Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice

Twenty-four atypical scrapie cases from sheep with different prion protein genotypes from Great Britain were transmitted to transgenic tg338 and/or TgshpXI mice expressing sheep PrP alleles, but failed to transmit to wild-type mice. Mean incubation periods were 200–300 days in tg338 mice and 300–500 days in TgshpXI mice. Survival times in C57BL/6 and VM/Dk mice were >700 days. Western blot analysis of mouse brain samples revealed similar multi-band, protease-resistant prion protein (PrPres) profiles, including an unglycosylated band at ∼8–11 kDa, which was shown by antibody mapping to correspond to the ∼93–148 aa portion of the PrP molecule. In transgenic mice, the incubation periods, Western blot PrPres profiles, brain lesion profiles and abnormal PrP (PrPSc) distribution patterns produced by the Great Britain atypical scrapie isolates were similar and compatible with the biological characteristics of other European atypical scrapie or Nor98 cases.

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Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

10.1101/2020.07.21.213488 ◽

2020 ◽

Author(s):

Manish Bhattacharjee ◽

Navin Adhikari ◽

Renu Sudhakar ◽

Zeba Rizvi ◽

Divya Das ◽

...

Keyword(s):

Mass Spectrometry ◽

Plasmodium Falciparum ◽

Western Blot ◽

Wild Type ◽

Malaria Parasites ◽

Post Translational Modifications ◽

Functional Conservation ◽

Cellular Processes ◽

Physiological Substrates

ABSTRACTA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (Δrub1) or NEDD8 conjugating E2 enzyme (ΔUbc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

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Neuroanatomical distribution of abnormal prion protein in naturally occurring atypical scrapie cases in Great Britain

Acta Neuropathologica ◽

10.1007/s00401-008-0433-8 ◽

2008 ◽

Vol 116 (5) ◽

pp. 547-559 ◽

Cited By ~ 38

Author(s):

Sarah Jo Moore ◽

Marion Simmons ◽

Melanie Chaplin ◽

John Spiropoulos

Keyword(s):

Great Britain ◽

Prion Protein ◽

Atypical Scrapie ◽

Naturally Occurring

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Transmission and Adaptation of Chronic Wasting Disease to Hamsters and Transgenic Mice: Evidence for Strains

Journal of Virology ◽

10.1128/jvi.02474-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 4305-4314 ◽

Cited By ~ 72

Author(s):

Gregory J. Raymond ◽

Lynne D. Raymond ◽

Kimberly D. Meade-White ◽

Andrew G. Hughson ◽

Cynthia Favara ◽

...

Keyword(s):

Transgenic Mice ◽

Prion Protein ◽

Chronic Wasting Disease ◽

Transmissible Spongiform Encephalopathy ◽

Wild Type Mouse ◽

Mouse Strains ◽

Spongiform Encephalopathy ◽

Wild Type ◽

Wasting Disease ◽

Incubation Periods

ABSTRACT In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.

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Characterization of proteinase K-resistant N- and C-terminally truncated PrP in Nor98 atypical scrapie

Journal of General Virology ◽

10.1099/vir.0.81618-0 ◽

2006 ◽

Vol 87 (6) ◽

pp. 1751-1760 ◽

Cited By ~ 47

Author(s):

Mikael Klingeborn ◽

Lotta Wik ◽

Magnus Simonsson ◽

Lena H. M. Renström ◽

Therese Ottinger ◽

...

Keyword(s):

Prion Protein ◽

Structural Gene ◽

Proteinase K ◽

Atypical Scrapie ◽

Antigenic Composition ◽

Electrophoretic Profile ◽

Brain Extracts ◽

Overlapping Region

An increasing number of scrapie cases with atypical characteristics, designated Nor98, have recently been recognized. Here, the proteinase K (PK)-resistant prion protein (PrP) fragments from two Swedish cases of Nor98 atypical scrapie have been characterized. The prominent, fast-migrating band in the distinct Nor98 Western immunoblot electrophoretic profile was determined to be of 7 kDa in size and was accordingly designated Nor98-PrP7. The antigenic composition of Nor98-PrP7, as assayed by a panel of anti-PrP antibodies, revealed that this fragment comprised a mid-region of PrP from around aa 85 to 148. N- and C-terminally truncated fragments spanning the mid-region of PrP have only been observed in the genetic prion disorder Gerstmann–Sträussler–Scheinker disease. It is shown here that the long-term PK resistance of Nor98-PrP7 is reduced compared with that of PrPres in classical scrapie. Enzymic deglycosylation did not change the distinct electrophoretic profile of Nor98-PrP7. A previously unidentified, PK-resistant, C-terminal PrP fragment of around 24 kDa was detected and its PK resistance was investigated. After deglycosylation, this fragment migrated as a 14 kDa polypeptide and was designated PrP-CTF14. Antigenic determination and the size of 14 kDa suggested a fragment spanning approximately aa 120–233. The existence of two PK-resistant PrP fragments, Nor98-PrP7 and PrP-CTF14, that share an overlapping region suggests that at least two distinct PrP conformers with different PK-resistant cores are present in brain extracts from Nor98-affected sheep. The structural gene of PrP in three Nor98-affected sheep was analysed, but no mutations were found that could be correlated to the aberrant PK-resistant profile observed.

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Immunological characterization of abnormal prion protein from atypical scrapie cases in sheep using a panel of monoclonal antibodies

Journal of General Virology ◽

10.1099/vir.0.81816-0 ◽

2006 ◽

Vol 87 (12) ◽

pp. 3715-3722 ◽

Cited By ~ 31

Author(s):

Anja Gretzschel ◽

Anne Buschmann ◽

Jan Langeveld ◽

Martin H. Groschup

Keyword(s):

Monoclonal Antibodies ◽

Prion Protein ◽

Proteinase K ◽

Atypical Scrapie ◽

Surveillance Programme ◽

Rapid Tests ◽

The Brain ◽

Sheep Scrapie ◽

Scrapie Case

After the implementation of an active surveillance programme for scrapie in sheep in the EU, the number of diagnosed classical scrapie cases rose sharply and a novel kind of so-called atypical scrapie case was discovered. These atypical scrapie cases display unusual features concerning the distribution of the abnormal prion protein (PrPSc) in the brain, a distinct electrophoretic profile of PrPSc and an inconsistent reaction pattern in the currently used rapid tests. In this report, PrPSc of two German atypical sheep scrapie cases was characterized by epitope mapping using a panel of 18 monoclonal antibodies that were directed against epitopes located throughout the prion protein. This analysis suggests that PrPSc derived from atypical scrapie cases and treated with proteinase K is largely composed of an 11 kDa fragment (previously referred to as the 12 kDa band) and of polymeric fragments thereof. The 11 kDa band corresponds to a prion protein fragment spanning approximately aa 90–153 and may therefore represent a novel PrPSc type.

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Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

Journal of Bacteriology ◽

10.1128/jb.00535-08 ◽

2008 ◽

Vol 190 (20) ◽

pp. 6589-6597 ◽

Cited By ~ 20

Author(s):

Maureen Varina ◽

Steven M. Denkin ◽

Andrew M. Staroscik ◽

David R. Nelson

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Protease Activity ◽

Western Blot Analysis ◽

Culture Supernatant ◽

Vibrio Anguillarum ◽

Wild Type ◽

Processing Protease ◽

Culture Supernatants

ABSTRACT The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ∼10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.

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