[Show abstract][Hide abstract] ABSTRACT: Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.
[Show abstract][Hide abstract] ABSTRACT: Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.
No preview · Article · Jan 2013 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Fatal neurodegenerative prion diseases are caused by the transmissible PrP(Sc) prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion.
[Show abstract][Hide abstract] ABSTRACT: Sex effect on the incubation period of variant Creutzfeldt-Jakob disease (vCJD) disease in human and ME-7 murine models was
investigated. In the 167 vCJD cases reported in the United Kingdom as of January 2009, age at onset was significantly lower
in female patients (by 2 years) than in male patients after stratification on birth cohort. In C57/Bl6N mice infected with
ME-7 scrapie strain, incubation was shorter in female than in male mice. The incubation period increased in castrated male
mice after intraperitoneal infection but not after intracerebral inoculation. In the absence of androgen receptors, the incubation
period for prion disease increased after intraperitoneal inoculation. In ovariectomized or estrogen receptor a–defective female
mice, no effect was observed on the incubation period of mouse prion disease. These results show that androgens influence
the prion diseases incubation period after inoculation at a peripheral site.
Full-text · Article · Aug 2010 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: A growing number of studies have investigated the interaction between C1q and PrP, but the oligomeric form of PrP involved
in this interaction remains to be determined. Aggregation of recombinant full-length murine PrP in the presence of 100 mm NaCl allowed us to isolate three different types of oligomers by size-exclusion chromatography. In contrast to PrP monomers
and fibrils, these oligomers activate the classical complement pathway, the smallest species containing 8–15 PrP protomers
being the most efficient. We used Thioflavine T fluorescence to monitor PrP aggregation and showed that, when added to the
reaction, C1q has a cooperative effect on PrP aggregation and leads to the formation of C1q-PrP complexes. In these complexes,
C1q interacts through its globular domains preferentially with the smallest oligomers, as shown by electron microscopy, and
retains the ability to activate the classical complement pathway. Using two cell lines, we also provide evidence that C1q
inhibits the cytotoxicity induced by the smallest PrP oligomers. The cooperative interaction between C1q and PrP could represent
an early step in the disease, where it prevents elimination of the prion seed, leading to further aggregation.
Full-text · Article · Jun 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains.
Full-text · Article · Dec 2008 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP(C), for "cellular prion protein") into an abnormal state (PrP(Sc), for "scrapie prion protein"). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP(C). In contrast to its homologue PrP(C), Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP(Sc)-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP(C) (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the alpha-helical monomer forms soluble beta-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.
No preview · Article · Feb 2008 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Mice defective for C1q complement factor show enhanced resistance to peripheral prion inoculation, and previous work demonstrated a direct interaction between C1q and conformationally modified PrP. However, the nature and physiological consequences of this interaction remain uncharacterized. PrP amino acids 141-159 has been identified as a potential C1q binding site; we show, by both surface plasmon resonance (SPR) spectroscopy and ELISA, that C1q and its globular region bind to PrP mutagenized in the region of interest with comparable efficiency to that of wild-type protein. To test PrP's ability to activate complement, soluble oligomers of the PrP constructs were made. Only PrP and mutagenized PrP oligomers activate the classical complement cascade while PrP monomer and the C-terminal domain, both in oligomeric and in monomeric form, failed to induce activation. This suggests that a conformational change in PrP, which occurs both when PrP is bound to an SPR sensor chip and when it undergoes oligomerization, is requisite for PrP/C1q interaction and activation of the complement cascade. We propose that C1q may act as a natural sensor for prions, leading to activation of the classical complement cascade, which could result in local inflammation and subsequent recruitment of the immune cells that prions initially infect.
Full-text · Article · Jan 2008 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.
Full-text · Article · Jan 2005 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Introduction Heparan sulfate (HS) is a cell surface and basement membrane, sulfated polysaccharide involved in a huge array of biological processes, such as cell proliferation, chemo-attraction, inflammation, matrix assembly, embryo development or viral attachment. These multiple functions stem from HS ability to bind and modulate the activity of a large number of proteins. The interaction of HS with its ligands is primarily, although not exclusively, of an electrostatic nature and involves the recognition of basic domains on the protein by specific motifs of the polysaccharide. Importantly, subtle differences in HS structure have been shown to dramatically affect the polysaccharide activity, as observed for HS regulation of bFGF signalling. Therefore, the identification of both protein and saccharide structural determinants implicated in HS/protein binding is critical for understanding the biological role of the interaction. In this study, we have developed a simple technique enabling the identification of HS-binding sites on proteins. Materials and methods Briefly, proteins were immobilized on heparin beads, using an adapted protocol of a zero-length two-step cross-linking method and submitted to proteolytic digestion. Peptides retained on beads, i.e. containing amino acids involved in heparin binding, were then identified by solid phase N-terminus protein sequencing performed directly on the heparin beads. Results This technique was used to analyse a number of HS protein ligands, including the envelope protein gC of a herpes virus (pseudorabies) and the chemokine RANTES. Results obtained pinpointed residues known to be critical for HS binding and thus validate the method. Conclusion This technique constitutes a novel tool that should greatly facilitate HS binding site cartography of the ever-expanding family of heparin-binding proteins and provides an attractive alternative to more complex approaches such as site-directed mutagenesis.
No preview · Article · Aug 2004 · International Journal of Experimental Pathology