ABSTRACT
The pathogenicity of group A Streptococcus (GAS) is mediated by direct bacterial invasivity and toxin-associated damage. Among the extracellular products, the exotoxin streptolysin O (SLO) is produced by almost all GAS strains. SLO is a pore forming toxin (PFT) hemolitically active and extremely toxic in vivo. Recent evidence suggests that human serum albumin (HSA), the most abundant protein in plasma, is a player in the innate immunity "orchestra." We previously demonstrated that HSA acts as a physiological buffer, partially neutralizing Clostridioides difficile toxins that reach the bloodstream after being produced in the colon. Here, we report the in vitro and ex vivo capability of HSA to neutralize the cytotoxic and hemolytic effects of SLO. HSA binds SLO with high affinity at a non-conventional site located in domain II, which was previously reported to interact also with C. difficile toxins. HSA:SLO recognition protects HEp-2 and A549 cells from cytotoxic effects and cell membrane permeabilization induced by SLO. Moreover, HSA inhibits the SLO-dependent hemolytic effect in red blood cells isolated from healthy human donors. The recognition of SLO by HSA may have a significant protective role in human serum and sustains the emerging hypothesis that HSA is an important constituent of the innate immunity system.
Subject(s)
Erythrocytes/immunology , Hemolysis/immunology , Immunity, Innate , Serum Albumin, Human/immunology , Streptococcus pyogenes/immunology , Streptolysins/immunology , A549 Cells , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Humans , Serum Albumin, Human/chemistry , Streptococcus pyogenes/chemistry , Streptolysins/chemistryABSTRACT
The aim of this study was to evaluate the performance of "Micro Biological Survey - MBS Test" in the enumeration of bacterial load in cow raw milk. The MBS test is based on a colorimetric method recently developed and patented by "Roma Tre" University, Italy. The evaluation of the performance of the MBS method was carried out by comparison with plate count at 30°C (gold standard) and flow cytometry. Thirteen independent set of experiments were performed analyzing a total of 104 samples of cow raw milk with the selected methods. Results obtained using the MBS method are comparable with those obtained with the plate count method at 30°C (CFU/mL) and flow cytometry technology; in particular, the results obtained with the MBS method are very close to plate count's at 30°C. On the other hand, there are statistically significant differences between these two methods' and flow cytometry technology's results that could be due to the different experimental conditions.
ABSTRACT
PURPOSE: Recently, it has been demonstrated that Raman spectroscopy is able to differentiate between healthy parathyroid tissues and parathyroid adenoma based on the basis of a specific molecular fingerprint. However, to our knowledge, no previous studies have been performed to evaluate the metabolic profile of parathyroid adenoma. Therefore, we designed a proof of concept study aimed to investigate the glucose/fatty acid metabolisms, in addition to the mitochondrial changes, in solitary parathyroid adenoma and in healthy parathyroid glands. METHODS: Nine females with primary hyperparathyroidism due to a solitary parathyroid adenoma and formal surgical indication for parathyroidectomy have been enrolled. At the time of surgery, the removed specimens were immediately submitted unfixed and a tissue slice of about 0.5 cm in diameter was obtained from the nodular lesion. The expression of selected metabolic enzymes and proteins has been evaluated by western blot analysis, using human parathyroid whole tissue lysates as control. RESULTS: Data obtained highlighted an increase, compared with the healthy group, of: (i) the glucose uptake by the GLUT-1 receptor and its phosphorylation by hexokinase II (HXKII); (ii) the expression of 3-phosphoglycerate dehydrogenase (3-PGDH) and glucose-6-phosphate dehydrogenase (G6PD); (iii) lipids biosynthesis; and (iv) cytochrome c expression. CONCLUSIONS: Our findings highlight for the first time the parathyroid adenoma metabolic hallmarks that could represent potential molecular targets usable for the development of new pharmacological treatments, allowing to reduce surgical parathyroidectomy.
Subject(s)
Adenoma , Parathyroid Neoplasms , Adenoma/surgery , Female , Humans , Metabolome , Parathyroid Glands , Parathyroid Hormone , Parathyroid Neoplasms/surgery , ParathyroidectomyABSTRACT
Neonicotinoids are a widely used class of insecticides that target the acetylcholine recognition site of the nicotinic acetylcholine receptors in the central nervous system of insects. Although neonicotinoids display a high specificity for insects, their use has been recently debated since several studies led to the hypothesis that they may have adverse ecological effects and potential risks to mammals and even humans. Due to their hydrophobic nature, neonicotinoids need specific carriers to allow their distribution in body fluids. Human serum albumin (HSA), the most abundant plasma protein, is a key carrier of endogenous and exogenous compounds. The in silico docking and ligand binding properties of acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam to HSA are here reported. Neonicotinoids bind to multiple fatty acid (FA) binding sites, preferentially to the FA1 pocket, with high affinity. Values of the dissociation equilibrium constant for neonicotinoid binding FA1 of HSA (i.e., calc Kn ) derived from in silico docking simulations (ranging between 3.9 × 10-5 and 6.3 × 10-4 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (i.e., exp Kn ; ranging between 2.1 × 10-5 and 6.9 × 10-5 M). Accounting for the HSA concentration in vivo (~7.5 10-4 M), values of Kn here determined suggest that the formation of the HSA:neonicotinoid complexes may occur in vivo. Therefore, HSA appears to be an important determinant for neonicotinoid transport and distribution to tissues and organs, particularly to the liver where they are metabolized.
Subject(s)
Neonicotinoids/metabolism , Serum Albumin, Human/metabolism , Humans , Insecticides/chemistry , Insecticides/metabolism , Insecticides/pharmacokinetics , Molecular Docking Simulation , Neonicotinoids/chemistry , Neonicotinoids/pharmacokinetics , Serum Albumin, Human/chemistry , ThermodynamicsABSTRACT
BACKGROUND: Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multi-domain macromolecule with at least nine binding sites for endogenous and exogenous ligands. HSA displays an extraordinary ligand binding capacity as a depot and carrier for many compounds including most acidic drugs. Consequently, HSA has the potential to influence the pharmacokinetics and pharmacodynamics of drugs. OBJECTIVE: In this review, the structural determinants of drug binding to the multiple sites of HSA are analyzed and discussed in detail. Moreover, insight into the allosteric and competitive mechanisms underpinning drug recognition, delivery, and efficacy are analyzed and discussed. CONCLUSION: As several factors can modulate drug binding to HSA (e.g., concurrent administration of drugs competing for the same binding site, ligand binding to allosteric-coupled clefts, genetic inherited diseases, and post-translational modifications), ligand binding to HSA is relevant not only under physiological conditions, but also in the pharmacological therapy management.
Subject(s)
Binding Sites , Serum Albumin, Human , Humans , Ligands , Protein BindingABSTRACT
Ruxolitinib is a type I JAK inhibitor approved by FDA for targeted therapy of Philadelphia-negative myeloproliferative neoplasms (MPNs), all characterized by mutations activating the JAK2/STAT signaling pathway. Treatment with ruxolitinib improves constitutional symptoms and splenomegaly. However, patients can become resistant to treatment and chronic therapy has only a mild effect on molecular/pathologic remissions. Drugs interaction with plasma proteins, i.e. human serum albumin (HSA), is an important factor affecting the intensity and duration of their pharmacological actions. Here, the ruxolitinib recognition by the fatty acid binding sites (FAs) 1, 6, 7, and 9 of HSA has been investigated from the bioinformatics, biochemical and/or biological viewpoints. Docking simulations indicate that ruxolitinib binds to multiple sites of HSA. Ruxolitinib binds to the FA1 and FA7 sites of HSA with high affinity (Kr = 3.1 µM and 4.6 µM, respectively, at pH 7.3 and 37.0 °C). Moreover, HSA selectively blocks, in a dose dependent manner, the cytotoxic activity of ruxolitinib in JAK2V617F+ cellular models for MPN, in vitro. Furthermore this event is accompanied by changes in the cell cycle, p27Kip1 and cyclin D3 levels, and JAK/STAT signaling. Given the high plasma concentration of HSA, ruxolitinib trapping may be relevant in vivo.
Subject(s)
Enzyme Inhibitors/metabolism , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Pyrazoles/metabolism , Serum Albumin, Human/chemistry , Serum Albumin, Human/metabolism , Amino Acid Substitution , Binding Sites , Cell Line , Computational Biology , Enzyme Inhibitors/pharmacology , Fatty Acids/chemistry , Fatty Acids/metabolism , Humans , In Vitro Techniques , Janus Kinase 2/antagonists & inhibitors , K562 Cells , Kinetics , Molecular Docking Simulation , Mutant Proteins/antagonists & inhibitors , Mutation, Missense , Myeloproliferative Disorders/blood , Myeloproliferative Disorders/drug therapy , Myeloproliferative Disorders/genetics , Nitriles , Pyrazoles/pharmacology , Pyrimidines , Signal Transduction , ThermodynamicsABSTRACT
This study aimed to assess the predictors of acute kidney injury (AKI) during colistin therapy in a cohort of patients with bloodstream infections (BSI) due to colistin-susceptible Gram-negative bacteria, focusing on the role of serum albumin levels. The study consisted of two parts: (1) a multicentre retrospective clinical study to assess the predictors of AKI during colistin therapy, defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria; and (2) bioinformatic and biochemical characterization of the possible interaction between human serum albumin and colistin. Among the 170 patients included in the study, 71 (42%), 35 (21%), and 11 (6%) developed KDIGO stage 1 (K1-AKI), KDIGO stage 2 (K2-AKI), and KDIGO stage 3 (K3-AKI), respectively. In multivariable analyses, serum albumin <2.5 g/dL was independently associated with K1-AKI (subdistribution hazard ratio [sHR] 1.85, 95% confidence interval [CI] 1.17-2.93, p = 0.009) and K2-AKI (sHR 2.37, 95% CI 1.15-4.87, p = 0.019). Bioinformatic and biochemical analyses provided additional information nurturing the discussion on how hypoalbuminemia favors development of AKI during colistin therapy. In conclusion, severe hypoalbuminemia independently predicted AKI during colistin therapy in a large cohort of patients with BSI due to colistin-susceptible Gram-negative bacteria. Further study is needed to clarify the underlying causal pathways.
Subject(s)
Acute Kidney Injury/blood , Acute Kidney Injury/diagnosis , Anti-Bacterial Agents/adverse effects , Colistin/adverse effects , Hypoalbuminemia/blood , Acute Kidney Injury/etiology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/therapeutic use , Colistin/chemistry , Colistin/therapeutic use , Computational Biology/methods , Female , Humans , Incidence , Male , Models, Molecular , Molecular Conformation , Retrospective Studies , Sepsis/blood , Sepsis/complications , Sepsis/drug therapy , Serum Albumin, Human/chemistry , Serum Albumin, Human/metabolism , Severity of Illness Index , Structure-Activity RelationshipABSTRACT
Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease. Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos. Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins. Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Clostridioides difficile/metabolism , Enterotoxins/metabolism , Serum Albumin, Human/metabolism , Animals , Caco-2 Cells , Cell Line, Tumor , Humans , Zebrafish/metabolismABSTRACT
Fipronil is a broad-spectrum pesticide widely used in agriculture, horticulture, and forestry. Because fipronil can cause a variety of toxic effects in animals and humans, its use is authorized as a pesticide in veterinary medicinal products for pets, but not for the treatment of livestock animals whose products are intended for consumption. Recently, however, the presence of fipronil residues has been detected in the eggs and meat of layer hens from farms located in different European countries. Given the relevance of fipronil toxicity for human health, it is important to gain information concerning its fate in the human body, including its binding mode to human serum albumin (HSA), the most abundant protein in plasma. Here, the inhibition of heme-Fe(III) binding to the fatty acid site 1 (FA1) of HSA by fipronil is reported. Docking simulations support functional data, indicating that the FA1 site is the preferential cleft for fipronil recognition by HSA. The affinity of fipronil for HSA (Kf = 1.9 × 10-6 M, at pH 7.3, and 20.0°C) may be relevant in vivo. Indeed, HSA could play a pivotal role in fipronil transport and scavenging, thus reducing the pesticide-free plasmatic levels, with consequent reduced systemic toxicity. In turn, fipronil binding to the FA1 site of HSA could impair the recognition of endogenous and exogenous molecules.
Subject(s)
Pesticides/chemistry , Protein Conformation/drug effects , Pyrazoles/chemistry , Serum Albumin, Human/chemistry , Allosteric Regulation/drug effects , Animals , Binding Sites/drug effects , Chickens , Fatty Acids , Humans , Kinetics , Pesticides/adverse effects , Pesticides/pharmacology , Protein Binding/drug effects , Pyrazoles/adverse effects , Pyrazoles/pharmacology , Serum Albumin, Human/drug effectsABSTRACT
Prenatal exposure to the antiepileptic and mood stabilizer valproic acid (VPA) is an environmental risk factor for autism spectrum disorders (ASD), although recent epidemiological studies show that the public awareness of this association is still limited. Based on the clinical findings, prenatal VPA exposure in rodents is a widely used preclinical model of ASD. However, there is limited information about the precise biochemical mechanisms underlying the link between ASD and VPA. Here, we tested the effects of increasing doses of VPA on behavioral features resembling core and secondary symptoms of ASD in rats. Only when administered prenatally at the dose of 500mg/kg, VPA induced deficits in communication and social discrimination in rat pups, and altered social behavior and emotionality in the adolescent and adult offspring in the absence of gross malformations. This dose of VPA inhibited histone deacetylase in rat embryos and favored the formation of DNA double strand breaks (DSB), but impaired their repair. The defective DSB response was no more visible in one-day-old pups, thus supporting the hypothesis that unrepaired VPA-induced DNA damage at the time of neural tube closure may underlie the autistic-like traits displayed in the course of development by rats prenatally exposed to VPA. These experiments help to understand the neurodevelopmental trajectories affected by prenatal VPA exposure and identify a biochemical link between VPA exposure during gestation and ASD.
Subject(s)
Autistic Disorder/chemically induced , Autistic Disorder/metabolism , DNA Damage/drug effects , DNA Repair/drug effects , Prenatal Exposure Delayed Effects , Valproic Acid/toxicity , Animals , Anxiety/chemically induced , Anxiety/genetics , Anxiety/metabolism , Autistic Disorder/genetics , DNA Damage/physiology , DNA Repair/physiology , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Histones/metabolism , Male , Pregnancy , Rats, Wistar , Social Behavior , Vocalization, AnimalABSTRACT
The endocannabinoid system is a unique neuromodulatory system that affects a wide range of biological processes and maintains the homeostasis in all mammal body systems. In recent years, several pharmacological tools to target endocannabinoid neurotransmission have been developed, including direct and indirect cannabinoid agonists and cannabinoid antagonists. Due to their hydrophobic nature, cannabinoid agonists and antagonists need to bind specific transporters to allow their distribution in body fluids. Human serum albumin (HSA), the most abundant plasma protein, is a key determinant of drug pharmacokinetics. As HSA binds both the endocannabinoid anandamide and the active ingredient of Cannabis sativa, Δ-9-tetrahydrocannabinol, we hypothesize that HSA can be the most important carrier of cannabinoid drugs. In silico docking observations strongly indicate that HSA avidly binds the indirect cannabinoid agonists URB597, AM5206, JZL184, JZL195, and AM404, the direct cannabinoid agonists WIN55,212-2 and CP55,940, and the prototypical cannabinoid antagonist/inverse agonist SR141716. Values of the free energy for cannabinoid drugs binding to HSA range between -5.4 kcal mol-1 and -10.9 kcal mol-1 . Accounting for the HSA concentration in vivo (â¼ 7.5 × 10-4 M), values of the free energy here determined suggest that the formation of the HSA:cannabinoid drug complexes may occur in vivo. Therefore, HSA appears to be an important determinant for cannabinoid efficacy and may guide the choice of the drug dose regimen to optimize drug efficacy and to avoid drug-related toxicity. © 2017 IUBMB Life, 69(11):834-840, 2017.
Subject(s)
Cannabinoid Receptor Agonists/metabolism , Cannabinoid Receptor Antagonists/metabolism , Carrier Proteins/metabolism , Endocannabinoids/metabolism , Serum Albumin, Human/metabolism , Animals , Binding Sites , Biological Transport , Cannabinoid Receptor Agonists/chemistry , Cannabinoid Receptor Antagonists/chemistry , Carrier Proteins/chemistry , Endocannabinoids/chemistry , Humans , Kinetics , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Serum Albumin, Human/chemistry , Substrate Specificity , Synaptic Transmission/physiology , ThermodynamicsABSTRACT
Cantharidin, a monoterpene isolated from the insect blister beetle, has long been used as a medicinal agent in the traditional Chinese medicine. Cantharidin inhibits a subgroup of serine/threonine phosphatases, thus inducing cell growth inhibition and cytotoxicity. Cantharidin has anticancer activity in vitro, since it is able of inducing p53-dependent apoptosis and double-strand breakage of DNA in cancer cells. Although the toxicity of cantharidin to the gastrointestinal and urinary tracts prevents its medical use, it is a promising lead compound for chemical modification to develop new anticancer therapeutics. In fact, cantharidin does not cause myelosuppression and displays anticancer activity against cells with a multidrug resistance phenotype. Here, the competitive inhibitory effect of cantharidin on heme-Fe(III) binding to the fatty acid site 1 (FA1) of human serum albumin (HSA) is reported. Docking and molecular dynamics simulations support functional data indicating the preferential binding of cantharidin to the FA1 site of HSA. Present results may be relevant in vivo as HSA could transport cantharidin, which in turn could affect heme-Fe(III) scavenging by HSA.
Subject(s)
Binding, Competitive , Cantharidin/pharmacology , Fatty Acids/metabolism , Heme/metabolism , Serum Albumin, Human/metabolism , Cantharidin/chemistry , Dansyl Compounds/chemistry , Dansyl Compounds/metabolism , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding/drug effects , Sarcosine/analogs & derivatives , Sarcosine/chemistry , Sarcosine/metabolism , Serum Albumin, Human/chemistry , ThermodynamicsABSTRACT
In 2000, the third member of the globin family was discovered in human and mouse brain and named neuroglobin (Ngb). Ngb is a monomeric 3/3 globin structurally similar to myoglobin and to the α- and ß-chains of hemoglobin, however it displays a bis-histidyl six-coordinate heme-Fe atom. Therefore, ligand binding to the Ngb metal center is limited from the dissociation of the distal His(E7)64-Fe bond. From its discovery, more than 500 papers on Ngb structure, expression, reactivity, and localization have been published to highlight its biochemical properties and its role(s) in health and disease. In vivo experiments have shown that increased levels of Ngb significantly protect both heart and brain from hypoxic/ischemic and oxidative stress-related insults, whereas decreased Ngb levels lead to an exacerbation of tissue injuries. Although some contradictory data emerged, human Ngb overexpression has been hypothesized to protect neurons from mitochondrial dysfunctions and neurodegenerative disorders such as Alzheimer's disease, and to play a shielding role in cancer cells. Recently, the recognition of Ngb interactors and inducers enlarges the functions of this stress-inducible globin, opening new therapeutic approaches to prevent neuronal cell death. Here, structural and functional aspects of human Ngb are examined critically to highlight its roles in health and disease.
Subject(s)
Disease Susceptibility , Globins/chemistry , Globins/physiology , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/physiology , Structure-Activity Relationship , Amino Acid Sequence , Animals , Brain/metabolism , Evolution, Molecular , Gene Expression Regulation , Humans , Ligands , Neuroglobin , Oxidation-Reduction , Polymorphism, Genetic , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Transport , Signal Transduction , ThermodynamicsABSTRACT
Lactoferrin is an iron-binding protein present in large quantities in colostrum and in breast milk, in external secretions and in polymorphonuclear leukocytes. Lactoferrin's main function is non-immune protection. Among several protective activities shown by lactoferrin, those displayed by orally administered lactoferrin are: (i) antimicrobial activity, which has been presumed due to iron deprivation, but more recently attributed also to a specific interaction with the bacterial cell wall and extended to viruses and parasites; (ii) immunomodulatory activity, with a direct effect on the development of the immune system in the newborn, together with a specific antinflammatory effects; (iii) a more recently discovered anticancer activity. It is worth noting that most of the protective activities of lactoferrin have been found, sometimes to a greater extent, also in peptides derived from limited proteolysis of lactoferrin that could be generated after lactoferrin ingestion. Lactoferrin could therefore be considered an ideal nutraceutic product because of its relatively cheap production from bovine milk and of its widely recognized tolerance after ingestion, along with its well demonstrated protective activities. The most important protective activities shown by orally administered bovine lactoferrin are reviewed in this article.
ABSTRACT
Lipocalins are an evolutionarily conserved family of proteins that bind and transport a variety of exogenous and endogenous ligands. Lipocalins share a conserved eight anti-parallel ß-sheet structure. Among the different lipocalins identified in humans, α-1-acid glycoprotein (AGP), apolipoprotein D (apoD), apolipoprotein M (apoM), α1-microglobulin (α1-m) and retinol-binding protein (RBP) are plasma proteins. In particular, AGP is the most important transporter for basic and neutral drugs, apoD, apoM, and RBP mainly bind endogenous molecules such as progesterone, pregnenolone, bilirubin, sphingosine-1-phosphate, and retinol, while α1-m binds the heme. Human serum albumin (HSA) is a monomeric all-α protein that binds endogenous and exogenous molecules like fatty acids, heme, and acidic drugs. Changes in the plasmatic levels of lipocalins and HSA are responsible for the onset of pathological conditions associated with an altered drug transport and delivery. This, however, does not necessary result in potential adverse effects in patients because many drugs can bind both HSA and lipocalins, and therefore mutual compensatory binding mechanisms can be hypothesized. Here, molecular and clinical aspects of ligand transport by plasma lipocalins and HSA are reviewed, with special attention to their role as alterative carriers in health and disease.
Subject(s)
Drug Carriers/metabolism , Lipocalins/metabolism , Serum Albumin/metabolism , Biological Transport , Drug Carriers/chemistry , Humans , Ligands , Lipocalins/chemistry , Models, Molecular , Pharmaceutical Preparations/administration & dosage , Pharmaceutical Preparations/metabolism , Protein Binding , Protein Conformation , Serum Albumin/chemistryABSTRACT
Iron plays a key role in a wide range of metabolic and signalling functions representing an essential nutrient for almost all forms of life. However, the ferric form is hardly soluble, whereas the ferrous form is highly toxic. Thus, in biological fluids, most of the iron is sequestered in iron- or haem-binding proteins and the level of free iron is low, making haem and iron acquisition a challenge for pathogenic bacteria during infections. Although toxic to the host, free haem is a major and readily available source of iron for several pathogenic microorganisms. Both Gram-positive and Gram-negative bacteria have developed several strategies to acquire free haem-Fe and protein-bound haem-Fe. Haemophores are a class of secreted and cell surface-exposed proteins promoting free-haem uptake, haem extraction from host haem proteins, and haem presentation to specific outer-membrane receptors that internalize the metal-porphyrins. Here, structural biology of bacterial haemophores is reviewed focusing on haem acquisition, haem internalization, and haem-degrading systems.
Subject(s)
Bacteria/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Heme/metabolism , Iron/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Bacteria/metabolism , Metabolic Networks and Pathways , Models, Biological , Models, Molecular , Protein ConformationABSTRACT
Ovotransferrin or conalbumin belong to the transferrin protein family and is endowed with both iron-transfer and protective activities. In addition to its well-known antibacterial properties, ovotransferrin displays other protective roles similar to those already ascertained for the homologous mammalian lactoferrin. These additional functions, in many cases not directly related to iron binding, are also displayed by the peptides derived from partial hydrolysis of ovotransferrin, suggesting a direct relationship between egg consumption and human health.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Conalbumin/pharmacology , Dietary Supplements , Egg White/chemistry , Functional Food , Diet , Humans , Iron/metabolism , Peptides/pharmacologyABSTRACT
Low-molecular-mass trypsin inhibitors from Arabidopsis thaliana, Brassica napus var. oleifera, and Sinapis alba L. (ATTI, RTI, and MTI, respectively) display more than 69% amino acid sequence identity. Among others, the amino acid sequence Cys-Ala-Pro-Arg-Ile building up the inhibitor reactive site, and the eight Cys residues forming four disulfide bridges are conserved. However, the disulfide bridge connectivity of RTI and MTI (C1-C3, C2-C4, C5-C6, and C7-C8) is different from that of ATTI Cys (C1-C8, C2-C5, C3-C6, and C4-C7). Despite the different disulfide bridge connectivity, the reactive site loop of ATTI, RTI, and MTI is solvent exposed permitting trypsin recognition. Structural considerations here reported suggest that proteins showing high amino acid sequence identity and common functional properties could display different three-dimensional structures. This may reflect high inhibitor plasticity in relation to plant-pathogen interactions, plant tissue development as well as the different redox potential of cell compartments.
Subject(s)
Brassicaceae/chemistry , Protein Folding , Trypsin Inhibitors/chemistry , Amino Acid Sequence , Disulfides/chemistry , Models, Molecular , Molecular Sequence Data , Plant Proteins/chemistry , Structural Homology, Protein , Trypsin Inhibitors/metabolism , Trypsinogen/chemistry , Trypsinogen/genetics , Trypsinogen/metabolismABSTRACT
Human serum albumin (HSA) represents an important determinant of plasma oncotic pressure and a relevant factor that modulates fluid distribution between the body compartments. Moreover, HSA (i) represents the depot and transporter of several compounds, both endogenous and exogenous, (ii) affects the pharmacokinetics of many drugs, (iii) regulates chemical modifications of some ligands, (iv) shows (pseudo-)enzymatic properties, (v) inactivates some toxic compounds, and (vi) displays anti-oxidant properties. HSA binding and (pseudo-)enzymatic properties are regulated competitively, allosterically, and by covalent modifications. While competitive inhibition of HSA binding properties is evident, allosteric mechanisms and covalent modifications affecting HSA reactivity are less clear. In several pathological conditions in which free heme-Fe levels increase, the buffering capacity of plasma hemopexin is overwhelmed and most of heme-Fe binds to the fatty acid site 1 of HSA. HSA-heme-Fe displays globin-like properties; in turn, heme-Fe modulates competitively and allosterically HSA binding and reactivity properties. Remarkably, heme-Fe-mediated HSA properties are time-dependent, representing a case for "chronosteric effects". Here, we review the drug-based modulation of (i) heme-Fe-recognition by HSA and (ii) heme-Fe-mediated reactivity.
Subject(s)
Heme/metabolism , Iron/metabolism , Pharmaceutical Preparations/metabolism , Serum Albumin/metabolism , Allosteric Regulation/physiology , Animals , Heme/chemistry , Humans , Iron/chemistry , Pharmaceutical Preparations/chemistry , Protein Binding/physiology , Protein Structure, Secondary , Serum Albumin/chemistryABSTRACT
The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of Ks, k+2, and k+2/Ks obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k+3 << k+2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.