ATP Release from Vascular Endothelia Occurs Across Cx43 Hemichannels and Is Attenuated during Hypoxia

Background: Extracellular ATP is an important signaling molecule for vascular adaptation to limited oxygen availability (hypoxia). Here, we pursued the contribution of vascular endothelia to extracellular ATP release under hypoxic conditions. Methodology, Principal Findings: We gained first insight from studying ATP release from endothelia (HMEC-1) pre-exposed to hypoxia. Surprisingly, we found that ATP release was significantly attenuated following hypoxia exposure (2 % oxygen, 2263 % after 48 h). In contrast, intracellular ATP was unchanged. Similarly, lactate-dehydrogenase release into the supernatants was similar between normoxic or hypoxic endothelia, suggesting that differences in lytic ATP release between normoxia or hypoxia are minimal. Next, we used pharmacological strategies to study potential mechanisms for endothelialdependent ATP release (eg, verapamil, dipyridamole, 18-alpha-glycyrrhetinic acid, anandamide, connexin-mimetic peptides). These studies revealed that endothelial ATP release occurs – at least in part- through connexin 43 (Cx43) hemichannels. A real-time RT-PCR screen of endothelial connexin expression showed selective repression of Cx43 transcript and additional studies confirmed time-dependent Cx43 mRNA, total and surface protein repression during hypoxia. In addition, hypoxia resulted in Cx43-serine368 phosphorylation, which is known to switch Cx43 hemi-channels from an open to a closed state. Conclusions/Significance: Taken together, these studies implicate endothelial Cx43 in hypoxia-associated repression o

Physiological roles for ecto-5’-nucleotidase (CD73)

Nucleotides and nucleosides influence nearly every aspect of physiology and pathophysiology. Extracellular nucleotides are metabolized through regulated phosphohydrolysis by a series of ecto-nucleotidases. The formation of extracellular adenosine from adenosine 5’-monophosphate is accomplished primarily through ecto-5’-nucleotidase (CD73), a glycosyl phosphatidylinositol-linked membrane protein found on the surface of a variety of cell types. Recent in vivo studies implicating CD73 in a number of tissue protective mechanisms have provided new insight into its regulation and function and have generated considerable interest. Here, we review contributions of CD73 to cell and tissue stress responses, with a particular emphasis on physiologic responses to regulated CD73 expression and function, as well as new findings utilizing Cd73-deficient animals

Dynamic purine signaling and metabolism during neutrophil–endothelial interactions

During episodes of hypoxia and inflammation, polymorphonuclear leukocytes (PMN) move into underlying tissues by initially passing between endothelial cells that line the inner surface of blood vessels (transendothelial migration, TEM). TEM creates the potential for disturbances in vascular barrier and concomitant loss of extravascular fluid and resultant edema. Recent studies have demonstrated a crucial role for nucleotide metabolism and nucleoside signaling during inflammation. These studies have implicated multiple adenine nucleotides as endogenous tissue protective mechanisms invivo. Here, we review the functional components of vascular barrier, identify strategies for increasing nucleotide generation and nucleoside signaling, and discuss potential therapeutic targets to regulate the vascular barrier during inflammation

The interaction of dengue virus capsid protein with negatively charged interfaces drives the in vitro assembly of nucleocapsid-like particles.

Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4' is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation

Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity

Submitted by Sandra Infurna ([email protected]) on 2019-05-02T15:36:16Z No. of bitstreams: 1 Marciano_Paes_etal_IOC_2019.pdf: 2757474 bytes, checksum: f484f68c0e57cb9176253927e0d15291 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-05-02T15:49:17Z (GMT) No. of bitstreams: 1 Marciano_Paes_etal_IOC_2019.pdf: 2757474 bytes, checksum: f484f68c0e57cb9176253927e0d15291 (MD5)Made available in DSpace on 2019-05-02T15:49:17Z (GMT). No. of bitstreams: 1 Marciano_Paes_etal_IOC_2019.pdf: 2757474 bytes, checksum: f484f68c0e57cb9176253927e0d15291 (MD5) Previous issue date: 2019Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Departamento de Biotecnologia Farmacêutica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biodiversidade e Sustentabilidade. Macaé, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisa Médica. Rio de Janeiro, RJ. Brasil.Universidade do Estado do Rio de Janeiro. Laboratório de Ultraestrutura e Biologia Tecidual. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Física Biológica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Física Biológica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations