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1.
Jiri G. Safar Xiangzhu Xiao Mohammad E. Kabir Shugui Chen Chae Kim Tracy Haldiman Yvonne Cohen Wei Chen Mark L. Cohen Witold K. Surewicz 《PLoS pathogens》2015,11(4)
The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrPSc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrPSc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD), determined their conformational stability and replication tempo in vitro, as well as characterized structural organization using recently emerged approaches based on hydrogen/deuterium (H/D) exchange coupled with mass spectrometry. Our data clearly demonstrate that these phenotypically distant prions differ in a major way with regard to their structural organization, both at the level of the polypeptide backbone (as indicated by backbone amide H/D exchange data) as well as the quaternary packing arrangements (as indicated by H/D exchange kinetics for histidine side chains). Furthermore, these data indicate that, in contrast to previous observations on yeast and some murine prion strains, the replication rate of sCJD prions is primarily determined not by conformational stability but by specific structural features that control the growth rate of prion protein aggregates. 相似文献
2.
Tracy Haldiman Chae Kim Yvonne Cohen Wei Chen Janis Blevins Liuting Qing Mark L. Cohen Jan Langeveld Glenn C. Telling Qingzhong Kong Jiri G. Safar 《The Journal of biological chemistry》2013,288(41):29846-29861
The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrPSc). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrPSc particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrPSc particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrPC substrate, the dominant PrPSc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrPSc is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrPSc conformers. 相似文献
3.
The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro 总被引:4,自引:0,他引:4
Nishina KA Deleault NR Mahal SP Baskakov I Luhrs T Riek R Supattapone S 《Biochemistry》2006,45(47):14129-14139
A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner. 相似文献
4.
Cellular PrP is actively cycled between the cell surface and the endosomal pathway. The exact site and mechanism of conversion from PrP(C) to PrP(Sc) remain unknown. We have previously used recombinant antibodies containing grafts of PrP sequence to identify three regions of PrP(C) (aa23-27, 98-110, and 136-158) that react with PrP(Sc) at neutral pH. To determine if any regions of PrP(C) react with PrP(Sc) at an acidic pH similar to that of an endosomal compartment, we tested our panel of grafted antibodies for the ability to precipitate PrP(Sc) in a range of pH conditions. At pH near or lower than 6, PrP-grafted antibodies representing the octapeptide repeat react strongly with PrP(Sc) but not PrP(C). Modified grafts in which the histidines of the octarepeat were replaced with alanines did not react with PrP(Sc). PrP(Sc) precipitated by the octapeptide at pH 5.7 was able to seed conversion of normal PrP to PrP(Sc) in vitro. However, modified PrP containing histidine to alanine substitutions within the octapeptide repeats was still converted to PrP(Sc) in N2a cells. These results suggest that once PrP has entered the endosomal pathway, the acidic environment facilitates the binding of PrP(Sc) to the octarepeat of PrP(C) by the change in charge of the histidines within the octarepeat. 相似文献
5.
6.
Julie M. Cramer Timothy Thompson Albert Geskin William LaFramboise Eric Lagasse 《PloS one》2015,10(3)
The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease. 相似文献
7.
A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrPC). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrPSc followed by detection with a europium-labelled anti-PrPC antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrPSc in cases of vCJD. 相似文献
8.
Background
Transmissible agents involved in prion diseases differ in their capacities to target different regions of the central nervous system and lymphoid tissues, which are also host-dependent.Methodology/Principal Findings
Protease-resistant prion protein (PrPres) was analysed by Western blot in the spleen of transgenic mice (TgOvPrP4) that express the ovine prion protein under the control of the neuron-specific enolase promoter, after infection by intra-cerebral route with a variety of transmissible spongiform encephalopathies (TSEs) from cattle and small ruminants. Splenic PrPres was consistently detected in classical BSE and in most natural scrapie sources, the electrophoretic pattern showing similar features to that of cerebral PrPres. However splenic PrPres was not detected in L-type BSE and TME-in-cattle, or in the CH1641 experimental scrapie isolate, indicating that some TSE strains showed reduced splenotropism in the ovine transgenic mice. In contrast with CH1641, PrPres was also consistently detected in the spleen of mice infected with six natural “CH1641-like” scrapie isolates, but then showed clearly different molecular features from those identified in the brains (unglycosylated PrPres at ∼18 kDa with removal of the 12B2 epitope) of ovine transgenic mice or of sheep. These features included different cleavage of the main PrPres cleavage product (unglycosylated PrPres at ∼19 kDa with preservation of the 12B2 epitope) and absence of the additional C-terminally cleaved PrPres product (unglycosylated form at ∼14 kDa) that was detected in the brain.Conclusion/Significance
Studies in a transgenic mouse model expressing the sheep prion protein revealed different capacities of ruminant prions to propagate in the spleen. They showed unexpected features in “CH1641-like” ovine scrapie suggesting that such isolates contain mixed conformers with distinct capacities to propagate in the brain or lymphoid tissues of these mice. 相似文献9.
Geoff Barnard Lee Hopkins Sowmiya Moorthie David Seilly Paul Tonks Reza Dabaghian Jonathan Clewley John Coward Ian McConnell 《朊病毒》2007,1(2):121-127
A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrPC). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrPSc followed by detection with a europium-labelled anti-PrPC antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrPSc in cases of vCJD.Key Words: BSE, scrapie and vCJD Diagnosis, DELFIA®, novel N terminal antibodies 相似文献
10.
Atsuko Takeuchi Atsushi Kobayashi James W. Ironside Shirou Mohri Tetsuyuki Kitamoto 《The Journal of biological chemistry》2013,288(30):21659-21666
To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype. 相似文献
11.
Alkes L. Price Arti Tandon Nick Patterson Kathleen C. Barnes Nicholas Rafaels Ingo Ruczinski Terri H. Beaty Rasika Mathias David Reich Simon Myers 《PLoS genetics》2009,5(6)
Identifying the ancestry of chromosomal segments of distinct ancestry has a wide range of applications from disease mapping to learning about history. Most methods require the use of unlinked markers; but, using all markers from genome-wide scanning arrays, it should in principle be possible to infer the ancestry of even very small segments with exquisite accuracy. We describe a method, HAPMIX, which employs an explicit population genetic model to perform such local ancestry inference based on fine-scale variation data. We show that HAPMIX outperforms other methods, and we explore its utility for inferring ancestry, learning about ancestral populations, and inferring dates of admixture. We validate the method empirically by applying it to populations that have experienced recent and ancient admixture: 935 African Americans from the United States and 29 Mozabites from North Africa. HAPMIX will be of particular utility for mapping disease genes in recently admixed populations, as its accurate estimates of local ancestry permit admixture and case-control association signals to be combined, enabling more powerful tests of association than with either signal alone. 相似文献
12.
Madhusudhan Budatha Simone Silva Teodoro Ignacio Montoya Ayako Suzuki Sheena Shah-Simpson Cecilia Karin Wieslander Masashi Yanagisawa Ruth Ann Word Hiromi Yanagisawa 《PloS one》2013,8(2)
Mice deficient for the fibulin-5 gene (Fbln5−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. 相似文献
13.
Yo Ching Cheng Samia Hannaoui Theodore R. John Sandor Dudas Stefanie Czub Sabine Gilch 《PloS one》2016,11(11)
Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrPSc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding. 相似文献
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15.
The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-β protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer''s disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders. 相似文献
16.
Xiangxiang Zhang Jianzheng Fang Bin Xu Shengli Zhang Shifeng Su Zhen Song Yunfei Deng Hainan Wang Dan Zhao Xiaobing Niu Zengjun Wang 《PloS one》2013,8(12)
Objective
Epididymal protease inhibitor (Eppin) was located on the surface of spermatozoa and modulates the liquefaction of human semen. Here, we identify the correlative protein partner of Eppin to explore the molecular mechanism of liquefaction of human semen.Methods
(1) Human seminal vesicle proteins were transferred on the membrane by Western blotting and separated by 2-D electrophoresis and incubated in recombinant Eppin. The correlative protein was identified by Mass Spectrometry (MS) (2). Western blotting was used to determine the relation of rEppin and rFibronectin(Fn); (3) Co-localization in spermatozoa were detected using immunofluorescence; (4) Correalation of Eppin and Fn was proved by co-immunoprecipitation.Results
Fn was identified as the binding partner of recombinant Eppin by MS. Recombinant of Eppin was made and demonstrated that the Eppin fragment binds the fn 607-1265 fragment. The Eppin-Fn complex presents on the sperm tail and particularly in the midpiece region of human ejaculated spermatozoa. Immunoprecipitation indicated that Eppin in the spermatozoa lysates was complexed with Fn.Conclusions
Our study demonstrates that Eppin and Fn bind to each other in human semen and on human ejaculated spermatozoa. Eppin-Fn complex may involve in semen coagulation, liquefaction and the survival and preparation of spermatozoa for fertility in the female reproductive tract. 相似文献17.
18.
Thomas Zögg Michael Sponring Sabrina Schindler Maria Koll Rainer Schneider Hans Brandstetter Bernhard Auer 《Structure (London, England : 1993)》2013,21(6):929-938
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19.
Quantitative Electroimmunoblotting Study of the Calcium-Activated Neutral Protease in Human Myelin 总被引:1,自引:1,他引:0
Degradation of myelin basic protein (MBP) in human man myelin was monitored by electroimmunoblotting. Problems of variation between, as well as within, electroimmunoblots were overcome by the introduction of an internal standard in each sample, thus allowing reproducible quantification of MBP. The Ca2+-dependent protease acting on MBP was active at endogenous levels of Ca2+ (congruent to 300 micrograms/g myelin) and was inhibited in the presence of Ca2+ chelators. Extensive degradation of MBP occurred rapidly in the presence of added Ca2+, reaching a plateau after a 1 h incubation (80-85% degradation). The proteolytic activity was not enhanced in the presence of 2-mercaptoethanol. It was most active at neutral pH and at temperatures approaching physiological conditions. No difference was observed between proteolytic activities of control and multiple sclerotic myelin. It is suggested that fluctuations in the accessibility of free Ca2+ to the protease may lead to the regulation of Ca2+-activated myelinolysis. 相似文献
20.
Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis
Ghada Al-Salih Nawwar Al-Attar Sandrine Delbosc Liliane Louedec Elisabeth Corvazier Stéphane Loyau Jean-Baptiste Michel Dominique Pidard Xavier Duval Olivier Meilhac 《PloS one》2012,7(9)