Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium.

Inositol 1,4,5-trisphosphate (InsP3) is a component of the phosphoinositide second-messenger system which mobilizes Ca2+ from intracellular stores. Recently, an InsP3 receptor binding protein from rat cerebellar membranes was solubilized and purified to homogeneity. The potent inhibition by Ca2+ of [3H]InsP3 binding to the InsP3 receptor in cellular membranes is not apparent in the purified receptor. The Ca2+-dependent inhibition of [3H]InsP3 binding in the crude homogenate (concn. giving 50% inhibition = 300 nM) can be restored by addition of solubilized cerebellar membranes to the purified receptor. In the present study, we further characterize the protein in solubilized membranes which confers Ca2+-sensitivity to the receptor, and which we term 'calmedin'. Calmedin appears to be a neutral membrane protein with an estimated Mr of 300,000 by gel filtration in the presence of Triton X-100. Calmedin confers a Ca2+-sensitivity to InsP3 receptor binding, which can be completely reversed by 10 min incubation with EDTA and therefore does not represent Ca2+-dependent proteinase action. Calmedin effects on the purified InsP3 receptor depend on Ca2+ binding to the calmedin, although Ca2+ also binds directly to the InsP3 receptor. The regional distribution of calmedin differs from that of the InsP3 receptor in the brain, suggesting that it also mediates other Ca2+-dependent functions. Calmedin activity in peripheral tissues is much lower than in brain.     

Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.     

Rat brain endoplasmic reticulum calcium pools are anatomically and functionally segregated.

Autoradiographic imaging can localize 45Ca2+ selectively accumulated via the Ca2+, Mg2(+)-ATPase into endoplasmic reticulum stores in rat brain slices. 45Ca2+ accumulation is markedly stimulated by oxalate and displays a heterogeneous distribution which resembles the mRNA distribution for a sarcoendoplasmic reticulum Ca2+, Mg2(+)-ATPase. Inositol 1,4,5-triphosphate [I(1,4,5)P3] inhibits 45Ca2+ accumulation selectively into regions corresponding to those enriched in I(1,4,5)P3 receptor-binding sites and in Ca2+, -Mg2(+)-ATPase mRNA. Thus rat brain endoplasmic reticulum calcium stores are anatomically and functionally differentiated.     

Cofactor Molecules Induce Structural Transformation during Infectious Prion Formation

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Identification of two prion protein regions that modify scrapie incubation time

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp(0/0), and Tg(MoPrP, Delta23-88)9949/Prnp(0/0) mice were approximately 50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Delta23-88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp(0/0) mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.     

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).     

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 PrP^Sc. A critical prediction of the protein-only hypothesis is that autocatalytic PrP^Sc molecules should be infectious. However, some autocatalytic recombinant PrP^Sc 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 >10⁵-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 PrP^C substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrP^C. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrP^C molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrP^Sc structure.     

Dissociation of infectivity from seeding ability in prions with alternate docking mechanism

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrP(Sc), suggesting that these domains of cellular prion protein (PrP(C)) serve as docking sites for PrP(Sc) during prion propagation. To examine the role of polybasic domains in the context of full-length PrP(C), we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ～5 rounds of sPMCA, PrP(Sc) molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrP(Sc) prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrP(Sc) molecules. Remarkably, ΔC-PrP(Sc) and other polybasic domain PrP(Sc) molecules displayed diminished or absent biological infectivity relative to wild-type PrP(Sc), despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrP(Sc) prions interact with PrP(C) molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrP(Sc) propagation. Furthermore, polybasic domain deficient PrP(Sc) molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrP(Sc) molecules may not depend on a single stereotypic mechanism, but that normal PrP(C)/PrP(Sc) interaction through polybasic domains may be required to generate prion infectivity.     

Comparable Attenuation of Aβ<Subscript>25–35</Subscript>-Induced Neurotoxicity by Quercitrin and 17β-Estradiol in Cultured Rat Hippocampal Neurons

In the present work, potential protective effects of quercitrin (a phytoestrogen) on Aβ-induced neurotoxicity in cultured rat hippocampal neurons were investigated in comparison with 17β-estradiol. Cell viability, oxidative status, and antioxidative potentials were used as comparative parameters. Co-exposure of cultured neurons to Aβ_25–35 with either quercitrin or 17β-estradiol (50–100 μM) for 72 h attenuated Aβ_25–35-induced neurotoxicity and lipid peroxidation, but not Aβ_25–35-induced ROS accumulation. However, only 17β-estradiol counteracted a reduction in glutathione content and only quercitrin counteracted a reduction in glutathione peroxidase activity. Both compounds displayed no effects on superoxide dismutase activity. A specific estrogen receptor antagonist, ICI 182780, did not abolish neuroprotective effects of quercitrin and 17β-estradiol. These findings suggested that quercitrin and 17β-estradiol attenuated Aβ_25–35-induced neurotoxicity in a comparable manner. Underlying neuroprotective mechanisms of both compounds were probably not related to estrogen receptor-mediated genomic mechanisms but might involve with their antioxidant and free radical scavenging properties.     

Variable clinical responses of a scrub typhus outbred mouse model to feeding by Orientia tsutsugamushi infected mites

Rodents are the natural hosts for Leptotrombidium mites that transmit Orientia tsutsugamushi, the causative agent of scrub typhus, a potentially fatal febrile human disease. Utilizing mite lines that included O. tsutsugamushi infected and non-infected Leptotrombidium species we investigated the varied infection response of outbred mice (ICR) exposed to L. chiangraiensis (Lc), L. imphalum (Li) and L. deliense (Ld). Each of six mite lines (Lc1, Lc5, Li3, Li4, Li7 and Ld) was separately placed in the inner ears of ICR mice either as a single individual (individual feeding, IF) or as a group of 2-4 individuals (pool feeding, PF). The species of infected chigger feeding on mice significantly affected mortality rates of the mice, with mite lines of Lc causing higher mean (±SE) mortality (90.7 ± 3.6 %) than mite lines of Li (62.9 ± 5.6 %) or Ld (53.6 ± 5.8 %). Mouse responses which included time to death, food consumption and total mice weight change depended on mite species and their O. tsutsugamushi genotype, more than on feeding procedure (IF vs. PF) except for mite lines within the Lc. Infected mite lines of Lc were the most virulent infected mites assessed whereas the infected Ld species was the least virulent for the ICR. Mice killed by various mite lines showed enlarged spleens and produced ascites. The results of this investigation of the clinical responses of ICR mice to feeding by various infected mite lines indicated that the different species of infected mites and their O. tsutsugamushi genotype produced different clinical presentations in ICR mice, a scrub typhus mouse model which mimics the natural transmission of O. tsutsugamushi that is critical for understanding scrub typhus disease in terms of natural transmission, host-pathogen-vector interaction and vaccine development.