Mechanism of Peroxynitrite Interaction with Ferric M. tuberculosis Nitrobindin: A Computational Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.

Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3-. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2- to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M-1 s-1, 9.3 M-1 s-1, 1.4 × 101 M-1 s-1, and 5.8 M-1 s-1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2--based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-ß-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-ß-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged.

Serum albumin and nucleic acids biodistribution: From molecular aspects to biotechnological applications.

Serum albumin (SA) is the most abundant protein in plasma and represents the main carrier of endogenous and exogenous compounds. Several evidence supports the notion that SA binds single and double-stranded deoxynucleotides and ribonucleotides at two sites, with values of the dissociation equilibrium constant (i.e., Kd ) ranging from micromolar to nanomolar values. This can be relevant from a physiological and pathological point of view, as in human plasma circulates cell-free nucleic acids (cfNAs), released by different tissues via apoptosis, necrosis, and secretions, circulates as single and double-stranded NAs. Albeit SA shows low hydrolytic reactivity toward DNA and RNA, the high plasma concentration of this protein and the occurrence of several SA receptors may be pivotal for sequestering and hydrolyzing cfNAs. Therefore, pathological conditions like cancer, characterized by altered levels of human SA or by altered SA post-translational modifications, may influence cfNAs distribution and metabolism. Besides, the stability, solubility, biocompatibility, and low immunogenicity make SA a golden share for biotechnological applications related to the delivery of therapeutic NAs (TNAs). Indeed, pre-clinical studies report the therapeutic potential of SA:TNAs complexes in precision cancer therapy. Here, the molecular and biotechnological implications of SA:NAs interaction are discussed, highlighting new perspectives on SA plasmatic functions.

Hydroxylamine-induced oxidation of ferrous nitrobindins.

Hemoglobin and myoglobin are generally taken as molecular models of all-α-helical heme-proteins. On the other hand, nitrophorins and nitrobindins (Nb), which are arranged in 8 and 10 ß-strands, respectively, represent the molecular models of all-ß-barrel heme-proteins. Here, kinetics of the hydroxylamine- (HA-) mediated oxidation of ferrous Mycobacterium tuberculosis, Arabidopsis thaliana, and Homo sapiens nitrobindins (Mt-Nb(II), At-Nb(II), and Hs-Nb(II), respectively), at pH 7.0 and 20.0 °C, are reported. Of note, HA displays antibacterial properties and is a good candidate for the treatment and/or prevention of reactive nitrogen species- (RNS-) linked aging-related pathologies, such as macular degeneration. Under anaerobic conditions, mixing the Mt-Nb(II), At-Nb(II), and Hs-Nb(II) solutions with the HA solutions brings about absorbance spectral changes reflecting the formation of the ferric derivative (i.e., Mt-Nb(III), At-Nb(III), and Hs-Nb(III), respectively). Values of the second order rate constant for the HA-mediated oxidation of Mt-Nb(II), At-Nb(II), and Hs-Nb(II) are 1.1 × 104 M-1 s-1, 6.5 × 104 M-1 s-1, and 2.2 × 104 M-1 s-1, respectively. Moreover, the HA:Nb(II) stoichiometry is 1:2 as reported for ferrous deoxygenated and carbonylated all-α-helical heme-proteins. A comparative look of the HA reduction kinetics by several ferrous heme-proteins suggests that an important role might be played by residues (such as His or Tyr) in the proximity of the heme-Fe atom either coordinating it or not. In this respect, Nbs seem to exploit somewhat different structural aspects, indicating that redox mechanisms for the heme-Fe(II)-to-heme-Fe(III) conversion might differ between all-α-helical and all-ß-barrel heme-proteins.

Binding of direct oral anticoagulants to the FA1 site of human serum albumin.

The anticoagulant therapy is widely used to prevent and treat thromboembolic events. Until the last decade, vitamin K antagonists were the only available oral anticoagulants; recently, direct oral anticoagulants (DOACs) have been developed. Since 55% to 95% of DOACs are bound to plasma proteins, the in silico docking and ligand-binding properties of drugs apixaban, betrixaban, dabigatran, edoxaban, and rivaroxaban and of the prodrug dabigatran etexilate to human serum albumin (HSA), the most abundant plasma protein, have been investigated. DOACs bind to the fatty acid (FA) site 1 (FA1) of ligand-free HSA, whereas they bind to the FA8 and FA9 sites of heme-Fe(III)- and myristic acid-bound HSA. DOACs binding to the FA1 site of ligand-free HSA has been validated by competitive inhibition of heme-Fe(III) recognition. Values of the dissociation equilibrium constant for DOACs binding to the FA1 site (ie, calc KDOAC ) derived from in silico docking simulations (ranging between 1.2 × 10-8 M and 1.4 × 10-6 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (ie, exp KDOAC ; ranging between 2.5 × 10-7 M and 2.2 × 10-6 M). In addition, this study highlights the inequivalence of rivaroxaban binding to mammalian serum albumin. Given the HSA concentration in vivo (~7.5 × 10-4 M), values of KDOAC here determined indicate that the formation of the HSA:DOACs complexes in the absence and presence of FAs and heme-Fe(III) may occur in vivo. Therefore, HSA appears to be an important determinant for DOACs transport.

Serum Albumin: A Multifaced Enzyme.

Human serum albumin (HSA) is the most abundant protein in plasma, contributing actively to oncotic pressure maintenance and fluid distribution between body compartments. HSA acts as the main carrier of fatty acids, recognizes metal ions, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays esterase, enolase, glucuronidase, and peroxidase (pseudo)-enzymatic activities. HSA-based catalysis is physiologically relevant, affecting the metabolism of endogenous and exogenous compounds including proteins, lipids, cholesterol, reactive oxygen species (ROS), and drugs. Catalytic properties of HSA are modulated by allosteric effectors, competitive inhibitors, chemical modifications, pathological conditions, and aging. HSA displays anti-oxidant properties and is critical for plasma detoxification from toxic agents and for pro-drugs activation. The enzymatic properties of HSA can be also exploited by chemical industries as a scaffold to produce libraries of catalysts with improved proficiency and stereoselectivity for water decontamination from poisonous agents and environmental contaminants, in the so called "green chemistry" field. Here, an overview of the intrinsic and metal dependent (pseudo-)enzymatic properties of HSA is reported to highlight the roles played by this multifaced protein.

Mycobacterial and Human Ferrous Nitrobindins: Spectroscopic and Reactivity Properties.

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural-functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

Proteomic and Bioinformatic Investigation of Altered Pathways in Neuroglobin-Deficient Breast Cancer Cells.

Neuroglobin (NGB) is a myoglobin-like monomeric globin that is involved in several processes, displaying a pivotal redox-dependent protective role in neuronal and extra-neuronal cells. NGB remarkably exerts its function upon upregulation by NGB inducers, such as 17ß-estradiol (E2) and H2O2. However, the molecular bases of NGB's functions remain undefined, mainly in non-neuronal cancer cells. Human MCF-7 breast cancer cells with a knocked-out (KO) NGB gene obtained using CRISPR/Cas9 technology were analyzed using shotgun label-free quantitative proteomics in comparison with control cells. The differential proteomics experiments were also performed after treatment with E2, H2O2, and E2 + H2O2. All the runs acquired using liquid chromatography-tandem mass spectrometry were elaborated within the same MaxQuant analysis, leading to the quantification of 1872 proteins in the global proteomic dataset. Then, a differentially regulated protein dataset was obtained for each specific treatment. After the proteomic study, multiple bioinformatics analyses were performed to highlight unbalanced pathways and processes. Here, we report the proteomic and bioinformatic investigations concerning the effects on cellular processes of NGB deficiency and cell treatments. Globally, the main processes that were affected were related to the response to stress, cytoskeleton dynamics, apoptosis, and mitochondria-driven pathways.

Neonicotinoid trapping by the FA1 site of human serum albumin.

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.

Contaminations in (meta)genome data: An open issue for the scientific community.

In recent years, the high throughput and the low cost of next-generation sequencing (NGS) technologies have led to an increase of the amount of (meta)genomic data, revolutionizing genomic research studies. However, the quality of sequencing data could be affected by experimental errors derived from defective methods and protocols. This represents a serious problem for the scientific community with a negative impact on the correctness of studies that involve genomic sequence analysis. As a countermeasure, several alignment and taxonomic classification tools have been developed to uncover and correct errors. In this critical review some of these integrated software tools and pipelines used to detect contaminations in reference genome databases and sequenced samples are reported. In particular, case studies of bacterial contaminations, contaminations of human origin, mitochondrial contaminations of ancient DNA, and cross contaminations are examined.

Kinetics of cyanide and carbon monoxide dissociation from ferrous human haptoglobin:hemoglobin(II) complexes.

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) trapping the αß dimers of Hb. In turn, the Hp:Hb complexes display heme-based reactivity. Here, the kinetics of cyanide and carbon monoxide dissociation from ferrous-ligated Hp:Hb complexes are reported at pH 7.0 and 20.0 °C. Cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb-CN- has been followed upon the dithionite-mediated conversion of ferric to ferrous-ligated Hp:Hb complexes. Values of kon for the dithionite-mediated reduction of Hp1-1:Hb(III)-CN- and Hp2-2:Hb(III)-CN- are (7.3 ± 1.1) × 106 M-1 s-1 and (6.2 ± 1.0) × 106 M-1 s-1, respectively. Values of the first-order rate constant (i.e., h) for cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb(II)-CN- are (1.2 ± 0.2) × 10-1 s-1 and (1.3 ± 0.2) × 10-1 s-1, respectively. CO dissociation from Hp:Hb(II)-CO complexes has been followed by replacing CO with NO. Values of the first-order rate constant (i.e., l) for CO dissociation from Hp1-1:Hb(II)-CO are (1.4 ± 0.2) × 10-2 s-1 and (6.2 ± 0.8) × 10-3 s-1, and those from Hp2-2:Hb(II)-CO are (1.3 ± 0.2) × 10-2 s-1 and (7.3 ± 0.9) × 10-3 s-1. Values of kon, h, and l correspond to those reported for the R-state of tetrameric Hb and isolated α and ß chains. This highlights the view that the conformation of the Hb αß-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. Furthermore, unlike ferric Hb(III), ligated ferrous Hb(II) does not show an assembly-linked structural change.

Ferric nitrosylated myoglobin catalyzes peroxynitrite scavenging.

Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of ·NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. ·NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., ·NOkon) is (6.8 ± 0.7) × 104 M-1 s-1 and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., ·NOkoff) is 3.1 ± 0.3 s-1. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., ·NOkoff/·NOkon = (4.6 ± 0.7) × 10-5 M) is virtually the same as that directly measured (i.e., ·NOK = (3.8 ± 0.5) × 10-5 M). In the absence of ·NO, Mb(III) catalyzes the conversion of peroxynitrite to NO3-, the value of the second-order rate constant (i.e., Pkon) being (1.9 ± 0.2) × 104 M-1 s-1. However, in the presence of ·NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (Pkon* = (2.8 ± 0.3) × 103 M-1 s-1). These data expand the multiple roles of ·NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and ·NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO.

NO Scavenging through Reductive Nitrosylation of Ferric Mycobacterium tuberculosis and Homo sapiens Nitrobindins.

Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins (Mt-Nb(III) and Hs-Nb(III), respectively). The conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is a monophasic process, suggesting that over the explored NO concentration range (between 2.5 × 10-5 and 1.0 × 10-3 M), NO binding is lost in the mixing time (i.e., NOkon ≥ 1.0 × 106 M-1 s-1). The pseudo-first-order rate constant for the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) (i.e., k) is not linearly dependent on the NO concentration but tends to level off, with a rate-limiting step (i.e., klim) whose values increase linearly with [OH-]. This indicates that the conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is limited by the OH--based catalysis. From the dependence of klim on [OH-], the values of the second-order rate constant kOH- for the reductive nitrosylation of Mt-Nb(III)-NO and Hs-Nb(III)-NO were obtained (4.9 (±0.5) × 103 M-1 s-1 and 6.9 (±0.8) × 103 M-1 s-1, respectively). This process leads to the inactivation of two NO molecules: one being converted to HNO2 and another being tightly bound to the ferrous heme-Fe(II) atom.

Potentiation of paclitaxel effect by resveratrol in human breast cancer cells by counteracting the 17ß-estradiol/estrogen receptor α/neuroglobin pathway.

Neuroglobin (NGB), an antiapoptotic protein upregulated by 17ß-estradiol (E2), is part of E2/estrogen receptor α (ERα) pathway pointed to preserve cancer cell survival in presence of microenvironmental stressors including chemotherapeutic drugs. Here, the possibility that resveratrol (Res), an anticancer plant polyphenol, could increase the susceptibility of breast cancer cells to paclitaxel (Pacl) by affecting E2/ERα/NGB pathway has been evaluated. In MCF-7 and T47D (ERα-positive), but not in MDA-MB 231 (ERα-negative) nor in SK-N-BE (ERα and ERß positive), Res decreases NGB levels interfering with E2/ERα-induced NGB upregulation and with E2-induced ERα and protein kinase B phosphorylation. Although Res treatment does not reduce cell viability by itself, this compound potentiates Pacl proapoptotic effects. Notably, the increase of NGB levels by NGB expression vector transfection prevents Pacl or Res/Pacl effects. Taken together, these findings indicate a new Res-based mechanism that acts on tumor cells impairing the E2/ERα/NGB signaling pathways and increasing cancer cell susceptibility to chemotherapeutic agent.

No lanthanides-based catalysis in eukaryotes.

In the "Hypothesis" paper entitled "Lanthanides-Based Catalysis in Eukaryotes" (IUBMB Life 2018 Nov;70(11):1067-1075), we analyzed the possibility that Ce3+ -dependent methanol dehydrogenases (MDHs) could be found not only in archaea and bacteria but also in eukaryotic organisms. This hypothesis was based on the observation that MDHs protein sequences carrying the signature of Ce3+ -based active sites could be found in genome-derived proteomes of the eukaryotes Plasmodium yoelii yoelii, Nephila clavipes, Hyalella azteca, Pantholops hodgsonii, and Homo sapiens. Data were analyzed following standard procedures employed in the study of phylogenetic relationships among members of protein families and their occurrence in diverse organisms. Furthermore, the study relied on current annotations of protein sequences in the nonredundant protein sequences database, which we did not have any element to doubt about. After the publication of this hypothesis, following analyses carried out by Prof. Huub Op den Camp (Department of Microbiology, Faculty of Science, Radboud University, 6500 GL Nijmegen, The Netherlands), evidence has emerged that the sequences of the putative eukaryotic homologs of bacterial lanthanide-dependent MDHs, identified in our work, either derive from wrong annotation in GenBank or from undetected and pervasive bacterial contamination of the corresponding genomes. Thus, even though our study was technically correct, we were induced to support the initial hypothesis due to annotation errors and undetected bacterial contamination of the relevant genomes in the nucleotide sequences database. Therefore, at present, the hypothesis put forward in our article is not backed up by the currently available data. On a different note, this issue calls for a much higher attention on the integrity/correctness of the data deposited in the sequence databases, a need already highlighted in the literature also for the opposite problem, that is, human contamination of genomic data of other organisms. © 2018 IUBMB Life, 71(3):398-399, 2019.

Reductive nitrosylation of ferric microperoxidase-11.

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c, which is considered as a heme-protein model. Here, the reductive nitrosylation of ferric MP11 (MP11(III)) under anaerobic conditions has been investigated between pH 7.4 and 9.2, at T = 20.0 °C. At pH ≤ 7.7, NO binds reversibly to MP11(III) leading to the formation of the MP11(III)-NO complex. However, between pH 8.2 and 9.2, the addition of NO to MP11(III) leads to the formation of ferrous nitrosylated MP11(II) (MP11(II)-NO). In fact, the transient MP11{FeNO}6 species is converted to ferrous deoxygenated MP11 (MP11(II)) by OH-- and H2O-based catalysis, which represents the rate-limiting step of the whole reaction. Then, MP11(II) binds NO very rapidly leading to MP11(II)-NO formation. Over the whole pH range explored, the apparent values of kon, koff, and K (= koff/kon) for MP11(III)(-NO) (de)nitrosylation are essentially pH independent, ranging between 5.8 × 105 M-1 s-1 and 1.6 × 106 M-1 s-1, between 1.9 s-1 and 3.7 s-1, and between 1.4 × 10-6 M and 4.6 × 10-6 M, respectively. Values of the apparent pseudo-first-order rate constant for the MP11{FeNO}6 conversion to MP11(II) (i.e., h) increase linearly with pH; the apparent values [Formula: see text] and [Formula: see text] are 7.2 × 102 M-1 s-1 and 2.5 × 10-4 s-1, respectively. Present data confirm that MP11 is a useful molecular model to highlight the role of the protein matrix on the heme-based reactivity.

Fluoride and azide binding to ferric human hemoglobin:haptoglobin complexes highlights the ligand-dependent inequivalence of the α and ß hemoglobin chains.

Haptoglobin (Hp) binds human hemoglobin (Hb), contributing to prevent extra-erythrocytic Hb-induced damage. Hp forms preferentially complexes with αß dimers, displaying heme-based reactivity. Here, kinetics and thermodynamics of fluoride and azide binding to ferric human Hb (Hb(III)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) are reported (pH 7.0 and 20.0 °C). Fluoride binds to Hp1-1:Hb(III) and Hp2-2:Hb(III) with a one-step kinetic and equilibrium behavior. In contrast, kinetics of azide binding to and dissociation from Hp1-1:Hb(III)(-N3-) and Hp2-2:Hb(III)(-N3-) follow a two-step process. However, azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) should reflect azide binding to the ferric ß and α chains, respectively, as also proposed for the similar behavior observed in Hb(III). Present results highlight the ligand-dependent kinetic inequivalence of Hb subunits in the ferric form, reflecting structural differences between the two subunits in the interaction with some ferric ligands.

Human Serum Albumin Is an Essential Component of the Host Defense Mechanism Against Clostridium difficile Intoxication.

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.

Dissecting the 17ß-estradiol pathways necessary for neuroglobin anti-apoptotic activity in breast cancer.

Neuroglobin (NGB) is a relatively recent discovered monomeric heme-protein, which behave in neurons as a sensor of injuring stimuli including oxidative stress, hypoxia, and neurotoxicity. In addition, the anti-apoptotic activity of overexpressed NGB has been reported both in neurons and in cancer cell lines. We recently demonstrated that, NGB functions as a compensatory protein of the steroid hormone 17ß-estradiol (E2) protecting cancer cells against the apoptotic death induced by oxidative stress. However, the E2-induced signaling pathways at the root of NGB over-expression and mitochondrial re-localization in breast cancer cells is still elusive. By using a kinase screening library, here, we report that: i) There is a strong positive correlation between NGB and ERα expression and activity in breast cancer cells; ii) The E2-activated phosphatidyl-inositol 3 kinase (PI3K)/protein kinase B (AKT) and protein kinase C (PKC) pathways are necessary to modulate the NGB protein levels; iii) The E2-induced persistent activation of AKT drive NGB to mitochondria; iv) Reactive oxygen species (ROS)-inducing compounds activating rapidly and transiently AKT does not affect the NGB mitochondrial level; and v) High level of NGB into mitochondria are necessary for the pro-survival and anti-apoptotic effect of this globin in cancer cells. As a whole, these results underline the E2 triggered pathways in E2-responsive breast cancer cells that involve NGB as a compensatory protein devoted to cancer cell survival.

Fipronil recognition by the FA1 site of human serum albumin.

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.