Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein particles, conferring anti-atherogenic properties through reverse cholesterol transport. In its lipid-free state, apoA-I self-associates, a process implicated in normal physiology as well as in pathological conditions, such as amyloidosis. Although various mechanisms have been proposed to explain its self-association, there is still no consensus, and its functional implications remain unclear. Here, we employed a multi-parametric fluorescent probe to investigate apoA-I self-association. We used three single cysteine mutants located in different helixes: K107C (H4), K133C (H5), and F225C (H10); and labelled them with pyrene as detailed previously (Tárraga et al. Arch Biochem Biophys 699 (2021) 108748). Our original experiments revealed excimer emission between helixes H5 and H10 and polarity changes also in H4. By exploiting specific pyrene band emissions to monitor dimer association (via excimer formation) and microenvironment polarity (P-value), we tracked apoA-I oligomerization as a function of protein concentration. Several mathematical models of self-association were developed and compared using selection criteria to identify the simplest model reproducing apoA-I's complex behaviour in aqueous media: a Sequential Association submodel limited to a tetramer as the highest order oligomeric species and considering both dimers and tetramers as responsible for the excimer's emission. Multi-equilibria models enhanced the titration analysis, allowing estimation of association constants (Ka) and oligomeric species distribution. Our results support previous evidence that contacts among helixes, which stabilize discoidal HDL particles, are already present in lipid-free apoA-I, with dimeric species predominating, and possible tetrameric too. Further investigation of these species is essential to elucidate their physiological and pathological roles, such as in atherosclerosis.

Apolipoprotein A-I (APOA1) oligomerization is thought to be essential for high-density lipoprotein (HDL) formation and metabolism. Naturally occurring mutations can disrupt normal APOA1 folding and self-association, leading to dysfunctional HDL formation and cardiovascular disease. The congenital APOA1 variant p.K131del (APOA1K107del) has been associated with cardiovascular pathologies such as low HDL-cholesterol levels and aortic amyloidosis, and multiple studies indicate structural changes in APOA1 conformation underlie associated dysfunction. In the current study, we confirmed that APOA1K107del exhibits no notable defect in lipid-binding. However, using polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC), we found that loss of lysine 107 resulted in a remarkable shift in the distribution of APOA1 oligomers with a much higher proportion of monomers present in APOA1K107del compared to wild-type APOA1. Further investigation using quantitative cross-linking revealed a major disruption of interactions in helical regions reported to participate in domain swaps necessary for proper self-association. This structural disruption appears to impair N- and C-termini interactions and dynamics that lead to non-specific aggregation. These findings support the hypothesis that lysine 107 is critical for proper folding and self-association of lipid-free APOA1 which could impact HDL biogenesis.

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (KA) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.

This article contains data for the self-association of pyrene-labelled single Cys-mutants of apolipoprotein A-I (apoA-I). Mathematical models were developed to characterise the self-association events at different cysteine positions on apoA-I obtained as a function of protein concentration based on the multi-parametric spectrum of pyrene, particularly P-value and excimer emissions. The present work complements data related to the article entitled “Analysis of pyrene-labelled apolipoprotein A-I oligomerisation in solution: Spectra deconvolution and changes in P-value and excimer formation” Tárraga et al. [1].

This article shows the dataset of clearance assays and the reconstitution of stable biological nano-complexes using both detergent-assisted and spontaneous solubilization of phospholipids by the recombinant purified apolipoprotein A-I (apoA-I). Protein was intra-chain crosslinked in order to introduce steric constrains. Then, native and crosslinked protein function was evaluated by a data collection of dimiristoyl phosphatidyl choline (DMPC) micellization curves. Additionally, resulting particles from spontaneous or detergent-assisted lipid solubilization were characterized by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and native polyacrylamide gel electrophoresis (PAGE). Here we set up an experimental design that may help study protein structure based on its function, since interaction with biological membranes and lipids is an intrinsic activity attributed to many proteins in circulation. In addition, by t-test analysis of collected-data, we examined the formation of lipoprotein particles by native and intra-chain crosslinked proteins under different conditions like temperature and time incubation. Thus, data shown here strengthen the usefulness of an easy, rapid, accessible and inexpensive approach to test protein flexibility related to its function.

Background: The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis. Methods: Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in native conformation or under oxidation or intramolecular crosslinking. Results: K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants. Conclusions: We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding. Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency. General significance: The results learnt from this variant strength a close association between amyloidosis and atherosclerosis.

En diciembre de 2019, se produjo un nuevo brote de enfermedad por coronavirus (COVID-19) en Wuhan, China. El síndrome respiratorio agudo severo-coronavirus-2 (SARS-CoV-2), que es el séptimo coronavirus conocido que infecta a los humanos, es altamente infeccioso y se ha expandido rápidamente en todo el mundo desde su descubrimiento. El diagnóstico de la infección por SARS-CoV-2 se basa en la detección del genoma viral (ARN) a través de técnicas de biología molecular. Con este fin, se extrae el ARN total para su posterior detección mediante PCR cuantitativa en tiempo real (RT-qPCR). Las pruebas cuantitativas de ácidos nucleicos se han convertido en el “estándar de oro” para el diagnóstico y guía en la toma de decisiones clínicas. Sin embargo, los ensayos de RT-qPCR dirigidos al SARS-CoV-2 tienen varios desafíos, especialmente en términos de diseño de cebadores / sondas y de desarrollo de metodologías que permitan estimar la carga viral en pacientes con diagnóstico de COVID-19.

The cholesterol-ester transfer protein (CETP) exchanges lipids between high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs). The excessive transport of lipids from HDLs to LDLs mediated by this protein can cause an alteration in the deposition of lipoproteins onto the arterial walls, thus promoting the development of arteriosclerosis. Different CETP inhibitors have been tested in recent years, but none has been confirmed as being effectively palliative for the disease. We employed in silico databases and molecular docking as a computational method to predict how potential CETP inhibitors could interact with the active site of the CETP protein. Upon previously comparing two computer software packages to determine which generated a greater number of accurate CETP-inhibitor-complex structures, we chose the more appropriate program for our studies. We then abstracted a series of databases of known CETP inhibitors and noninhibitors exhibiting different 50% concentrations of CETP-inhibitory (INH) activity, to generate virtual structures for docking with different combinations of the CETP receptor. From this process, we obtained as the most suitable structure 4F2A_1OB_C_PCW-it accordingly having a greater area under the receiver operating characteristic curve. The molecular docking of known compounds in comparison with the respective conformation of this inhibitor enabled us to obtain ΔGs (in kcal/mol) from which data we made a first exploration of unknown compounds for CETP-INH activity. Thus, the 4F2A_1OB_C_PCW structure was docked with DrugBank-Approved commercial compounds in an extensive database, whose status had already been established from pharmacokinetics and toxicology. In this study, we present a group of potential compounds as CETP-inhibitor candidates.

Rodolfo R. Brenner, died on 3rd July 2018, two weeks before his 96th birthday. His memory will remain forever with all of us who had the privilege of knowing him.

Background and aims: Data about glucocorticoids role in the development of atherosclerosis are controversial showing different effects in human than in experimental animal models. Atherosclerosis is the result of a chronic inflammatory response to an injured endothelium where an uncontrolled uptake of OxLDL by macrophages triggers the development of foam cells, the main component of fatty streaks in atherosclerotic plaque. There are few data about the direct effect of glucocorticoids in macrophages of atherosclerotic plaque. The aim of the study was to elucidate the role of glucocorticoids in the development of foam cells in atherosclerosis initiation.Methods: For this purpose we used THP1 cells differentiated to macrophages with phorbol esters and incubated with OxLDL alone or with cortisol or cortisone. THP1 cells were also incubated with cortisone plus an inhibitor of 11 beta-hydroxysteroid dehydrogenase 1 (11bHSD1) activity to determine the role of this enzyme on glucocorticoid action in this process.Results: Ours results showed that cortisol and cortisone decreased significantly the inflammation promoted by OxLDL, and also diminished the expression of genes involved in influx and efflux of cholesterol resulting in a reduced lipid accumulation. Likewise cortisol and cortisone decreased 11 beta HSD1 expression in THP1 cells. The presence of the inhibitor of 11 beta HSD1 abolished all the effects elicited by cortisone.Conclusion: Our results indicate a direct effect of glucocorticoids on macrophages braking atherosclerosis initiation, reducing pro-inflammatory markers and OxLDL uptake and cholesterol re-esterification, but also inhibiting cholesterol output. These effects appear to be mediated, at least in part, by 11 beta HSD1 activity. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

This article supports experimental evidence on the time-dependent effect on gene expression related to inflammation and cholesterol deposition in lipid-loaded cells. The cells employed were human monocytes THP1 line transformed into macrophages by treatment with phorbol esters. Macrophages were treated at different times with oxidized low density lipoprotein (Ox-LDL) and then gene expression was measured. We also include data about the different types of oxidized lipoprotein obtained (low, media or high oxidation) for differential exposure with Cu ions. These data include characterization to lipid and protein peroxidative damage and also quantification of cell viability by exposure to native and modified LDL. The present article complements data published in "Decreased OxLDL uptake and cholesterol efflux in THP1 cells elicited by cortisol and by cortisone through 11β-hydroxysteroid dehydrogenase type 1" Ledda et al. (in press) [1].

La arteriosclerosis es una patología que está asociada a un estado inflamatorio crónico con implicación de un gran número de citoquinas proinflamatorias durante su evolución. El cortisol es un glucocorticoide con acción antiinflamatoria que actúa en la resolución de la inflamación mediante, la activación de macrófagos por la vía alternativa M2c antiinflamatorios y la inhibición de citoquinas pro-inflamatorias entre otras acciones. Si bien la mayor parte de los glucocorticoides son sintetizados en las glándulas suprarrenales, a nivel de tejidos periféricos, entre ellos las células del sistema inmune, la señal glucocorticoide puede verse amplificada o inhibida por la actividad de las enzimas 11b-hidroxiesteroide deshidrogenasa de tipo 1 (11bHSD1) y de tipo 2 (11bHSDH2), encargadas de sintetizar cortisol y cortisona respectivamente. El objetivo de este trabajo fue comparar el efecto del cortisol y de la cortisona sobre la expresión de genes vinculados al proceso inflamatorio, genes de las enzimas involucradas en la interconversión de cortisol-cortisona y genes del metabolismo del colesterol (Col) implicados en el almacenamiento y remoción del mismo. Monocitos humanos de la línea celular THP1 fueron transformados en macrófagos por adición de ésteres de forbol (PMA) en el medio de cultivo. Lipoproteínas de baja densidad (LDLs) fueron aisladas de plasma humano y peroxidadas in vitro con Cu++ para ser transformadas en LDL oxidadas (LDLOx). Los macrófagos THP1 fueron tratados por 24 horas con LDLOx y concentraciones crecientes de cortisol o cortisona (0.1 a 1000 nM). La cortisona fue añadida en presencia de un inhibidor (BVT 2733) de la enzima 11ßHSD1 con el propósito de evitar una posible conversión de la misma a cortisol. La expresión de genes marcadores de macrófagos y de inflamación (F4/80, TNFa, MMR y diferentes interleuquinas), de internalización de LDLOx (FAT/CD36), de esterificación de Col (ACAT) y de remoción de Col (LXRa, ABC-A1, ABC-G1 y ApoE) fueron evaluados por real time PCR (RT-PCR) como indicadores de inflamación y acumulación lipídica. También, los genes de las deshidrogenasas de tipo I y II fueron analizados. La presencia de LDLOx incrementó la expresión génica de todos los genes estudiados con excepción del MMR (marcador de macrófagos antiinflamatorios). El cortisol promovió una disminución dosis dependiente en la expression de todos los genes menos para MMR el cuál tuvo un marcado incremento. La cortisona no tuvo ningún efecto sobre los genes estudiados. La la cortisona solo mimetiza el efecto del cortisol únicamente cuando fué administrada a la dosis más alta posiblemente por su conversion a cortisol. La presencia de BTV 2733 previene el efecto de la cortisona en todos los casos. Nuestros resultados indican un efecto directo del cortisol sobre los macrófagos, disminuyendo por un lado la entrada de LDLOx y la re-esterificación del Col y por el otro la remoción del Col. La actividad de la 11bHSD1en macrófagos podría tener un rol relevante en la progresión de la arteriosclerosis.

Introducción Los glucocorticoides son antiinflamatorios que actúan en la resolución de la inflamación mediante la activación de macrófagos por la vía alternativa M2c anti-inflamatorios, entre otras acciones. Se considera que los glucocorticoides están implicados en el desencadenamiento y/o mantenimiento de la obesidad, tal como se ha postulado para la aterosclerosis. A nivel de tejidos periféricos, entre ellos las células del sistema inmune, la señal glucocorticoide puede verse amplificada o inhibida por la actividad de las enzimas 11β-hidroxiesteroide deshidrogenasa de tipo 1 (11βHSDH1) y de tipo 2 (11βHSDH2), respectivamente. La expresión de 11βHSD1 está involucrada en la reducción de cortisona a cortisol (forma activa del glucocorticoide) y la 11βHSDH2 transforma al cortisol en cortisona (glucocorticoide inactivo).

Discoidal high-density lipoproteins (D-HDL) are critical intermediates in reverse cholesterol transport.Most of the present knowledge of D-HDL is based on studies with reconstituted lipoprotein complexes of apolipoprotein A-I (apoA-I) obtained by cholate dialysis (CD).D-HDL can also be generated by the direct microsolubilization (DM) of phospholipid vesicles at the gel/fluid phase transition temperature, a process mechanistically similar to the "in vivo" apoAI lipidation via ABCA1.We compared the apoA-I configuration in D-HDL reconstituted with dimyristoylphosphatidylcholine by both procedures using fluorescence resonance energy transfer measurements with apoA-I tryptophan mutants and fluorescently labeled cysteine mutants.Results indicate that apoA-I configuration in D-HDL depends on the reconstitution process and are consistent with a "double belt" molecular arrangement with different helix registry.As reported by others, a configuration with juxtaposition of helices 5 of each apoAI monomer (5/5 registry) predominates in D-HDL obtained by CD.However, a configuration with helix 5 of one monomer juxtaposed with helix 2 of the other (5/2 registry) would predominate in D-HDL generated by DM.Moreover, we also show that the kinetics of cholesterol efflux from macrophage cultures depends on the reconstitution process, suggesting that apoAI configuration is important for this HDL function.