High-density lipoproteins (HDL) are crucial for cardiovascular health. HDL dysfunction is strongly linked to atherosclerosis and other diseases. Although HDL is often treated as a single entity, it comprises a spectrum of subfractions differing in size, structure, and composition. Understanding the ultrastructure and function of these subfractions is essential for uncovering the molecular mechanisms behind HDL dysfunction and improving disease risk prediction. We employed Small-Angle X-ray Scattering (SAXS) and cryogenic-electron tomography (cryo-ET) to analyze the structural features of total HDL and its subfractions: HDL2b, HDL2a, and HDL3. We present updated, detailed structural models showing that the structure of HDL changes dramatically from the smallest to the largest subfraction, with HDL3 partially exposing the core to the particle's surface. Moreover, total HDL reflects the weighted sum of its subfractions, emphasizing that analysis of total HDL alone may be misleading. HDL subfractions were also studied in serum samples from 16 individuals classified as low- or high-risk for atherosclerosis. Even though biochemical differences appeared mainly in HDL2, structural differences were most pronounced in HDL3 addressing that particle composition alone cannot fully distinguish HDL dysfunction. Multimodal analysis integrating structural and biochemical data was able to separate risk groups and revealed correlations between structural parameters and cardiovascular risk status. Our findings support the importance of analyzing HDL subfractions to uncover molecular drivers of disease risk and suggest candidate biomarkers for atherosclerosis upon validation in larger cohorts.

Atherosclerosis is the leading cause of cardiovascular diseases and remains a global health challenge. Low-density lipoproteins are crucial in atherogenesis, with plasma levels as significant independent predictors of the condition. Lipoprotein(a), Lp(a), is an LDL variant considered a stand-alone atherosclerosis predictor. Although structurally similar in composition to LDL, Lp(a) contains an additional protein, apolipoprotein(a), covalently linked to apolipoprotein-B100 via a disulfide bond. This distinguishing protein is believed to confer Lp(a) its distinctive properties, including marked heterogeneity. While many studies have structurally characterised LDL particles, Lp(a) remains understudied. In this study, we isolated LDL particles from serum of normolipidemic, healthy individuals with either low or high Lp(a) levels. Our study provides biochemical, structural and functional characterisation of low and high Lp(a)-total LDL fractions. Fourier transform infrared spectroscopy (FTIR) revealed that high Lp(a) LDL exhibited a reduced ability to remove lipids from model membranes compared to low Lp(a) LDL. However, this functional difference could not be sufficiently associated with structural differences in total LDL fractions, as determined by small-angle X-ray scattering (SAXS), except for a significant difference in the particles' core-to-shell scattering mass (SM) ratio. Western blot analysis further revealed a higher abundance of Lp(a) in a small-dense subfraction, LDL6, leading us to hypothesise that structural differences might be more evident in these subfractions than in total fractions. Supporting this hypothesis, SAXS measurements on LDL6 subfractions from two subjects, with low and high Lp(a), revealed an increased protein shell thickness in high Lp(a) LDL6, a feature not directly observed in total fractions. Our data thus suggests a key role of LDL6 in LDL dysfunction.

Lipoproteins, key mediators of lipid transport, facilitate the bidirectional transfer of lipids such as fatty acids, triglycerides, and cholesterol between soluble particles and cell membranes. High-density lipoproteins (HDL) primarily engage in reverse cholesterol transport, while low-density lipoproteins (LDL) predominantly deposit lipids, affecting cardiovascular health with a well-known role in the formation of the atherosclerotic plaque. In addition, lipoproteins play an important role in neutralizing bacterial lipopolysaccharides (LPS), the major component of Gram-negative bacterial outer membranes, which act as potent TLR4 agonists and can trigger severe immune responses. Lipoproteins bind LPS in plasma, with HDL showing strong binding affinity and LDL contributing to LPS clearance under specific conditions. Here, we explore the interaction of LDL and human serum albumin (HSA), another serum lipid-binding protein, with model lipid bilayers containing either phospholipids or LPS. Using neutron reflectometry and attenuated total reflection infrared spectroscopy, we characterize lipid transfer processes influenced by calcium levels and lipid composition. Calcium plays a key role in receptor-mediated LDL binding, but less is known on its effect on LDL-mediated lipid transfer in the absence of LDL receptors. Our results show that elevated calcium levels enhance stable LDL adsorption onto mammalian phospholipid-cholesterol membranes, promoting lipid cargo deposition despite the absence of specific LDL-receptors. Conversely, LDL showed no stable binding to LPS reconstituted in asymmetric outer membrane models but was able to deposit phospholipids in the membrane. In contrast, HSA removed lipids from mammalian membranes and exhibited minimal interaction with LPS-containing models. The findings elucidate the distinct lipid exchange mechanisms of LDL and HSA and their roles in modulating lipid transfer at membrane interfaces. Receptor-free enhanced LDL lipid deposition in calcium-enriched environments may have implications for cardiovascular disease progression. Conversely, the minimal interaction of LDL with bacterial LPS suggests a limited ability to extract LPS from membrane environments. This study provides structural insights into the interplay between lipoproteins, calcium, and membrane composition, with relevance to atherosclerosis and systemic endotoxemia.

High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.

Lipoproteins play a crucial role in lipid metabolism, serving as carriers for lipids such as cholesterol and triglycerides in the bloodstream. Atherosclerosis is a complex cardiovascular disease characterized by the accumulation of cholesterol-rich plaques in arterial walls, leading to narrowed and hardened arteries. Recently, the spike protein from the SARS-CoV-2 virus, responsible for COVID-19, has been the subject of research concerning its potential impact on lipid metabolism and its association with cardiovascular disease. Understanding the interaction between lipoproteins and the spike protein influence on lipid metabolism could have implications for our knowledge of cardiovascular health.

In this research, we investigated the ultrastructure of HDL from individuals with different lipid profiles as well as the interaction of mature HDL and model of nascent HDL with model membranes. Understanding these differences will help create novel rHDL particles with superior lipid-removing and CVD-treating properties. Finally, the interaction between the spike protein and HDL in model cell membranes to study potential imbalances in lipid metabolism. To achieve the different objectives, lipid deposition, exchange and removal were followed by techniques such as Neutron reflection and attenuated total reflection Fourier transformation infrared spectroscopy while, the ultrastructure was unravelled by small-angle X-ray scattering.

En la sangre humana, las lipoproteínas sirven como transportador de lípidos como el colesterol y los triglicéridos. Cuando se acumula demasiado colesterol en las arterias, puede causar aterosclerosis, una condición que puede afectar al corazón.

Ahora se ha hablado mucho sobre la proteína “spike” del virus que causó la pandemia de COVID-19. Los investigadores encuentran una conexión entre cómo el virus y esta proteína en particular podrían afectar la forma en que el cuerpo humano maneja las grasas. Esto es importante porque podría mostrar una relación entre la enfermedad causada por este virus y las enfermedades cardiovasculares.

En esta tesis, analicé de cerca la estructura del HDL, el tipo de lipoproteína también conocida como “colesterol bueno”. Correlacioné su estructura con cómo el HDL intercambia grasas de Membranas Celulares modelo, que consisten en un modelo simplificado de las paredes de nuestras células. Mi intención era descubrir estas correlaciones porque podrían ayudarnos a producir HDL artificiales que pueden ser usadas para eliminar grasas y tratar problemas cardíacos.

Por último, observé cómo se comporta la proteína “spike” del virus que causa la enfermedad COVID-19 y el HDL cuando se encuentran juntos en las paredes celulares modelo. Esto podría decirnos si esta proteína afecta como nuestro cuerpo maneja la grasa.

Para estudiar todo esto, utilicé algunas técnicas sofisticadas como la reflexión de neutrones y la espectroscopia infrarroja para rastrear cómo se mueven las grasas, y también rayos X para tomar "fotografías" de la estructura del HDL. 

I mänskligt blod arbetar lipoproteiner som viktiga bärare av fetter som kolesterol och triglycerider. När för mycket kolesterol ansamlas i artärerna kan det orsaka ateroskleros, ett sjukdomstillstånd som påverkar hjärtat.

De senaste åren har det varit mycket diskussioner om "spike-proteinet" från det virus som orsakade COVID-19 pandemin. Forskare har funnit ett samband mellan hur viruset och detta protein kan påverka hur den mänskliga kroppen hanterar fett. Detta skulle kunna vara viktigt eftersom det kan visa på ett samband mellan sjukdomen orsakad av viruset och hjärt-kärlsjukdomar. I denna avhandling har jag tittat närmare på strukturen för HDL, den typ av lipoprotein som är känd som "det goda kolesterolet". Jag korrelerade dess struktur med hur HDL utbyter fett från modellmembran, en förenklad modell av våra cellväggar. Jag hade för avsikt att utreda dessa korrelationer eftersom de skulle kunna hjälpa oss att skapa skräddarskydd HDL som är ännu bättre på att avlägsna fett och behandla hjärt-kärlsjukdom.

Slutligen tittade jag på hur "spike-proteinet" från viruset som orsakade COVID19 och HDL uppför sig när de möts vid ett modellmembran. Detta skulle kunna berätta för oss huruvida detta protein påverkar hur våra kroppar hanterar fett.

För att undersöka allt detta har jag använt tjusiga metoder som neutronreflektion och infraröd spektroskopi för att spåra hur fett rör sig och röntgen för att ta "bilder" på HDLs struktur. 

Cholesterol has been shown to affect the extent of coronavirus binding and fusion to cellular membranes. The severity of Covid-19 infection is also known to be correlated with lipid disorders. Furthermore, the levels of both serum cholesterol and high-density lipoprotein (HDL) decrease with Covid-19 severity, with normal levels resuming once the infection has passed. Here we demonstrate that the SARS-CoV-2 spike (S) protein interferes with the function of lipoproteins, and that this is dependent on cholesterol. In particular, the ability of HDL to exchange lipids from model cellular membranes is altered when co-incubated with the spike protein. Additionally, the S protein removes lipids and cholesterol from model membranes. We propose that the S protein affects HDL function by removing lipids from it and remodelling its composition/structure.

High-density lipoproteins (HDL), also known as "good cholesterol," are essential for preserving cardiovascular health. Understanding the different functions and how each HDL subfraction contributes to general health requires a comprehension of the structural complexity of HDL subfractions. Subpopulations of HDL particles with distinctive lipid and protein contents include HDL2b, HDL2a, and HDL3. These subfractions have various affinities for lipids and cholesterol, which affects how well they mediate reverse cholesterol transport (RCT) and how effective they might be at preventing a therosclerosis. Clarifying these subfractions' unique functions in cholesterol metabolism and cardiovascular prevention requires a thorough investigation of their structural characteristics. Here, we reveal structural details of total HDL fraction and its subfractions by Small-Angle X-ray Scattering (SAXS), a powerful technique to determine particle size, shape, and internal structure without the need for freezing. This property makes SAXS very attractive for analysing the dynamic properties in HDL. Briefly, we found remarkable differences between total HDL (HDLT) and the subpopulations HDL3, HDL2a and HDL2b regarding their biochemical composition and structural characteristics when comparing between individuals presenting a high or low risk of developing cardiovascular disease(CVD).