Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP^ARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP^ARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP^ARR and PrP^ARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP^ARR form was more toxic than the scrapie susceptible PrP^ARQ variant. Moreover, the α-helical conformation of PrP^ARR was less stable than that of PrP^ARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP^ARQ variant displays a higher propensity to form large aggregates than PrP^ARR. Interestingly, in the presence of small amounts of PrP^ARR, PrP^ARQ aggregability was reduced to levels similar to that of PrP^ARR. Thus, in contrast to PrP^ARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP^ARQ that allows the formation of large amyloid fibrils.     

Intracellular accumulation of a mild-denatured monomer of the human PrP fragment 90-231, as possible mechanism of its neurotoxic effects

Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre-fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90–231 (hPrP90–231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild-denatured hPrP90–231 (incubated for 1 h at 53°C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90–231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.     

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.     

Ruta graveolens L. Induces Death of Glioblastoma Cells and Neural Progenitors,but Not of Neurons,via ERK 1/2 and AKT Activation

Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens’ effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue’s noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.     

The renaissance of mitochondrial calcium transport.

Although the capacity of mitochondria for accumulating Ca2+ down the electrical gradient generated by the respiratory chain has been known for over three decades, the physiological significance of this phenomenon has been re-evaluated only recently. Indeed, it was long believed that the low affinity of the mitochondrial Ca2+ transporters would allow significant uptake only in conditions of cellular Ca2+ overload. Conversely, the direct measurement of [Ca2+] in the mitochondrial matrix revealed major [Ca2+] increases upon agonist stimulation. In this review, we will summarize: (a) the mechanisms that allow this large response, reconciling the biochemical properties of the transporters and the large amplitude of the mitochondrial [Ca2+] rises, and (b) the biological role of mitochondrial Ca2+ signalling, that encompasses the regulation of mitochondrial function and the modulation of the spatio-temporal pattern of cytosolic [Ca2+] increases.     

Identification and Analysis of Proteins Sharing Dehydroascorbate Reductase Activity

Dehydroascorbate (DHA)-reducing proteins were observed using a native-PAGE/activity staining method, and identified in soybean and rice by Western analysis using antibodies against homologous protein known to catalyse DHA reduction. Administration of 1 mM DHA apparently did not trigger DHA reductase activity in lupin and onion, whereas activity was increased in rice and barley.     

Mechanism of active shrinkage in mitochondria. II. Coupling between strong electrolyte fluxes.

1. Addition of succinate to valinomycin-treated mitochondria incubated in KCL causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner. 2. A passive penetration of K+ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude. 3. Passive swelling and active shrinkage occurs also when K+ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration. 4. Active shrinkage, either with K+ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%. 5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.     

Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+

The intracellularly trappable fluorescent Ca2+ indicator quin-2 was used to measure free cytosolic Ca2+, [Ca2+]i, in the two highly dedifferentiated tumor cell lines, Ehrlich and Yoshida ascites carcinomas. It was found that these carcinoma cells can trap quin-2 similarly to normal cells, but [Ca2+]i was apparently significantly lower than in any normal cell tested previously with this method. By using a new lipid-soluble heavy metal chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), which crosses artificial and natural membranes, it was found that endogenous heavy metals are responsible for partially quenching quin-2 fluorescence trapped inside the cells. Although the quenching of intracellular quin-2 fluorescence is quantitatively more relevant in these ascites carcinomas, TPEN was effective also in normal cells like lymphocytes and granulocytes. Both in the normal and especially in the malignant cell lines [Ca2+]i can be grossly underestimated at low intracellular quin-2 concentrations. Endogenous heavy metal quenching is thus a potential source of artifact when [Ca2+]i is measured with quin-2. When corrected for quin-2 fluorescence quenching by intracellular heavy metals, [Ca2+]i and basic regulatory mechanisms of [Ca2+]i homeostasis in Ehrlich and Yoshida carcinomas are similar to those of nontransformed cells.