Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca2+ release in permeabilized pancreatic acinar cells

Inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca2+ release induced with 0.18 μM IP3 is half maximally inhibited with approximately 5 μM decavanadate. Complete inhibition is achieved with around 20 μM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP3, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca2+ uptake into intracellular stores, does not influence IP3 induced Ca2+ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP3 to the same cellular preparations was investigated. We found that binding of IP3 to a high affinity receptor site (Kd approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 μM decavanadate, IP3 binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP3 with the same affinity (Kd approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP3 was displaced from these subcellular membranes with similar kinetics by unlabelled IP3 and decavanadate. The data suggest that the inhibitory action of decavanadate on IP3 induced Ca2+ release is a consequence of its effect on binding of IP3 to its receptor. EGTA, ethylene-glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PEG, polyethylene glycol; IP3, inositol 1,4,5-trisphosphate; MOPS, morpholinopropane sulfonic acid; HEPES, N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid; Tris, tris(hydroxymethyl)-aminomethan

Comparative Analysis of Mutant Huntingtin Binding Partners in Yeast Species.

Huntington\u27s disease is caused by the pathological expansion of a polyglutamine (polyQ) stretch in Huntingtin (Htt), but the molecular mechanisms by which polyQ expansion in Htt causes toxicity in selective neuronal populations remain poorly understood. Interestingly, heterologous expression of expanded polyQ Htt is toxic in Saccharomyces cerevisiae cells, but has no effect in Schizosaccharomyces pombe, a related yeast species possessing very few endogenous polyQ or Q/N-rich proteins. Here, we used a comprehensive and unbiased mass spectrometric approach to identify proteins that bind Htt in a length-dependent manner in both species. Analysis of the expanded polyQ-associated proteins reveals marked enrichment of proteins that are localized to and play functional roles in nucleoli and mitochondria in S. cerevisiae, but not in S. pombe. Moreover, expanded polyQ Htt appears to interact preferentially with endogenous polyQ and Q/N-rich proteins, which are rare in S. pombe, as well as proteins containing coiled-coil motifs in S. cerevisiae. Taken together, these results suggest that polyQ expansion of Htt may cause cellular toxicity in S. cerevisiae by sequestering endogenous polyQ and Q/N-rich proteins, particularly within nucleoli and mitochondria

Isolation of Phosphatidylethanolamine as a Solitary Cofactor for Prion Formation in the Absence of Nucleic Acids

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrP(Sc)) can be produced de novo from a mixture of the normal conformer (PrP(C)) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nuclease-resistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrP(Sc) molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrP(Sc) formation in the absence of RNA. PE facilitated the propagation of PrP(Sc) molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrP(Sc) formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrP(Sc) molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions

Anti-prion drug mPPIg5 inhibits PrP(C) conversion to PrP(Sc).

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future

Cofactor Molecules Maintain Infectious Conformation and Restrict Strain Properties in Purified Prions

Prions containing misfolded prion protein (PrP(Sc)) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrP(Sc) structure accompanied by a reduction in specific infectivity of >10(5)-fold, to undetectable levels, despite the ability of adapted “protein-only” PrP(Sc) molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrP(Sc) structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrP(Sc) conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by \u3e105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half-maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by \u3e105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure

Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux

AbstractThe immunosuppressant drug FK506 binds to the immunophilin protein FKBP12 and inhibits its prolyl isomerase activity. Immunosuppresive actions, however, are mediated via an FK506-FKBP12 inhibition of the Ca2+-activated phosphatase calcineurin. Physiologic cellular roles for FKBP12 have remained unclear. FKBP12 is physically associated with the RyR and IP3R Ca2+ channels in the absence of FK506, with added FK506 disrupting these complexes. Dissociation of FKBP12 results in alteration of channel Ca2+ conductance in both cases. We now report that calcineurin is physiologically associated with the IP3R-FKBP12 and RyR-FKBP12 receptor complexes and that this interaction can be disrupted by FK506 or rapamycin. Calcineurin anchored to the IP3R via FKBP12 regulates the phosphorylation status of the receptor, resulting in a dynamic Ca2+-sensitive regulation of IP3-mediated Ca2+ flux

Identification of a Homology-Independent Linchpin Domain Controlling Mouse and Bank Vole Prion Protein Conversion

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion