Jorge Bernardino de la Serna

Like cholesterol, protein crowding has been shown to alter the temperature dependence of lateral diffusion in biomembranes, eventually eliminating the distinct liquid-gel phase transition. Here, we resolve the molecular origin of this protein effect with coarse-grained and all-atom simulations of protein-crowded phosphatidylcholine bilayers, which give access to lipid structure and dynamics on both sides of the transition temperature. Unlike cholesterol, which is known to induce a continuous liquid ordered phase by having opposite effects on the two phases, we find that membrane proteins decrease lipid order at all temperatures studied. The disordering is stronger at low temperatures, so that once the proteins cover roughly half of the membrane area the low- and high-temperature phases become alike and the transition is lost. Our results suggest that in addition to local cholesterol concentration, local protein concentration may also help dictate membrane fluidity and especially its response to temperature change.

E-cigarettes have attracted considerable attention as a safer substitute for tobacco smoking. While replacing conventional cigarettes, it introduced new inhalable toxicants, including benzaldehyde-propylene glycol acetal (BPGA), a flavour-derived chemical adduct present in e-cigarette vapour. The health risks associated with such flavour‐derived acetals remain insufficiently elucidated at the cellular level. This study investigated the pro fibrotic-related changes in alveolar epithelial cells (A549 cells) caused by BPGA. The cells exposed to various concentrations of BPGA were evaluated for cytotoxicity, intracellular reactive oxygen species (ROS), pro-fibrotic responses like epithelial to mesenchymal transition (EMT) and apoptosis. BPGA exposure resulted in a significant increase of intracellular ROS production and triggered upregulation of antioxidant enzymes as a compensatory protective response. However, elevated ROS levels disrupted cellular homeostasis and initiated partial apoptotic responses. These molecular and cellular alterations eventually led to pro-fibrotic responses, such as changes in the expression of EMT markers like α-smooth muscle actin and fibronectin. Collectively, these findings demonstrate that BPGA can induce oxidative stress in alveolar epithelial cells and partial apoptotic responses that can finally lead to inflammation. The EMT-related changes indicate the mechanism by which e-cigarette exposure could lead to lung injury and pulmonary fibrosis.

E-cigarettes have attracted significant attention as a safer substitute for conventional tobacco smoking. However, they have introduced new inhalable toxicants, including benzaldehyde-propylene glycol acetal (BPGA)—a chemical adduct produced by cherry-flavoured e-cigarettes. The health risks associated with such flavour-derived acetals remain insufficiently elucidated at the cellular level. This study investigated the role of BPGA in the progression of epithelial-to-mesenchymal transition (EMT)-like changes in alveolar epithelial cells (A549 cells). A549 cells exposed to various concentrations of BPGA were analysed for cell viability, morphology, mitochondrial function, lysosomal health, and cytoskeletal integrity using viability assays and fluorescence imaging. Intracellular reactive oxygen species (ROS) production was quantified using the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Antioxidant enzyme expression, inflammatory responses, and EMT-associated phenotypic alterations were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence (IF) assays. Exposure of alveolar epithelial cells to BPGA caused a concentration-dependent decrease in cell viability. BPGA exposure resulted in mitochondrial membrane depolarisation, lysosomal damage, cytoskeletal changes, and stress fibre formation, which altered cell morphology. It significantly increased intracellular ROS production. As a result, antioxidant enzyme levels were upregulated as a protective response. However, during severe oxidative stress, this response was overwhelmed. Excess ROS disrupted cellular homeostasis and initiated apoptosis, though not completely. ROS also acted as a signalling molecule, promoting the upregulation of inflammatory mediators. These changes were associated with altered EMT marker expression, suggesting that BPGA might drive EMT-like remodelling. In conclusion, BPGA, a chemical adduct from e-cigarette vapour, induces alveolar injury by promoting oxidative stress, inflammation, and EMT-related changes, which may explain a mechanism by which e-cigarette exposure could lead to lung injury and pulmonary fibrosis. Abstract Figure: Graphical abstract

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable, with GLP1R gene variation potentially linked to therapeutic outcomes. A GLP1R natural missense variant, A316T, protects against T2D and cardiovascular disease. Here, we generated and characterized a human GLP1R A316T mouse model. Human GLP1R ^A316T/A316T mice displayed lower fasting blood glucose versus wild-type littermates even under metabolic stress, as well as slower weight gain and alterations in islet cytoarchitecture, glucagon secretion, and liver metabolism under a high-fat, high-sucrose diet. This was however associated with blunted responses to pharmacological GLP-1RAs in vivo. Further investigations in β cell models demonstrated that human GLP1R A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R–targeting therapies.

Background: Atopic dermatitis (AD) is a highly prevalent inflammatory skin disease, affecting up to 30% of children at some point in their life and frequently persisting into adulthood. Insufficiency in the late epidermal protein filaggrin is frequently observed in the lesional skin of patients, with direct and indirect impact on the skin barrier quality and function. We hypothesized that filaggrin reduction influences intracellular, surface, and derived extracellular membranes of keratinocytes with multiple impacts on the cell function. Results: Using filaggrin knockdown keratinocytes generated by shRNA interference (shFLG), we determined that the physical characteristics of the cellular membranes (reported by refractive index) are changed on a filaggrin insufficiency background. Using proteomics, protein binding modeling, and functional assays, we established that filaggrin insufficiency in keratinocytes results in changes in both organelles comprised of internal cellular membranes (i.e., small extracellular vesicles, sEVs) and the plasma membrane. We detected increased association of anti-adhesive proteins (tenascin-C and matrilin-2) with sEVs, resulting in a reduction of the fibronectin-1-mediated sEV uptake by dendritic cell subsets. At the same time, dysregulation of the tight junction and cell adhesion molecules at the level of the cell increased keratinocyte adhesiveness to reconstituted basement membrane substratum as well as faster gap closure in the wound healing assay. We also independently confirmed the findings on sEV uptake and wound healing in filaggrin knockout N/TERT-2G keratinocytes, which more closely resemble primary cells. Conclusions: We conclude that the alterations in different membrane compartments in filaggrin insufficiency are reflected in changes in keratinocyte functions of relevance to AD pathology, and strategies to target those could open up new therapeutic approaches.

The deficiency of α 1 -antitrypsin protein is a genetic disorder characterized by the accumulation of misfolded protein aggregates within hepatocytes, leading to liver dysfunction. In the lung, it is found in macrophages, bronchial and epithelial alveolar cells type 2, leading to pulmonary emphysema. Despite extensive research, the precise mechanism underlying the formation of α 1 -antitrypsin inclusion bodies remain elusive. In this study, we combine equilibrium and non-equilibrium molecular dynamics simulations to elucidate the intricate process of α 1 -antitrypsin condensate formation and maturation. Our mechanistic model explains cluster accumulation—specifically the onset of this pathogenesis—through the emergence of phase-separated liquid-like protein droplets, which subsequently undergo inter-protein β -sheet transitions between misfolded variants, resulting in solid-like clusters. We find that this mechanism only applies to the misfolded variant, Z- α 1 -antitrypsin, which phase-separates driven by its disordered C-terminus. In contrast, the native protein, M- α 1 -antitrypsin, shows much lower propensity to phase-separate and later form kinetically trapped aggregates. Furthermore, we explore how Z- α 1 -antitrypsin exhibits an increased capacity to form condensates near external walls with different types of interactions. Such conditions can be similar to those found within the endoplasmic reticulum membrane, where phase separation and hardening take place. Overall, our results shed light on the molecular basis of α 1 -antitrypsin-related disorders and provide valuable microscopic insights for the development of therapeutic strategies targeting protein misfolding and aggregation-related disorders.

The suite of techniques encompassing optical super-resolution microscopy can facilitate detailed visualization of biological structures and biochemical transformations at unprecedented levels of resolution and contrast; however, they depend on imaging probes with specific biophysical and photophysical properties. In this context, metal complexes with tuneable photo-excited states and stability towards photobleaching are promising candidates for advanced imaging techniques. This Review illustrates how, by selecting appropriate optical properties and luminescence responses, metal complexes can be utilized as probes for a range of super-resolution microscopy techniques, including multimodal imaging, to study subcellular architecture and dynamics with nanoscale resolution. Limitations and challenges of the existing molecular probes are also discussed. By highlighting these recent innovations and providing suggestions for future directions, this Review further underscores the importance of optical probes in pushing the boundaries of super-resolution microscopy and advancing our understanding of complex biological systems.

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviours, clustering, trafficking and signalling in pancreatic beta cells and primary islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study 1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; 2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; 3) validates their functional relevance in beta cells; and 4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.