid packing and me... more The degree of lipid saturation is a key feature of membranes that determines lipid packing and membrane fluidity. It must be tightly regulated to guarantee 508a Wednesday,
F1000Research, 2017
Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which ... more Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts-cytochrome P450-reductase and cytochrome b 5 -are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s) played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed...
Scientific reports, Jan 10, 2017
Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP45... more Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. The...
Journal of the American Chemical Society, Apr 28, 2017
Cytochrome P450-Reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It i... more Cytochrome P450-Reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature dependent Fluorescence Recovery After Photobleaching (FRAP). The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramati...
Plant Physiology, 2016
Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ult... more Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137 P-X-P-X 140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.
Steroids, 2016
In this work we studied the effect of polyunsaturated fatty acids (PUFAs) methyl esters on choles... more In this work we studied the effect of polyunsaturated fatty acids (PUFAs) methyl esters on cholesterol photo-induced oxidation. The oxidative routes were modeled with a chemical reaction network (CRN), which represents the first application of CRN to the oxidative degradation of a food-related lipid matrix. Docosahexaenoic acid (DHA, T-I), eicosapentaenoic acid (EPA, T-II) and a mixture of both (T-III) were added to cholesterol using hematoporphyrin as sensitizer, and were exposed to a fluorescent lamp for 48 h. High amounts of Type I cholesterol oxidation products (COPs) were recovered (epimers 7α-and 7β-OH, 7-keto and 25-OH), as well as 5β,6β-epoxy. Fitting the experimental data with the CRN allowed characterizing the associated kinetics. DHA and EPA exerted different effects on the oxidative process. DHA showed a protective effect to 7-hydroxy derivatives, whereas EPA enhanced side-chain oxidation and 7β-OH kinetic rates. The mixture of PUFAs increased the kinetic rates several fold, particularly for 25-OH. With respect to the control, the formation of β-epoxy was reduced, suggesting potential inhibition in the presence of PUFAs.
Food and Chemical Toxicology
Autophagy is a catabolic pathway required for the clearance and recycling of cytoplasmic material... more Autophagy is a catabolic pathway required for the clearance and recycling of cytoplasmic materials. Upregulation and dysfunction of autophagy contributes to the pathology of cancer and neurogenerative diseases, respectively. To define the molecular mechanisms that control autophagic flux it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of 9 cell lines expressing HaloTagged autophagy factors from their endogenous loci, we systematically analyze the abundance, single-molecule dynamics, and autophagosome association kinetics of a wide variety of autophagy proteins involved in the initiation and maturation of the autophagosome. Our results reveal that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles and tethering of these ATG9 vesicles to donor membranes by ATG2 is a key step in phagophore maturation. In addition, we demonstrate that the overall lifetime of an autophagosome is...
Up to the 60% of the Western diet is characterized by consumption of high amounts of Ultra-proces... more Up to the 60% of the Western diet is characterized by consumption of high amounts of Ultra-processed foods (UPFs). From a nutritional standpoint, the high consumption of UPFs, including Fast-foods and Ready-to-Eat (RTE), emerges as a critical topic for public health linking nutritional quality and food safety. In the present work, we provide a systematic database of fatty acids composition of the most consumed UPFs in the US mid-western region. Saturate (SFA) and monounsaturated (MUFA) fatty acids were predominant in both FF and RTE, while health-beneficial polyunsaturated (PUFA) fatty acids were abundant only in seafood meals. Nutritional and non-nutritional attributes were correlated, finding positive correlation between caloric and sodium content. The significance of this study relies on providing new quantitative data for the most consumed UPFs, with the opportunity to define priority interventions for more advanced precision nutrition, especially for vulnerable populations.
Advances in Experimental Medicine and Biology, 2019
Flavonoids are plant secondary metabolites that act as protectants against harmful effects of UV-... more Flavonoids are plant secondary metabolites that act as protectants against harmful effects of UV-B radiation inasmuch as biotic stress, conferring at the same time pigmentation of fruits and leaves [67]. The term "flavonoid" refers to phenolics having a basic skeleton of diphenylpropane (C6-C3-C6), which consists of two aromatic rings linked through three carbons that usually form an oxygenated heterocycle [25, 52]. Flavonoids are broken down into several different sub-categories based on their chemical structure. The main subclasses commonly found in food items are: flavonols, flavones, flavanones, flavan-3-ols, proanthocyanidins, and anthocyanins [44, 67]. Figure 19.1 depicts the major classification of flavonoids according to their chemical structure. Their occurrence in food matrices has been extensively reviewed [39, 44], and has been subject of extensive research in the last decades. Table 19.1 contains a few examples of compounds from each of the subcategory, with the fruit (berry) in which they are commonly found. The monomeric unit of flavonoids can dimerize and polymerize to form other important high molecular weight molecules; this is the case of proanthocyanidins, that are polymers of flavan-3-ols or flavanols. Not only do these compounds act as plant protectants, but they can also be very beneficial to human health. Cohorts studies performed in the early '90 have shown that dietary consumption of flavonoids was inversely associated with morbidity and mortality from coronary heart disease [31, 32]. These findings have opened an intensive field of research on the effects of flavonoids and flavonoids-rich food extracts in cardiovascular diseases (CVD) progression, particularly in the modulating CVD-associated oxidative stress and inflammation. In this short review, we will summarize the current findings in flavonoids beneficial effects in preventing CVD through inhibition of initial stages of CVD progression. Given the magnitude of scientific literature in the field, we will focus on two strictly mechanistic aspects: inhibition of chemical-induced LDL oxidation, and the effect of flavonoids in the monocyte/macrophages activation pathways.
medRxiv, 2020
Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutrition... more Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutritional source during the first six months of life. IF is available in a variety of commercial presentations, although from a manufacturing standpoint, they can be categorized in powder- (PIF) or liquid- (LIF) based formulations. Herein, thirty commercial IFs were analyzed in their oxidative and non-oxidative lipidomics profiles. Results show that LIFs have a characteristic lipidomic fingerprint, enriched in an oxidated form of cholesterol, and a lower load of phytosterols. We identified 7-ketocholesterol - a major end-product of cholesterol oxidation - as a potential biomarker of IF manufacturing. Our data allowed re-classification of IF based on their metabolomic fingerprint, resulting in three groups assigned with low-to-high oxidative status. Finally, we modeled the dietary intake for cholesterol, sterols, and 7-ketocholesterol in the first year of life. The database provided in this stud...
Journal of Clinical Lipidology, 2020
Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. Ho... more Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in animal models. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique in determining that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic stimulation activation at a single partic...
Biophysical Journal, 2018
HIV enters human T cells through the fusion of viral and host-cell membranes. This fusion process... more HIV enters human T cells through the fusion of viral and host-cell membranes. This fusion process is mediated by a surface protein, gp41, and the platform provided by the cholesterol-rich viral membrane. The membrane-bound region of gp41 plays critical roles in this fusion process and is a major target of anti-gp41 antibodies and vaccine design. Here, EPR and spin-labeling techniques were used to dissect the interactions between the viral membrane and the membrane-bound region of gp41, including the membrane proximal ectodomain region (MPER) and the transmembrane domain (TM). The analyses revealed a helix-hinge-helix structure of the MPER sitting on the membrane headgroup region in a manner impacted by para-hinge and other residues. The MPER exerts its fusogenic activity by perturbing membrane properties and by inducing significant lipid order and membrane permeability changes. The MPER-induced membrane property changes are modulated by cholesterol content and the TM. In addition, anti-gp41 neutralizing antibodies disrupt MPER-lipid interactions by altering MPER conformation on the membrane and by abolishing the membrane-perturbing activity of the MPER. These findings suggest that the membrane-bound region of gp41 facilitates HIV infection through its interaction with the cholesterol-rich viral membrane, and this interaction is disrupted by anti-gp41 neutralizing antibodies.
Chemical communications (Cambridge, England), Jan 14, 2018
Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the s... more Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.
Chemphyschem : a European journal of chemical physics and physical chemistry, Oct 19, 2018
Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, and re... more Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, and requires enzymatic partners as well as environmental factors that tune its complex dynamic activity. P450 and its reducing counterparts are membrane-bound proteins which are believed to dynamically interact to form functional complexes. Increasing experimental evidence signifies the role (s) of protein-lipid interactions in P450's catalytic function and efficiency. The challenges posed by the membrane have severely limited high-resolution understanding of the molecular interfaces of these interactions. Nevertheless, recent NMR studies have provided piercing insights into the dynamic structural interactions that enable the function of P450. In this review, we will discuss different biomimetic approaches relevant to unveil molecular interplays at the membrane, focusing on our recent work on lipid-nanodiscs. We also highlight the need to expand the use of nanodiscs, and the power of a com...
Food and Chemical Toxicology, 2018
Food consumption can lead to the accumulation of certain chemical compounds able to exert toxic a... more Food consumption can lead to the accumulation of certain chemical compounds able to exert toxic activities against humans. Of mayor interests are those molecules generated during food processing and handling, since their occurrence and distribution depend of many intrinsic and extrinsic factors. Cholesterol-a lipid constituent of mammalian cells-is the precursor of several toxic molecules known as cholesterol oxidation products (COPs). In the last decades, it has been demonstrated that food processing can dramatically trigger COPs accumulation in meats, eggs, dairy products, fish and poultry. On the other hand, countless scientific evidences have pointed out the highly toxic and pathogenic activities of COPs, from cancer stimulation to neurodegenerative disorders, via molecular mechanisms that are largely unexplored. The aim of this review is to merge the evidence on COPs accumulation in foods and their toxic activities through dietary intake, as from in vivo and in vitro studies. We consider that is imperative to systematically monitor the formation of COPs and bridge these quantitative efforts with risk exposure assessment on sensitive populations.
Angewandte Chemie (International ed. in English), Jan 19, 2018
Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, th... more Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.
Biophysical Journal, 2017
The degree of lipid saturation is a key feature of membranes that determines lipid packing and me... more The degree of lipid saturation is a key feature of membranes that determines lipid packing and membrane fluidity. It must be tightly regulated to guarantee 508a Wednesday,
F1000Research, 2017
Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which ... more Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts-cytochrome P450-reductase and cytochrome b 5 -are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s) played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed...
Scientific reports, Jan 10, 2017
Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP45... more Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. The...
Journal of the American Chemical Society, Apr 28, 2017
Cytochrome P450-Reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It i... more Cytochrome P450-Reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature dependent Fluorescence Recovery After Photobleaching (FRAP). The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramati...
Plant Physiology, 2016
Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ult... more Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137 P-X-P-X 140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.
Steroids, 2016
In this work we studied the effect of polyunsaturated fatty acids (PUFAs) methyl esters on choles... more In this work we studied the effect of polyunsaturated fatty acids (PUFAs) methyl esters on cholesterol photo-induced oxidation. The oxidative routes were modeled with a chemical reaction network (CRN), which represents the first application of CRN to the oxidative degradation of a food-related lipid matrix. Docosahexaenoic acid (DHA, T-I), eicosapentaenoic acid (EPA, T-II) and a mixture of both (T-III) were added to cholesterol using hematoporphyrin as sensitizer, and were exposed to a fluorescent lamp for 48 h. High amounts of Type I cholesterol oxidation products (COPs) were recovered (epimers 7α-and 7β-OH, 7-keto and 25-OH), as well as 5β,6β-epoxy. Fitting the experimental data with the CRN allowed characterizing the associated kinetics. DHA and EPA exerted different effects on the oxidative process. DHA showed a protective effect to 7-hydroxy derivatives, whereas EPA enhanced side-chain oxidation and 7β-OH kinetic rates. The mixture of PUFAs increased the kinetic rates several fold, particularly for 25-OH. With respect to the control, the formation of β-epoxy was reduced, suggesting potential inhibition in the presence of PUFAs.