Toward the molecular basis of inherited prion diseases: NMR structure of the human prion protein with V210I mutation

The development of transmissible spongiform encephalopathies (TSEs) is associated with the conversion of the cellular prion protein (PrP^C) into a misfolded, pathogenic isoform (PrP^Sc). Spontaneous generation of PrP^Sc in inherited forms of disease is caused by mutations in gene coding for PrP (PRNP). In this work, we describe the NMR solution-state structure of the truncated recombinant human PrP (HuPrP) carrying the pathological V210I mutation linked to genetic Creutzfeldt-Jakob disease. The three-dimensional structure of V210I mutant consists of an unstructured N-terminal part (residues 90-124) and a well-defined C-terminal domain (residues 125-228). The C-terminal domain contains three α-helices (residues 144-156, 170-194 and 200-228) and a short antiparallel β-sheet (residues 129-130 and 162-163). Comparison with the structure of the wild-type HuPrP revealed that although two structures share similar global architecture, mutation introduces some local structural differences. The observed variations are mostly clustered in the α₂-α₃ inter-helical interface and in the β₂-α₂ loop region. Introduction of bulkier Ile at position 210 induces reorientations of several residues that are part of hydrophobic core, thus influencing α₂-α₃ inter-helical interactions. Another important structural feature involves the alteration of conformation of the β₂-α₂ loop region and the subsequent exposure of hydrophobic cluster to solvent, which facilitates intermolecular interactions involved in spontaneous generation of PrP^Sc. The NMR structure of V210I mutant offers new clues about the earliest events of the pathogenic conversion process that could be used for the development of antiprion drugs.     

Cell signaling in NMDA preconditioning and neuroprotection in convulsions induced by quinolinic acid

The search for novel, less invasive therapeutic strategies to treat neurodegenerative diseases has stimulated scientists to investigate the mechanisms involved in preconditioning. Preconditioning has been report to occur in many organs and tissues. In the brain, the modulation of glutamatergic transmission is an important and promising target to the use of effective neuroprotective agents. The glutamatergic excitotoxicity is a factor common to neurodegenerative diseases and acute events such as cerebral ischemia, traumatic brain injury and epilepsy. In this review we focus on the neuroprotection and preconditioning by chemical agents. Specially, chemical preconditioning models using N-methyl-d-aspartate (NMDA) pre-treatment, which has demonstrated to lead to neuroprotection against seizures and damage to neuronal tissue induced by quinolinic acid (QA). Here we attempted to gather important results obtained in the study of cellular and molecular mechanisms involved in NMDA preconditioning and neuroprotection.     

Megasporogenesis and programmed cell death in <Emphasis Type="Italic">Tillandsia</Emphasis> (Bromeliaceae)

The degeneration of three of four meiotic products is a very common process in the female gender of oogamous eukaryotes. In Tillandsia (and many other angiosperms), the surviving megaspore has a callose-free wall in chalazal position while the other three megaspores are completely embedded in callose. Therefore, nutrients and signals can reach more easily the functional megaspore from the nucellus through the chalazal pole with respect to the other megaspores. The abortion of three of four megaspores was already recognized as the result of a programmed cell death (PCD) process. We investigated the process to understand the modality of this specific type of PCD and its relationship to the asymmetric callose deposition around the tetrad. The decision on which of the four megaspores will be the supernumerary megaspores in angiosperms, and hence destined to undergo programmed cell death, appears to be linked to the callose layer deposition around the tetrad. During supernumerary megaspores degeneration, events leading to the deletion of the cells do not appear to belong to a single type of cell death. The first morphological signs are typical of autophagy, including the formation of autophagosomes. The TUNEL positivity and a change in morphology of mitochondria and chloroplasts indicate the passage to an apoptotic-like PCD phase, while the cellular remnants undergo a final process resembling at least partially (ER swelling) necrotic morphological syndromes, eventually leading to a mainly lipidic cell corpse still separated from the functional megaspore by a callose layer.     

Monomer dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.     

IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans

In the murine model of Cryptococcus neoformans infection Th1 (IL-12/IFN-gamma) and Th17 (IL-23/IL-17) responses are associated with protection, whereas an IL-4-dependent Th2 response exacerbates disease. To investigate the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13-overexpressing transgenic (IL-13Tg(+)), IL-13-deficient (IL-13(-/-)), and wild-type (WT) mice were infected intranasally. Susceptibility to C. neoformans infection was found when IL-13 was induced in WT mice or overproduced in IL-13Tg(+) mice. Infected IL-13Tg(+) mice had a reduced survival time and higher pulmonary fungal load as compared with WT mice. In contrast, infected IL-13(-/-) mice were resistant and 89% of these mice survived the entire period of the experiment. Ag-specific production of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with a significant type 2 cytokine shift but only minor changes in IFN-gamma production. Consistent with enhanced type 2 cytokine production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected in susceptible WT and IL-13Tg(+) mice. Interestingly, expression of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with reduced IL-17 production. IL-13 was found to induce formation of alternatively activated macrophages expressing arginase-1, macrophage mannose receptor (CD206), and YM1. In addition, IL-13 production led to lung eosinophilia, goblet cell metaplasia and elevated mucus production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes to fatal allergic inflammation during C. neoformans infection.