Thermal effects and drugs competition on the palmitate binding capacity of human serum albumin.

Human serum albumin (HSA) is the most abundant plasma protein of the circulatory system. It is a multidomain, multifunctional protein that, combining diverse affinities and wide specificity, binds, stores, and transports a variety of biological compounds, pharmacores, and fatty acids. HSA is finding increasing uses in drug-delivery due to its ability to carry functionalized ligands and prodrugs. All this raises the question of competition for binding sites occupancy in case of multiple ligands, which in turn influences the protein structure/dynamic/function relationship and also has an impact on the biomedical applications. In this work, the effects of interactive binding of palmitic acid (PA), warfarin (War) and ibuprofen (Ibu) on the thermal stability of HSA were studied using DSC, ATR-FTIR, and EPR. PA is a high-affinity physiological ligand, while the two drugs are widely used for their anticoagulant (War) and anti-inflammatory (Ibu) efficacy, and are exogenous compounds that accommodate in the deputed drug site DS1 and DS2, respectively overlapping with some of the fatty acid binding sites. The results indicate that HSA acquires the highest thermal stability when it is fully saturated with PA. The binding of this physiological ligand does not hamper the binding of War or Ibu to the native state of the protein. In addition, the three ligands bind simultaneously, suggesting a synergic cooperative influence due to allosteric effects. The increased thermal stability subsequent to binary and multiple ligands binding moderates protein aggregation propensity and restricts protein dynamics. The biophysics findings provide interesting features about protein stability, aggregation, and dynamics in interaction with multiple ligands and are relevant in drug-delivery.

A Linear Predictor Based on FTIR Spectral Biomarkers Improves Disease Diagnosis Classification: An Application to Multiple Sclerosis.

Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system that can lead to long-term disability. The diagnosis of MS is not simple and requires many instrumental and clinical tests. Sampling easily collected biofluids using spectroscopic approaches is becoming of increasing interest in the medical field to integrate and improve diagnostic procedures. Here we present a statistical approach where we combine a number of spectral biomarkers derived from the ATR-FTIR spectra of blood plasma samples of healthy control subjects and MS patients, to obtain a linear predictor useful for discriminating between the two groups of individuals. This predictor provides a simple tool in which the contribution of different molecular components is summarized and, as a result, the sensitivity (80%) and specificity (93%) of the identification are significantly improved compared to those obtained with typical classification algorithms. The strategy proposed can be very helpful when applied to the diagnosis of diseases whose presence is reflected in a minimal way in the analyzed biofluids (blood and its derivatives), as it is for MS as well as for other neurological disorders.

Palmitate-Induced Cardiac Lipotoxicity Is Relieved by the Redox-Active Motif of SELENOT through Improving Mitochondrial Function and Regulating Metabolic State.

Cardiac lipotoxicity is an important contributor to cardiovascular complications during obesity. Given the fundamental role of the endoplasmic reticulum (ER)-resident Selenoprotein T (SELENOT) for cardiomyocyte differentiation and protection and for the regulation of glucose metabolism, we took advantage of a small peptide (PSELT), derived from the SELENOT redox-active motif, to uncover the mechanisms through which PSELT could protect cardiomyocytes against lipotoxicity. To this aim, we modeled cardiac lipotoxicity by exposing H9c2 cardiomyocytes to palmitate (PA). The results showed that PSELT counteracted PA-induced cell death, lactate dehydrogenase release, and the accumulation of intracellular lipid droplets, while an inert form of the peptide (I-PSELT) lacking selenocysteine was not active against PA-induced cardiomyocyte death. Mechanistically, PSELT counteracted PA-induced cytosolic and mitochondrial oxidative stress and rescued SELENOT expression that was downregulated by PA through FAT/CD36 (cluster of differentiation 36/fatty acid translocase), the main transporter of fatty acids in the heart. Immunofluorescence analysis indicated that PSELT also relieved the PA-dependent increase in CD36 expression, while in SELENOT-deficient cardiomyocytes, PA exacerbated cell death, which was not mitigated by exogenous PSELT. On the other hand, PSELT improved mitochondrial respiration during PA treatment and regulated mitochondrial biogenesis and dynamics, preventing the PA-provoked decrease in PGC1-α and increase in DRP-1 and OPA-1. These findings were corroborated by transmission electron microscopy (TEM), revealing that PSELT improved the cardiomyocyte and mitochondrial ultrastructures and restored the ER network. Spectroscopic characterization indicated that PSELT significantly attenuated infrared spectral-related macromolecular changes (i.e., content of lipids, proteins, nucleic acids, and carbohydrates) and also prevented the decrease in membrane fluidity induced by PA. Our findings further delineate the biological significance of SELENOT in cardiomyocytes and indicate the potential of its mimetic PSELT as a protective agent for counteracting cardiac lipotoxicity.

The interaction of tryptophan enantiomers with model membranes is modulated by polar head type and physical state of phospholipids.

The mutual influence of chiral bioactive molecules and supramolecular assemblies is currently being studied in many research fields, including medical-pharmaceutical applications. Model membranes of phospholipids, such as the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the anionic dipalmitoylphosphatidylglycerol (DPPG), interact with a variety of chiral compounds that include amino acids. In this work, the interaction of tryptophan enantiomers, L-Trp and D-Trp, on DPPC and DPPG bilayers was investigated by using differential scanning calorimetry, attenuated total reflectance-Fourier transform infrared and spin-label electron spin resonance spectroscopies as well as molecular docking simulations. The results show that Trp enantiomers slightly perturb the bilayer thermotropic phase transitions. For both membranes, O atoms in the carbonyl groups have a propensity to act as acceptors of a (weak) hydrogen bond. The Trp chiral forms also promote formation of hydrogen bonds and/or hydration in the PO2- moiety of the phosphate group, especially for the DPPC bilayer. In contrast, they interact more closely with the glycerol group of DPPG polar head. Only for DPPC bilayers, both enantiomers increase the packing of the first hydrocarbon chain segments for temperatures through the gel state, whereas they do not affect the lipid chain order and mobility in the fluid state. The results are consistent with a Trp association in the upper region of the bilayers without permeation in the innermost hydrophobic region. The findings suggest that neutral and anionic lipid bilayers are differently sensitive to amino acid chirality.

ATR-FTIR spectroscopy of plasma supported by multivariate analysis discriminates multiple sclerosis disease.

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases showing various symptoms both of physical and cognitive type. In this work, we used attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy to analyze plasma samples for discriminating MS patients from healthy control individuals, and identifying potential spectral biomarkers helping the diagnosis through a quick non-invasive blood test. The cohort of the study consists of 85 subjects, including 45 MS patients and 40 healthy controls. The differences in the spectral features both in the fingerprint region (1800-900 cm-1) and in the high region (3050-2800 cm-1) of the infrared spectra were highlighted also with the support of different chemometric methods, to capture the most significant wavenumbers for the differentiation. The results show an increase in the lipid/protein ratio in MS patients, indicating changes in the level (metabolism) of these molecular components in the plasma. Moreover, the multivariate tools provided a promising rate of success in the diagnosis, with 78% sensitivity and 83% specificity obtained through the random forest model in the fingerprint region. The MS diagnostic tools based on biomarkers identification on blood (and blood component, like plasma or serum) are very challenging and the specificity and sensitivity values obtained in this work are very encouraging. Overall, the results obtained suggest that ATR-FTIR spectroscopy on plasma samples, requiring minimal or no manipulation, coupled with statistical multivariate approaches, is a promising analytical tool to support MS diagnosis through the identification of spectral biomarkers.

Low-temperature librations and dynamical transition in proteins at differing hydration levels.

Hydration of water affects the dynamics and in turn the activity of biomacromolecules. We investigated the dependence of the librational oscillations and the dynamical transition on the hydrating conditions of two globular proteins with different structure and size, namely ß-lactoglobulin (ßLG) and human serum albumin (HSA), by spin-label electron paramagnetic resonance (EPR) in the temperature range of 120-270 K. The proteins were spin-labeled with 5-maleimide spin-label on free cysteins and prepared in the lyophilized state, at low (h = 0.12) and full (h = 2) hydration levels in buffer. The angular amplitudes of librations are small and almost temperature independent for both lyophilized proteins. Therefore, in these samples, the librational dynamics is restricted and the dynamical transition is absent. In the small and compact beta-structured ßLG, the angular librational amplitudes increase with temperature and hydrating conditions, whereas hydration-independent librational oscillations whose amplitudes rise with temperature are recorded in the large and flexible alpha-structured HSA. Both ßLG and HSA at low and fully hydration levels undergo the dynamical transition at about 230 K. The overall results indicate that protein librational dynamics is activated at the low hydration level h = 0.12 and highlight biophysical properties that are common to other biosamples at cryogenic temperatures.

Interactive multiple binding of oleic acid, warfarin and ibuprofen with human serum albumin revealed by thermal and fluorescence studies.

Human serum albumin binds a wide variety of drugs with different structure and affinity to two main binding sites, drug site 1 (DS1) and drug site 2 (DS2), which partially or totally overlap with fatty acid (FA) sites. Although multiple binding sites are available for endogenous compounds, FAs are the primary physiological ligands of albumin and their competition in the occupancy of DS1 and DS2 affects the binding of exogenous molecules, with a possible impact on drug delivery. In this work, we have investigated the simultaneous binding of oleic acid, warfarin and ibuprofen to albumin using differential scanning calorimetry and fluorescence to evaluate the impact on the conformational stability of the protein. The two drugs are widely used for their anticoagulant (warfarin) and anti-inflammatory (ibuprofen) properties, and can be also considered as site markers to probe DS1 and DS2, respectively. Oleic acid is one of the most important fatty acids from a physiological point of view for its role as a source of energy for cells, and also it binds albumin with the highest association constant. When complexed with oleic acid the calorimetric profile of albumin shows a biphasic trend whose line shape depends on the ligand concentration. The binding capacity of either warfarin or ibuprofen to albumin is modulated by oleate molecules in a concentration-dependent mode being synergic cooperative (warfarin) or competitive-like (ibuprofen). The overall results provide insights on the dynamics of albumin/ligands complex, which in turn may have important pharmacokinetic and pharmacodynamic implications.

A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation.

The phosphomimetic mutation S82D in the cancer-associated, FAD-dependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation.

Estrogen receptor variant ERα46 and insulin receptor drive in primary breast cancer cells growth effects and interleukin 11 induction prompting the motility of cancer-associated fibroblasts.

Among the prognostic and predictive biomarkers of breast cancer (BC), the role of estrogen receptor (ER)α wild-type has been acknowledged, although the action of certain ERα splice variants has not been elucidated. Insulin/insulin receptor (IR) axis has also been involved in the progression and metastasis of BC. For instance, hyperinsulinemia, which is often associated with obesity and type 2 diabetes, may be a risk factor for BC. Similarly, an aberrant expression of IR or its hyperactivation may correlate with aggressive BC phenotypes. In the present study, we have shown that a novel naturally immortalized BC cell line (named BCAHC-1) is characterized by a unique expression of 46 kDa ERα splice variant (ERα46) along with IR. Moreover, we have shown that a multifaceted crosstalk between ERα46 and IR occurs in BCAHC-1 cells upon estrogen and insulin exposure for growth and pulmonary metastasis. Through high-throughput RNA sequencing analysis, we have also found that the cytokine interleukin-11 (IL11) is the main factor linking BCAHC-1 cells to breast cancer-associated fibroblasts (CAFs). In particular, we have found that IL11 induced by estrogens and insulin in BCAHC-1 cells regulates pro-tumorigenic genes of the "extracellular matrix organization" signaling pathway, such as ICAM-1 and ITGA5, and promotes both migratory and invasive features in breast CAFs. Overall, our results may open a new scientific avenue to identify additional prognostic and therapeutic targets in BC.

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study.

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D2O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.

Thermal Liquid Biopsy (TLB) of Blood Plasma as a Potential Tool to Help in the Early Diagnosis of Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is frequently characterized by a variety of clinical signs, often exhibiting little specificity. The diagnosis requires a combination of medical observations and instrumental tests, and any support for its objective assessment is helpful. OBJECTIVE: Herein, we describe the application of thermal liquid biopsy (TLB) of blood plasma samples, a methodology for predicting the occurrence of MS with a noninvasive, quick blood test. METHODS: TLB allows one to define an index (TLB score), which provides information about overall real-time alterations in plasma proteome that may be indicative of MS. RESULTS: This pilot study, based on 85 subjects (45 MS patients and 40 controls), showed good performance indexes (sensitivity and specificity both around 70%). The diagnostic methods better discriminate between early stage and low-burden MS patients, and it is not influenced by gender, age, or assumption of therapeutic drugs. TLB is more accurate for patients having low disability level (≤ 3.0, measured by the expanded disability status scale, EDSS) and a relapsing-remitting diagnosis. CONCLUSION: Our results suggest that TLB can be applied to MS, especially in an initial phase of the disease when diagnosis is difficult and yet more important (in such cases, accuracy of prediction is close to 80%), as well as in personalized patient periodic monitoring. The next step will be determining its utility in differentiating between MS and other disorders, in particular in inflammatory diseases.

Binding of warfarin differently affects the thermal behavior and chain packing of anionic, zwitterionic and cationic lipid membranes.

Warfarin is a coumarin derivative drug widely used for its anticoagulant properties. The interaction of warfarin with fully hydrated lipid bilayers has been studied by combining differential scanning calorimetry, spectrophotometry, electron spin resonance of chain-labelled lipids and molecular docking. Bilayers formed by lipids with different chemico-physical properties were considered, namely dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), and dioleoyltrimethyl-ammoniumpropane (DOTAP). We observed in all cases the binding of warfarin in proximity of the surface of the bilayers, leading to a variety of distinct effects on key molecular properties of the membranes. The drug associates with the lipid bilayers in the deprotonated open chain form, with an association constant similar for DMPC and DMPG (1.27·104 and 2.82·104 M-1, respectively) and lower for DOTAP (0.46·104 M-1). In DMPC bilayers, which are zwitterionic and with saturated symmetrical chains, warfarin at 10 mol% suppresses the pre-transition, slightly stabilizes the fluid state and reduces the cooperativity of the main transition. Moreover, it alters the lateral packing density of the chain segments close to the polar/apolar interface at any temperature through the gel phase. In anionic DMPG bilayers, the drug slightly perturbs the thermotropic phase behavior, and at 10 mol% markedly loosens the compact gel phase packing of the first chain segments. In cationic DOTAP bilayers, possessing unsaturated acyl chains, the drug induces a slightly higher degree of order and motional restriction in the outer hydrocarbon region in the frozen state. In all cases, as a surface adsorbed molecule, warfarin does not affect the segmental chain order and dynamics for temperatures in the fluid phase. The overall results provide an outline of the action of warfarin on membranes formed by lipids of different types.

Computational Approaches for the Discovery of GPER Targeting Compounds.

Estrogens exert a panel of biological activities mainly through the estrogen receptors α and ß, which belong to the nuclear receptor superfamily. Diverse studies have shown that the G protein-coupled estrogen receptor 1 (GPER, previously known as GPR30) also mediates the multifaceted effects of estrogens in numerous pathophysiological events, including neurodegenerative, immune, metabolic, and cardiovascular disorders and the progression of different types of cancer. In particular, GPER is implicated in hormone-sensitive tumors, albeit diverse issues remain to be deeply investigated. As such, this receptor may represent an appealing target for therapeutics in different diseases. The yet unavailable complete GPER crystallographic structure, and its relatively low sequence similarity with the other members of the G protein-coupled receptor (GPCR) family, hamper the possibility to discover compounds able to modulate GPER activity. Consequently, a reliable molecular model of this receptor is required for the design of suitable ligands. To date, convergent approaches involving structure-based drug design and virtual ligand screening have led to the identification of several GPER selective ligands, thus providing important information regarding its mode of action and function. In this survey, we summarize results obtained through computer-aided techniques devoted to the assessment of GPER ligands toward their usefulness in innovative treatments of different diseases.

The IL1ß-IL1R signaling is involved in the stimulatory effects triggered by hypoxia in breast cancer cells and cancer-associated fibroblasts (CAFs).

BACKGROUND: Hypoxia plays a relevant role in tumor-related inflammation toward the metastatic spread and cancer aggressiveness. The pro-inflammatory cytokine interleukin-1ß (IL-ß) and its cognate receptor IL1R1 contribute to the initiation and progression of breast cancer determining pro-tumorigenic inflammatory responses. The transcriptional target of the hypoxia inducible factor-1α (HIF-1α) namely the G protein estrogen receptor (GPER) mediates a feedforward loop coupling IL-1ß induction by breast cancer-associated fibroblasts (CAFs) to IL1R1 expression by breast cancer cells toward the regulation of target genes and relevant biological responses. METHODS: In order to ascertain the correlation of IL-ß with HIF-1α and further hypoxia-related genes in triple-negative breast cancer (TNBC) patients, a bioinformatics analysis was performed using the information provided by The Invasive Breast Cancer Cohort of The Cancer Genome Atlas (TCGA) project and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) datasets. Gene expression correlation, statistical analysis and gene set enrichment analysis (GSEA) were carried out with R studio packages. Pathway enrichment analysis was evaluated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. TNBC cells and primary CAFs were used as model system. The molecular mechanisms implicated in the regulation of IL-1ß by hypoxia toward a metastatic gene expression profile and invasive properties were assessed performing gene and protein expression studies, PCR arrays, gene silencing and immunofluorescence analysis, co-immunoprecipitation and ChiP assays, ELISA, cell spreading, invasion and spheroid formation. RESULTS: We first determined that IL-1ß expression correlates with the levels of HIF-1α as well as with a hypoxia-related gene signature in TNBC patients. Next, we demonstrated that hypoxia triggers a functional liaison among HIF-1α, GPER and the IL-1ß/IL1R1 signaling toward a metastatic gene signature and a feed-forward loop of IL-1ß that leads to proliferative and invasive responses in TNBC cells. Furthermore, we found that the IL-1ß released in the conditioned medium of TNBC cells exposed to hypoxic conditions promotes an invasive phenotype of CAFs. CONCLUSIONS: Our data shed new light on the role of hypoxia in the activation of the IL-1ß/IL1R1 signaling, which in turn triggers aggressive features in both TNBC cells and CAFs. Hence, our findings provide novel evidence regarding the mechanisms through which the hypoxic tumor microenvironment may contribute to breast cancer progression and suggest further targets useful in more comprehensive therapeutic strategies.

Electrochromic behaviour of Ir(iii) bis-cyclometalated 1,2-dioxolene tetra-halo complexes: fully reversible catecholate/semiquinone redox switches.

Neutral cyclometalated Ir(iii) complexes of general formula [Ir(ppy)2(O^O)squi], where ppy = 2-phenylpyridine and (O^O)squi = TBC (tetrabromocatechol) or TCC (tetrachlorocatechol) in their semiquinone (squi) monoanionic redox state, were synthesized by chemically oxidizing the anionic parent complexes NBu4[Ir(ppy)2(O^O)cat], in which (O^O)cat represents the corresponding ancillary dioxolene ligand in its dianionic catecholate (cat) redox state. This chemical oxidation leads to the modification of both the photophysical and the magnetic properties of the complexes. While the NBu4[Ir(ppy)2(O^O)cat] complexes are diamagnetic (D) and yellow-orange solids, the corresponding oxidized complexes [Ir(ppy)2(O^O)squi] display paramagnetic (P) properties and are characterized by a dark-green color. The conversion between the two forms (squi vs. cat) is electrochemically and chemically fully reversible. Indeed, the anionic NBu4[Ir(ppy)2(O^O)cat] complexes are quantitatively restored by chemical reduction of the neutral [Ir(ppy)2(O^O)squi] parents. These complexes therefore represent interesting redox based switches between multi-parameter states since they allow switching from a neutral paramagnetic to an anionic diamagnetic form together with a significant change in chromicity. Taking advantage of the significant color difference between the oxidized and the reduced form, an electrochromic cell was prepared with [Ir(ppy)2(TBC)squi] and its spectroelectrochemical properties are reported.

Anionic versus neutral Pt(II) complexes: The relevance of the charge for human serum albumin binding.

The focus of this work is pointing out the different behavior of two structurally related Pt(II) complexes, the anionic cyclometalated NBu4[(Bzq)Pt(Thio)], 1 and the neutral [(Phen)Pt(Thio)], 2, (Bzq = benzo[h]quinoline, Phen = 1,10-phenantroline, Thio = 1,2-benzenedithiolate), on the interaction with human serum albumin (HSA), a key drug-delivery protein in the bloodstream. Being very limited the number of anionic Pt(II) complexes reported to date, this is a pioneering example of report on a protein-ligand interaction involving a negatively charged platinum compound. The study was carried out by using fluorescence spectroscopy, differential scanning calorimetry and molecular docking simulations. The results revealed a strong binding affinity between the anionic compound and the protein, whereas a weak/moderate binding interaction was highlighted for the neutral one. Comparative studies with site specific ligands (warfarin and ibuprofen), allowed us to identify the protein binding sites of the two compounds. The work aims to shed light on the relevance of the charge in designing new drugs with a favorable binding affinity for HSA, which strongly contributes to influence their pharmacological and toxicological profile.

Effects of Polar Head Nature and Tail Length of Single-Chain Lipids on the Conformational Stability of ß-Lactoglobulin.

Interaction between ß-lactoglobulin and single-chain lipids, differing for either the length of the aliphatic chain or the molecular properties of the headgroup, was investigated at neutral and acidic pH to determine the impact on the thermal stability of the protein. Differential scanning calorimetry results with different fatty acids (from C10:0 to C18:0) show a correlation of both melting temperature and unfolding enthalpy of the protein with the ligand binding affinity, and the maximum effect was found for palmitic acid (PLM). The influence of the lipid polar head was investigated by comparing PLM with lyso-palmitoylphosphatidylcholine (LPC), which possesses the same aliphatic chain. At neutral pH, the stabilizing effect of LPC is less favorable compared to PLM. However, fluorescence results revealed that LPC can bind into the protein calyx even at acidic pH, at variance with fatty acids. Molecular dynamics simulations indicated that this difference is due to the ability of the polar head of LPC to interact with the protein loop that regulates the shift (Tanford transition) between open and closed state of the binding site of ß-lactoglobulin. The results provide a rationale for how a ligand has the ability to access the protein active site at acidic conditions by overcoming the Tanford transition, and they demonstrate that ß-lactoglobulin can deliver ligands with tailored properties of the polar head in a wide pH range.

Warfarin increases thermal resistance of albumin through stabilization of the protein lobe that includes its binding site.

Protein-drug interaction is of prominent interest in determining the pharmacokinetic and pharmacodynamic consequences on drug delivery. Warfarin is a widely used anticoagulant drug in the treatment of venous thrombosis and pulmonary embolism and is carried in the blood almost exclusively by human serum albumin. The effects of the binding of warfarin to the native state of albumin were characterized by UV-vis absorption, conventional and synchronous fluorescence, isothermal titration calorimetry, differential scanning calorimetry and molecular dynamics simulation. The overall results indicate that, under physiological condition, the binding of warfarin in site DS1 of albumin promotes local stabilization with resulting effects on the global protein dynamics. The increase of the protein stability has both an enthalpic and entropic character. Under denaturing condition, the stabilizing effect of warfarin is evidenced by an increase of both the melting temperature and unfolding enthalpy of albumin with the drug/protein molar ratio. More importantly, thermal resistance is increased due to selective effect on the specific protein lobe that includes the main drug binding site. The comparison of the thermal behavior of the protein-warfarin complex with that in the presence of a typical ligand of the other main protein binding site, i.e. drug site DS2, provides key insight on domain-specific stabilization effects on albumin.

AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs).

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.

Unsaturated lipid bilayers at cryogenic temperature: librational dynamics of chain-labeled lipids from pulsed and CW-EPR.

Fully hydrated bilayers of monounsaturated palmitoyloleoylphosphatidylcholine (POPC) and diunsaturated dioleoylphosphatidylcholine (DOPC) lipids have low main phase transition temperatures (271 K for POPC and 253 K for DOPC). Two-pulse echo detected spectra, combined with continuous wave electron paramagnetic resonance spectroscopy, are employed to study the low-temperature lamellar phases of the POPC and DOPC unsaturated bilayers that are usually studied in the fluid state. Phosphatidylcholine spin-labeled at C-5 and C-16 carbon atom positions along the acyl chain were used and the temperature varied over the range 77-270 K. Segmental chain librational oscillations of small amplitude and with correlation time in the subnanosecond to nanosecond range are found in both membranes. The mean-square angular amplitude, α2, of librations increases with temperature, is larger close to the bilayer midplane than close to the first acyl chain segments, and is larger in diunsaturated than in monounsaturated bilayers. In the inner hydrocarbon region of both lipid matrices, α2 increases first slowly and linearly with temperature and then more rapidly, and a dynamical transition is detected in the range 190-210 K. Compared to dipalmitoylphosphatidylcholine bilayers of fully saturated symmetric chain lipids, the presence of double bonds in the acyl chain enhances the intensity of librational motion which is characterized by larger angular variations at the terminal methyl ends. These findings highlight biophysical properties of unsaturated bilayers in the frozen state, including a detailed characterization of segmental chain dynamics and the evidence of a dynamical transition that appears to be a generic feature in hydrated macromolecular systems. These results can also be relevant in regulating membrane physical properties and function at higher physiological temperatures.