Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma pre-kallikrein and are members of the PAN module superfamily

AbstractMicronemes are specialised organelles, found in all apicomplexan parasites, which secrete molecules that are essential for parasite attachment to and invasion of host cells. Regions of several microneme proteins have sequence similarity to the Apple domains (A-domains) of blood coagulation factor XI (FXI) and plasma pre-kallikrein (PK). We have used mass spectrometry on a recombinant-expressed, putative A-domain from the microneme protein EtMIC5 from Eimeria tenella, to demonstrate that three intramolecular disulphide bridges are formed. These bridges are analogous to those that stabilise A-domains in FXI and PK. The data confirm that the apicomplexan domains are structural homologues of A-domains and are therefore novel members of the PAN module superfamily, which also includes the N-terminal domains of members of the plasminogen/hepatocyte growth factor family. The role of A-domains/PAN modules in apicomplexan parasites is not known, but their presence in the microneme suggests that they may be important for mediating protein–protein or protein–carbohydrate interactions during parasite attachment and host cell invasion

The P2X(7 )receptor is a candidate product of murine and human lupus susceptibility loci: a hypothesis and comparison of murine allelic products

Systemic lupus erythematosus and its murine equivalent, modelled in the New Zealand Black and New Zealand White (NZB × NZW)F(1 )hybrid strain, are polygenic inflammatory diseases, probably reflecting an autoimmune response to debris from cells undergoing programmed cell death. Several human and murine loci contributing to disease have been defined. The present study asks whether the proinflammatory purinergic receptor P2X(7), an initiator of a form of programmed cell death known as aponecrosis, is a candidate product of murine and human lupus susceptibility loci. One such locus in (NZB × NZW)F(1 )mice is lbw3, which is situated at the distal end of NZW chromosome 5. We first assess whether NZB mice and NZW mice carry distinct alleles of the P2RX(7 )gene as expressed by common laboratory strains, which differ in sensitivity to ATP stimulation. We then compare the responses of NZB lymphocytes, NZW lymphocytes and (NZB × NZW)F(1 )lymphocytes to P2X(7 )stimulation. NZB and NZW parental strains express the distinct P2X(7)-L and P2X(7)-P alleles of P2RX(7), respectively, while lymphocytes from these and (NZB × NZW)F(1 )mice differ markedly in their responses to P2X(7 )receptor stimulation. NZB mice and NZW mice express functionally distinct alleles of the proinflammatory receptor, P2X(7). We show that current mapping suggests that murine and human P2RX(7 )receptor genes lie within lupus susceptibility loci lbw3 and SLEB4, and we argue that these encode a product with the functional characteristics consistent with a role in lupus. Furthermore, we argue that aponecrosis as induced by P2X(7 )is a cell death mechanism with characteristics that potentially have particular relevance to disease pathogenesis

Manipulating adenovirus hexon hypervariable loops dictates immune neutralisation and coagulation factor X-dependent cell interaction in vitro and in vivo

Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting

Heme oxygenase (HO)-1 induction prevents Endoplasmic Reticulum stress-mediated endothelial cell death and impaired angiogenic capacity

Most of diabetic cardiovascular complications are attributed to endothelial dysfunction and impaired angiogenesis. Endoplasmic Reticulum (ER) and oxidative stresses were shown to play a pivotal role in the development of endothelial dysfunction in diabetes. Hemeoxygenase-1 (HO-1) was shown to protect against oxidative stress in diabetes; however, its role in alleviating ER stress-induced endothelial dysfunction remains not fully elucidated. We aim here to test the protective role of HO-1 against high glucose-mediated ER stress and endothelial dysfunction and understand the underlying mechanisms with special emphasis on oxidative stress, inflammation and cell death.Human Umbilical Vein Endothelial Cells (HUVECs) were grown in either physiological or intermittent high concentrations of glucose for 5days in the presence or absence of Cobalt (III) Protoporphyrin IX chloride (CoPP, HO-1 inducer) or 4-Phenyl Butyric Acid (PBA, ER stress inhibitor). Using an integrated cellular and molecular approach, we then assessed ER stress and inflammatory responses, in addition to apoptosis and angiogenic capacity in these cells.Our results show that HO-1 induction prevented high glucose-mediated increase of mRNA and protein expression of key ER stress markers. Cells incubated with high glucose exhibited high levels of oxidative stress, activation of major inflammatory and apoptotic responses [nuclear factor (NF)-κB and c-Jun N-terminal kinase (JNK)] and increased rate of apoptosis; however, cells pre-treated with CoPP or PBA were fully protected. In addition, high glucose enhanced caspases 3 and 7 cleavage and activity and augmented cleaved poly ADP ribose polymerase (PARP) expression whereas HO-1 induction prevented these effects. Finally, HO-1 induction and ER stress inhibition prevented high glucose-induced reduction in NO release and impaired the angiogenic capacity of HUVECs, and enhanced vascular endothelial growth factor (VEGF)-A expression.Altogether, we show here the critical role of ER stress-mediated cell death in diabetes-induced endothelial dysfunction and impaired angiogenesis and underscore the role of HO-1 induction as a key therapeutic modulator for ER stress response in ischemic disorders and diabetes. Our results also highlight the complex interplay between ER stress response and oxidative stress.This work was supported with grants to Dr Abdelali Agouni from the Royal Society, the Physiological Society, and Qatar University (grant QUUG-CPH-CPH-15/16-6). Mr Maamoun and Ms. Zachariah are supported by doctoral scholarships from Egyptian cultural bureau and Government of Botswana, respectively

The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Databases: Important resources for haemostasis clinicians and researchers

Haemophilia published by John Wiley & Sons Ltd Introduction: Advances in genomic sequencing have facilitated the sequencing of genes associated with disorders of haemostasis. The identification of variants within genes and access to curated data incorporating structural, functional, evolutionary as well as phenotypic data has become increasingly important in order to ascribe pathogenicity. Aim: The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Database Project aims to provide a single port of entry to a web-accessible resource for variants in genes involved in clinical bleeding disorders. Results: New databases have evolved from previously developed single gene variant coagulation database projects, incorporating new data, new analysis tools and a new common database architecture with new interfaces and filters. These new databases currently present information about the genotype, phenotype (laboratory and clinical) and structural and functional effects of variants described in the genes of factor (F) VII (F7), FVIII (F8), FIX (F9) and von Willebrand factor (VWF). Conclusion: The project has improved the quality and quantity of information available to the haemostasis research and clinical communities, thereby enabling accurate classification of disease severity in order to make assessments of likely pathogenicity

Inhibition of Thrombin Receptor Signaling on alpha-Smooth Muscle Actin(+) CD34(+) Progenitors Leads to Repair After Murine Immune Vascular Injury

OBJECTIVE: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). METHODS AND RESULTS: BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. CONCLUSIONS: Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage