The Thermal Structural Transition of Alpha-Crystallin Modulates Subunit Interactions and Increases Protein Solubility

Background: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract), is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K). Methods/Results: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Dm) and the interfacial tension (c) of the aggregating phase, that characterize subunit interactions. Conclusions/General Significance: The decrease of both Dm and c at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing

Cytoprotective Effect of Idebenone through Modulation of the Intrinsic Mitochondrial Pathway of Apoptosis in Human Retinal Pigment Epithelial Cells Exposed to Oxidative Stress Induced by Hydrogen Peroxide

Idebenone is a ubiquinone short-chain synthetic analog with antioxidant properties, which is believed to restore mitochondrial ATP synthesis. As such, idebenone is investigated in numerous clinical trials for diseases of mitochondrial aetiology and it is authorized as a drug for the treatment of Leber’s hereditary optic neuropathy. Mitochondria of retinal pigment epithelium (RPE) are particularly vulnerable to oxidative damage associated with cellular senescence. Therefore, the aim of this study was to explore idebenone’s cytoprotective effect and its underlying mechanism. We used a human-RPE cell line (ARPE-19) exposed to idebenone pre-treatment for 24 h followed by conditions inducing H2O2 oxidative damage for a further 24 h. We found that idebenone: (a) ameliorated H2O2-lowered cell viability in the RPE culture; (b) activated Nrf2 signaling pathway by promoting Nrf2 nuclear translocation; (c) increased Bcl-2 protein levels, leaving unmodified those of Bax, thereby reducing the Bax/Bcl-2 ratio; (d) maintained the mitochondrial membrane potential (ΔΨm) at physiological levels, preserving the functionality of mitochondrial respiratory complexes and counteracting the excessive production of ROS; and (e) reduced mitochondrial cytochrome C-mediated caspase-3 activity. Taken together, our findings show that idebenone protects RPE from oxidative damage by modulating the intrinsic mitochondrial pathway of apoptosis, suggesting its possible role in retinal epitheliopathies associated with mitochondrial dysfunction

Automatic Detection of Aerobic Threshold through Recurrence Quantification Analysis of Heart Rate Time Series

During exercise with increasing intensity, the human body transforms energy with mechanisms dependent upon actual requirements. Three phases of the body’s energy utilization are recognized, characterized by different metabolic processes, and separated by two threshold points, called aerobic (AerT) and anaerobic threshold (AnT). These thresholds occur at determined values of exercise intensity(workload) and can change among individuals. They are considered indicators of exercise capacities and are useful in the personalization of physical activity plans. They are usually detected by ventilatory or metabolic variables and require expensive equipment and invasive measurements. Recently, particular attention has focused on AerT, which is a parameter especially useful in the overweight and obese population to determine the best amount of exercise intensity for weight loss and increasing physical fitness. The aim of study is to propose a new procedure to automatically identify AerT using the analysis of recurrences (RQA) relying only on Heart rate time series, acquired from a cohort of young athletes during a sub-maximal incremental exercise test (Cardiopulmonary Exercise Test, CPET) on a cycle ergometer. We found that the minima of determinism, an RQA feature calculated from the Recurrence Quantification by Epochs (RQE) approach, identify the time points where generic metabolic transitions occur. Among these transitions, a criterion based on the maximum convexity of the determinism minima allows to detect the first metabolic threshold. The ordinary least products regression analysis shows that values of the oxygen consumption VO2 , heart rate (HR), and Workload correspondent to the AerT estimated by RQA are strongly correlated with the one estimated by CPET (r > 0.64). Mean percentage differences are <2% for both HR and VO2 and <11% for Workload. The Technical Error for HR at AerT is <8%; intraclass correlation coefficients values are moderate (≥0.66) for all variables at AerT. This system thus represents a useful method to detect AerT relying only on heart rate time series, and once validated for different activities, in future, can be easily implemented in applications acquiring data from portable heart rate monitors

Breast cancer cells and fibroblasts in co-culture: reciprocal influences on cell adhesion, membrane fluidity and migration

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs determined a definite increment in tumor cell migration velocity, even with a marked enhancement of the migration directionality induced by CAFs. Our results demonstrate a reciprocal influence of mammary cancer and stromal cells on various adhesiveness/invasiveness features. In particular, an overall pro-tumoral/-invasive effect of CAFs on both well- and poorly differentiated mammary cancer cells was exteriorized by reduction of cell adhesion, induction of membrane fluidity, and migration velocity and directionality, along with a promotion of epithelial-mesenchymal transition

Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage

Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies

Red blood cells membrane micropolarity as a novel diagnostic indicator of type 1 and type 2 diabetes

Classification of the category of diabetes is extremely important for clinicians to diagnose and select the correct treatment plan. Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins, as well as impairment in cholesterol homeostasis, can alter lipid density, packing, and interactions of Red blood cells (RBC) plasma membranes in type 1 and type 2 diabetes, thus varying their membrane micropolarity. This can be estimated, at a submicrometric scale, by determining the membrane relative permittivity, which is the factor by which the electric field between the charges is decreased relative to vacuum. Here, we employed a membrane micropolarity sensitive probe to monitor variations in red blood cells of healthy subjects (n=16) and patients affected by type 1 (T1DM, n=10) and type 2 diabetes mellitus (T2DM, n=24) to provide a cost-effective and supplementary indicator for diabetes classification. We find a less polar membrane microenvironment in T2DM patients, and a more polar membrane microenvironment in T1DM patients compared to control healthy patients. The differences in micropolarity are statistically significant among the three groups (p<0.01). The role of serum cholesterol pool in determining these differences was investigated, and other factors potentially altering the response of the probe were considered in view of developing a clinical assay based on RBC membrane micropolarity. These preliminary data pave the way for the development of an innovative assay which could become a tool for diagnosis and progression monitoring of type 1 and type 2 diabetes. Keywords: Diabetes mellitus, Membrane micropolarity, Red blood cells, Fluorescence lifetime microscopy, Metabolic imaging, Personalized medicin

Investigation of DHA-Induced Regulation of Redox Homeostasis in Retinal Pigment Epithelium Cells through the Combination of Metabolic Imaging and Molecular Biology

Diabetes-induced oxidative stress leads to the onset of vascular complications, which are major causes of disability and death in diabetic patients. Among these, diabetic retinopathy (DR) often arises from functional alterations of the blood-retinal barrier (BRB) due to damaging oxidative stress reactions in lipids, proteins, and DNA. This study aimed to investigate the impact of the ω3-polyunsaturated docosahexaenoic acid (DHA) on the regulation of redox homeostasis in the human retinal pigment epithelial (RPE) cell line (ARPE-19) under hyperglycemic-like conditions. The present results show that the treatment with DHA under high-glucose conditions activated erythroid 2-related factor Nrf2, which orchestrates the activation of cellular antioxidant pathways and ultimately inhibits apoptosis. This process was accompanied by a marked increase in the expression of NADH (Nicotinamide Adenine Dinucleotide plus Hydrogen) Quinone Oxidoreductase 1 (Nqo1), which is correlated with a contextual modulation and intracellular re-organization of the NAD+/NADH redox balance. This investigation of the mechanisms underlying the impairment induced by high levels of glucose on redox homeostasis of the BRB and the subsequent recovery provided by DHA provides both a powerful indicator for the detection of RPE cell impairment as well as a potential metabolic therapeutic target for the early intervention in its treatment

Fatty acid-related modulations of membrane fluidity in cells: detection and implications

Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for ?-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and ?-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells

Stearoyl-CoA desaturase 1 and paracrine signal involvement in the promotion of breast cancer cell migration induced by cancer-associated fibroblasts

Despite the acknowledged impact of the tumor stroma on breast cancer development and progression, the molecular basis of such effects remain partially unexplained. We previously reported that breast cancer-associated fibroblasts (CAFs) induced epithelial-mesenchymal transition and an increase in cell membrane fluidity and migration speed in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. More recently, in order to better define the mechanisms responsible for the CAF-promoted tumor cell migration, we investigated the role of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, and demonstrated its CAF-triggered up-regulation as well as its crucial role in the migratory ability of the above tumor cells. Besides SCD1 induction, a CAF-promoted enhancement in the protein level and/or activity of the SCD1 transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), was observed. Moreover, the influence of stroma-derived signals in cancer cell migration speed was proved by cell tracking analysis in the presence of neutralizing antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor, where a marked reduction or abolishment of the fibroblast-triggered increase in cancer cell migration speed was observed. In the last part of this study, in order to verify if soluble CAF-derived factors stimulate breast cancer cell migration in a SCD1-dependent manner, tumor cells were exposed to CAF-conditioned medium (CM) and their migration evaluated by scratch assay in the presence of a small molecule inhibitor of SCD1. Moreover, to assess if the induction of SCD1 expression by CAFs might occur via SREBP1, the desaturase levels were also determined in SREBP1-inhibited tumor cells. These latest investigations indicate that SCD1 contributes to the promotion of breast cancer cell migration by CAF-derived soluble factors, since the desaturase inhibition completely suppressed the stimulatory effect of CAF-CM on tumor cell migration. SREBP1 inhibition impaired CAF-mediated up-regulation of SCD1 in poorly invasive but not in highly invasive tumor cells, in which SREBP1-independent mechanisms may account for the enhancement of SCD1 levels. These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic strategies