Permeability of phospholipid membranes and human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a SfRBM 28th Annual ConferenceHydrogen peroxide (H2O2) is an oxygen-derived oxidant involved in multiple redox processes in the cell, ranging from physiological signaling pathways to oxidative damage reactions when it is found at higher concentrations. In the vascular system, H2O2 is metabolized mainly by red blood cells (RBC) due to their very efficient antioxidant systems and high membrane permeability. However, the information regarding H2O2 transport in the human RBC membrane is limited, as neither the exact value of the permeability coefficient (Pm) nor the permeation mechanisms are known. To explore whether H2O2 permeates through the lipid fraction or protein channels, we studied H2O2 solubility in organic solvents and its permeability in lipid membranes, in order to compare with the RBC membrane. Through measurements of partition constants, we found that H2O2 is 14 and 122000 times less soluble in octanol and hexadecane than in water, anticipating a large thermodynamic barrier to H2O2 permeation by lipid membranes. The Pm in phospholipid membranes of different compositions, determined using the catalase-latency method, varied from 4×10-4 to 5×10-3 cm s-1, at 37°C. On the other hand, in human RBC we determined a Pm of 1.6×10-3 cm s-1. After obtaining these results, we evaluated the potential role of aquaporins as H2O2 transporters by checking the effect of aquaporin inhibitors in H2O2 consumption by RBC, and also by studying H2O2 permeability in RBC devoid of either aquaporin 1 or aquaporin 3. Surprisingly, we could not detect any differences in H2O2 permeability in any case. Altogether, these results provide new information on lipid membrane permeability to H2O2 and a new value for the Pm in human RBC, which was previously unknown. Additionally, they indicate that H2O2 is not transported by aquaporins in human RBC membranes, suggesting simple diffusion or a still unidentified membrane protein as a more probable pathway.ANII: ANII: FMV_1_2019_15559

Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells.

International audienceChronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration

ATPe Dynamics in Protozoan Parasites: Adapt or Perish

In most animals, transient increases of extracellular ATP (ATPe) are used for physiological signaling or as a danger signal in pathological conditions. ATPe dynamics are controlled by ATP release from viable cells and cell lysis, ATPe degradation and interconversion by ecto-nucleotidases, and interaction of ATPe and byproducts with cell surface purinergic receptors and purine salvage mechanisms. Infection by protozoan parasites may alter at least one of the mechanisms controlling ATPe concentration. Protozoan parasites display their own set of proteins directly altering ATPe dynamics, or control the activity of host proteins. Parasite dependent activation of ATPe conduits of the host may promote infection and systemic responses that are beneficial or detrimental to the parasite. For instance, activation of organic solute permeability at the host membrane can support the elevated metabolism of the parasite. On the other hand ecto-nucleotidases of protozoan parasites, by promoting ATPe degradation and purine/pyrimidine salvage, may be involved in parasite growth, infectivity, and virulence. In this review, we will describe the complex dynamics of ATPe regulation in the context of protozoan parasite–host interactions. Particular focus will be given to features of parasite membrane proteins strongly controlling ATPe dynamics. This includes evolutionary, genetic and cellular mechanisms, as well as structural-functional relationships.Instituto de Investigaciones Bioquímicas de La Plat

Effects of Erythrocytes Treated with Alpha Hemolysin of E.Coli on Endothelial Cells

Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations might interfere with host-cell-signaling pathways, causing apoptosis. In the present investigation we demonstrate that a relatively low concentration of HlyA induces morphological changes and phosphatidylserine (PS) externalization of human erythrocytes. On the other hand, the unacylated nonhemolytic form of HlyA, ProHlyA induces similar morphological changes but no PS externalization. We performed osmoscan experiments to test the effect of both proteins on erythrocytes structure.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Estudios Inmunológicos y Fisiopatológico

TSPO ligands stimulate ZnPPIX transport and ROS accumulation leading to the inhibition of P. falciparum growth in human blood

After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2-VDAC complex, which leads to an accumulation of ROS

From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals

Erythropoiesis occurs mostly in bone marrow and ends in blood stream. Mature red blood cells are generated from multipotent hematopoietic stem cells, through a complex maturation process involving several morphological changes to produce a highly functional specialized cells. In mammals, terminal steps involved expulsion of the nucleus from erythroblasts that leads to the formation of reticulocytes. In order to produce mature biconcave red blood cells, organelles and ribosomes are selectively eliminated from reticulocytes as well as the plasma membrane undergoes remodeling. The mechanisms involved in these last maturation steps are still under investigation. Enucleation involves dramatic chromatin condensation and establishment of the nuclear polarity, which is driven by a rearrangement of actin cytoskeleton and the clathrin-dependent generation of vacuoles at the nuclear-cytoplasmic junction. This process is favored by interaction between the erythroblasts and macrophages at the erythroblastic island. Mitochondria are eliminated by mitophagy. This is a macroautophagy pathway consisting in the engulfment of mitochondria into a double-membrane structure called autophagosome before degradation. Several mice knock-out models were developed to identify mitophagy-involved proteins during erythropoiesis, but whole mechanisms are not completely determined. Less is known concerning the clearance of other organelles, such as smooth and rough ER, Golgi apparatus and ribosomes. Understanding the modulators of organelles clearance in erythropoiesis may elucidate the pathogenesis of different dyserythropoietic diseases such as myelodysplastic syndrome, leukemia and anemia

An Overview of Different Strategies to Recreate the Physiological Environment in Experimental Erythropoiesis

Human erythropoiesis is a complex process leading to the production of mature, enucleated erythrocytes (RBCs). It occurs mainly at bone marrow (BM), where hematopoietic stem cells (HSCs) are engaged in the early erythroid differentiation to commit into erythroid progenitor cells (burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E)). Then, during the terminal differentiation, several erythropoietin-induced signaling pathways trigger the differentiation of CFU-E on successive stages from pro-erythroblast to reticulocytes. The latter are released into the circulation, finalizing their maturation into functional RBCs. This process is finely regulated by the physiological environment including the erythroblast-macrophage interaction in the erythroblastic island (EBI). Several human diseases have been associated with ineffective erythropoiesis, either by a defective or an excessive production of RBCs, as well as an increase or a hemoglobinization defect. Fully understanding the production of mature red blood cells is crucial for the comprehension of erythroid pathologies as well as to the field of transfusion. Many experimental approaches have been carried out to achieve a complete differentiation in vitro to produce functional biconcave mature RBCs. However, the various protocols usually fail to achieve enough quantities of completely mature RBCs. In this review, we focus on the evolution of erythropoiesis studies over the years, taking special interest in efforts that were made to include the microenvironment and erythroblastic islands paradigm. These more physiological approaches will contribute to a deeper comprehension of erythropoiesis, improve the treatment of dyserythropoietic disorders, and break through the barriers in massive RBCs production for transfusion

Construction and validation of an atomic model for bacterial TSPO from electron microscopy density, evolutionary constraints, and biochemical and biophysical data.

International audienceThe 18kDa protein TSPO is a highly conserved transmembrane protein found in bacteria, yeast, animals and plants. TSPO is involved in a wide range of physiological functions, among which the transport of several molecules. The atomic structure of monomeric ligand-bound mouse TSPO in detergent has been published recently. A previously published low-resolution structure of Rhodobacter sphaeroides TSPO, obtained from tubular crystals with lipids and observed in cryo-electron microscopy, revealed an oligomeric structure without any ligand. We analyze this electron microscopy density in view of available biochemical and biophysical data, building a matching atomic model for the monomer and then the entire crystal. We compare its intra- and inter-molecular contacts with those predicted by amino acid covariation in TSPO proteins from evolutionary sequence analysis. The arrangement of the five transmembrane helices in a monomer of our model is different from that observed for the mouse TSPO. We analyze possible ligand binding sites for protoporphyrin, for the high-affinity ligand PK 11195, and for cholesterol in TSPO monomers and/or oligomers, and we discuss possible functional implications