Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

The blood–brain barrier (BBB ) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make‐up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15‐fold enrichment of endothelial cell marker Glut1 mRNA , whereas markers for other cell types were not enriched. Filter‐aided sample preparation was shown to be superior to in‐solution sample preparation (10251 peptides vs. 7533 peptides). Label‐free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP‐binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

     

Crystal structure of the HIV-2 neutralizing Fab fragment 7C8 with high specificity to the V3 region of gp125

7C8 is a mouse monoclonal antibody specific for the third hypervariable region (V3) of the human immunodeficiency virus type 2 (HIV-2)-associated protein gp125. The three-dimensional crystal structure of the Fab fragment of 7C8, determined to 2.7 Å resolution, reveals a deep and narrow antigen-binding cleft with architecture appropriate for an elongated epitope. The highly hydrophobic cleft is bordered on one side by the negatively charged second complementarity determining region (CDR2) and the unusually long positively charged CDR3 of the heavy chain and, on the other side, by the CDR1 of the light chain. Analysis of 7C8 in complex with molecular models of monomeric and trimeric gp125 highlights the importance of a conserved stretch of residues FHSQ that is localized centrally on the V3 region of gp125. Furthermore, modeling also indicates that the Fab fragment neutralizes the virus by sterically impairing subsequent engagement of the gp125 trimer with the co-receptor on the target cell.     

MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the beta 2-microglobulin subunit

NK cell recognition of targets is strongly affected by MHC class I specific receptors. The recently published structure of the inhibitory receptor Ly49A in complex with H-2Dd revealed two distinct sites of interaction in the crystal. One of these involves the alpha1, alpha2, alpha3, and beta2-microglobulin (beta2m) domains of the MHC class I complex. The data from the structure, together with discrepancies in earlier studies using MHC class I tetramers, prompted us to study the role of the beta2m subunit in MHC class I-Ly49 interactions. Here we provide, to our knowledge, the first direct evidence that residues in the beta2m subunit affect binding of MHC class I molecules to Ly49 receptors. A change from murine beta2m to human beta2m in three different MHC class I molecules, H-2Db, H-2Kb, and H-2Dd, resulted in a loss of binding to the receptors Ly49A and Ly49C. Analysis of the amino acids involved in the binding of Ly49A to H-2Dd in the published crystal structure, and differing between the mouse and the human beta2m, suggests the cluster formed by residues Lys3, Thr4, Thr28, and Gln29, as a potentially important domain for the Ly49A-H-2Dd interaction. Another possibility is that the change of beta2m indirectly affects the conformation of distal parts of the MHC class I molecule, including the alpha1 and alpha2 domains of the heavy chain.     

Dissection of the interaction of the human cytomegalovirus-derived US2 protein with major histocompatibility complex class I molecules: prominent role of a single arginine residue in human leukocyte antigen-A2

Human cytomegalovirus encodes several proteins that interfere with expression of major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The unique short protein 2 (US2) binds to many MHC class I allomorphs in the endoplasmic reticulum, preventing cell surface expression of the class I molecule in question. The molecular interactions underlying US2 binding to MHC class I molecules and its allele specificity have not been fully clarified. In the present study, we first compared the sequences and the structures of US2 retained versus non-retained human leukocyte antigen (HLA) class I allomorphs to identify MHC residues of potential importance for US2 binding. On the basis of this analysis, 18 individual HLA-A2 mutants were generated and the ability of full-length US2 to bind wild-type and mutated HLA-A2 complexes was assessed. We demonstrate that Arg181 plays a critical role in US2-mediated inhibition of HLA-A2 cell surface expression. The structural comparison of all known crystal structures of HLA-A2 either alone, or in complex with T cell receptor or the CD8 co-receptor, indicates that binding of US2 to HLA-A2 results in a unique, large conformational change of the side chain of Arg181. However, although the presence of Arg181 seems to be a prerequisite for US2 binding to HLA-A2, it is not sufficient for binding to all MHC class I alleles.     

Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta2-microglobulin

beta(2)-Microglobulin (beta(2)m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human beta(2)m (hbeta(2)m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse beta(2)m (mbeta(2)m) with hbeta(2)m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific beta(2)m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2D(b) in complex with LCMV-derived gp33 peptide and either hbeta(2)m or mbeta(2)m. The conformation of the gp33 peptide is not affected by the beta(2)m species. Comparison of the interface between beta(2)m and the alpha(1)alpha(2) domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hbeta(2)m and the alpha(1)alpha(2) domains of H-2D(b). We propose that the positioning of two hydrogen bond rich regions at the hbeta(2)m/alpha(1)alpha(2) interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2D(b)/hbeta(2)m complex. These two regions act as bridges, holding and stabilizing the underside of the alpha(1) and alpha(2) helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2D(b) in complex with either mbeta(2)m or hbeta(2)m provides a structural explanation for the differential binding of H-2D(b)/hbeta(2)m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of beta(2)m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hbeta(2)m impairs the recognition of Ly49C by H-2D(b)/gp33/hbeta(2)m. Finally, comparison of the two H-2D(b) crystal structures implies that the beta(2)m species may affect the strength of TCR recognition by affecting CD8 binding.     

Effects of sarcolectin (SCL) on human peripheral blood mononuclear cells

Sarcolectin (SCL) is a tissue growth factor found in various human or animal tissues, functioning in balance with interferons (IFNs) that can inhibit growth and affect cell differentiation. Like somatotropin, SCL is found in the pituitary gland. In humans, the SCL gene is located on chromosome 12 (q12-q13) and expressed as a 55 kDa protein consisting of 469 amino-acids. After a single activation of peripheral blood mononuclear cells (PBMC) obtained from more than 30 individuals, highly significant cell proliferation was found to peak after 7 days in culture. The presence of adherent cells was necessary for cell proliferation. SCL induced over-expression of alpha-IL-2 receptor (CD25) leading to proliferation of CD3+/CD4+/CD45RO+ T cells. Thus in PBMC, SCL induced CD4+ T cell growth and expression of inflammatory cytokine genes, including TNF-alpha, IL-1beta, IL-6 and IL-8. IFNs are also produced following activation as a feedback response which is maintained for about 20 days.     

Immune modulation by the human cytomegalovirus-encoded molecule UL18, a mystery yet to be solved

Human cytomegalovirus infects human populations at a high frequency worldwide. During the long coevolution of virus and host, a fine balance has developed between viral immune evasion strategies and defense mechanisms of the immune system. Human cytomegalovirus encodes multiple proteins involved in the evasion of immune recognition, among them UL18, a MHC class I homologue. Despite almost 20 years of research and the discovery of a broadly expressed inhibitory receptor for this protein, its function in immune modulation is not clear yet. Recent data suggest that besides inhibitory effects on various immune cells, UL18 may also act as an activating component during CMV infection. In this review, we provide an overview of the biology of UL18 and discuss several attempts to shed light on its function.     

Single and combined genotoxic and cytotoxic effects of two xenobiotics widely used in intensive aquaculture

Several chemicals are used in aquaculture to prevent or to treat disease outbreaks. These substances are mainly administered by two different routes: by prolonged immersion or by mixing into the diet. In the case of intensive aquaculture, the chemicals that are most frequently applied by immersion are formaldehyde (FA) 37% and oxytetracycline (OTC). The first is highly effective against most protozoa, as well as some of the most common parasites such as monogenetic trematodes. OTC presents a large spectrum of antibacterial activities and is used to treat systemic bacterial infections that affect fish. Under therapeutic use, FA (37%) is applied prophylactically at 200ml/m(3), whereas OTC is used curatively at 40g/m(3). The goal of the present study is to assess genotoxic and cytotoxic effects associated with exposure of the European sea bass (Dicentrarchus labrax) to FA37% and OTC under the same conditions as those applied in intensive aquaculture systems. To this end the micronucleus (MN) assay was applied in erythrocytes. Our results show that both tested chemicals present genotoxic and cytotoxic potential following a time-dependent pattern. Remarkably, the combined treatment induces a cumulative effect, which is particularly pronounced after 15 days of exposure. This suggests the critical hazards associated with exposure to FA and OTC when applied or released together.     

UHRF1 recruits the histone acetyltransferase Tip60 and controls its expression and activity

Tat-interactive protein, 60 kDa (Tip60) is a histone acetyltransferase with specificity toward lysine 5 of histone H2A (H2AK5) and plays multiple roles in chromatin remodeling processes. Co-immunoprecipitation experiments performed on Jurkat cells, showed that Tip60 is present in the same macro-molecular complex as UHRF1 (Ubiquitin-like containing PHD and RING domain 1), DNMT1 (DNA methyltransferase 1), and HDAC1 (histone deacetylase 1). Furthermore, immunocytochemistry experiments confirmed that Tip60 co-localizes with the UHRF1/DNMT1 complex. Although down-regulation of UHRF1 by RNA interference enhanced Tip60 expression, a significant decrease of the level of acetylated H2AK5 was observed. Consistently, we have observed that down-regulation of Tip60 and DNMT1 by RNA interference, dramatically reduced the levels of acetylated H2AK5. Altogether, these results suggest that Tip60 is a novel partner of the epigenetic integration platform interplayed by UHRF1, DNMT1 and HDAC1 involved in the epigenetic code replication.