Development of a rapid and comprehensive proteomics-based arboviruses detection system

Okamoto, Kenta; Endo, Yushirou; Inoue, Satoshi; Nabeshima, Takeshi; Nga, Phan Thi; Guillermo, Posadas H.; Yu, Fangfang; Phuong, Loan; Trang, Bui Minh; Natividad, Filipinas F.; Hasebe, Futoshi; Morita, Kouichi

doi:10.1016/j.jviromet.2010.03.006

Cited by 12 publications

(9 citation statements)

References 26 publications

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“…To identify EBOV VP30-associated kinase(s), we adopted a proteomics approach by employing different versions of VP30: wild-type VP30 (VP30 wt ); a nonphosphorylatable mutant of VP30 in which all six major phosphorylation sites at the N-terminus are mutated to alanine (VP30 6A ); a mutant that mimics fully phosphorylated VP30 via replacement of the six serines to negatively charged aspartic acid residues (VP30 6D ); and VP30 29S (Figure 1A) (10, 19). Ectopically expressed FLAG-tagged VP30 mutants were immunoprecipitated, and coprecipitating cellular proteins were eluted (16) and digested with trypsin prior to analysis by liquid chromatography-tandem mass spectrometry (Supplementary Figure 1) (23). A number of kinases coprecipitated with VP30 (Figure 1B, Figure supplement 1); of these, we focused on SRPK1, interferon-induced double-stranded RNA-activated protein kinase (PKR) and serine/threonine-protein kinase RIO2 (RIOK2).…”

Section: Resultsmentioning

confidence: 99%

Serine-arginine protein kinase 1 regulates Ebola virus transcription

Takamatsu

Krähling

Kolesnikova

et al. 2019

Preprint

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word count: 148) 13Ebola virus (EBOV) causes a severe and often fatal disease for which no approved 14 vaccines or antivirals are currently available. EBOV transcription requires the sequential 15 phosphorylation and dephosphorylation of the viral transcription factor VP30. While 16 dephosphorylation is carried out by phosphatases PP2A and PP1, the VP30-specific 17 kinase is unknown. Here, we report that serine-arginine protein kinase 1 and 2 (SRPK1 18 and SRPK2) phosphorylate serine-29 of VP30, which is located in an N-terminal R26xxS29 19 motif. Interaction with VP30 via the R26xxS29 motif recruits SRPK1 into EBOV-induced 20 inclusion bodies, the sites of viral RNA synthesis and an inhibitor of SRPK1/SRPK2 21 downregulates primary viral transcription. When the SRPK1 recognition motif of VP30 22 was mutated in a recombinant EBOV, virus replication was severely impaired. It is 23 presumed that the interplay between SRPK1 and PP2A in the EBOV inclusions provides 24 a comprehensive regulatory circuit to ensure the activity of VP30 in EBOV transcription. 25 26 inhibit ectopically expressed SRPK1 ( Figure 2B, lanes 2 and 4). Quantification of three 130 independent experiments confirmed the statistical significance of the observations 131 ( Figure 2B, graph). In summary, SRPK1 specifically phosphorylates VP30 wt and VP30 29S 132 in vitro and in cultured cells and regulates the phosphorylation state of VP30 together 133 with OA-sensitive phosphatases, likely PP2A and/or PP1. 134 SRPK1 regulates EBOV genome transcription/replication

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Section: Resultsmentioning

confidence: 99%

Serine-arginine protein kinase 1 regulates Ebola virus transcription

Takamatsu

Krähling

Kolesnikova

et al. 2019

Preprint

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“…1E) were obtained using the NanoFrontier nLC and NanoFrontier eLD Liquid Chromatography Mass Spectrometer (Hitachi High-technologies, Tokyo, Japan). The nanoLiquid Chromatography/ ElectroSpray Ionization/ Linear Ion Trap/ Time of Flight (nLC-ESI/LIT/TOF) and collision induced dissociation (CID) modes were used for MS detection and peptide fragmentation as described [32]. In the NanoFrontier nLC, the trypsinized peptides (1–10 µL) suspended in 0.3% formic acid were trapped on monolith trap column [C18-50-150 column, (0.05 mm I.D.…”

Section: Methodsmentioning

confidence: 99%

Immunoproteomics Identification of Major IgE and IgG4 Reactive Schistosoma japonicum Adult Worm Antigens Using Chronically Infected Human Plasma

et al. 2012

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Immunoepidemiological studies from endemic areas have revealed age-dependent resistance correlation with increased level of IgE and decreased level of IgG4 antibodies in responses to schistosomes’ soluble worm antigen. However, there have been limited studies on analyses of major antigens that provoke IgE and IgG4 immune response during chronic stage of schistosomiasis. In this study, for the first time, immunoproteomics approach has been applied to identify S. japonicum worm antigens in liquid fractions that are recognized by IgE and IgG4 antibody using plasma from chronically infected population. ProteomeLabPF 2D fractionated 1-D and 2-D fractions of SWA antigens were screened using pooled high IgE/IgG4 reactive plasma samples by dot-blot technique. In 1-D fractions, IgE isotype was detected by fewer antigenic fractions (43.2%). The most recognized isotype was IgG3 (79.5%) followed by IgG1 (75.0%) and IgG4 (61.4%). Liquid chromatography MS/MS protein sequencing of reactive 2-D fractions revealed 18 proteins that were identified, characterized and gene ontology categories determined. 2-D fractions containing proteins such as zinc finger, RanBP2-type, domain-containing protein were strongly recognized by IgE and moderately by IgG4 whereas fractions containing proteins such as ubiquitin-conjugating enzyme and cytosolic II 5'-nucleotidase strongly recognizing by IgG subclasses (IgG1, IgG3 and IgG4) but not IgE. By this study, a simple and reproducible proteomic method has been established to identify major immunoreactive S. japonicum antigens. It is anticipated that this will stimulate further research on the immunogenicity and protective potential of proteins identified as well as discovery of novel compounds that have therapeutic importance.

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“…In addition, western blotting was conducted using mouse anti-His tag monoclonal antibody (Novagen). Where the size of the recombinant protein did not correspond to the expected size on SDS-PAGE, the peptide sequence was analyzed by trypsin digestion and liquid chromatography-mass spectrometry (LC-MS/MS) following the detailed protocol described elsewhere [51]. After measuring the concentration of the recombinant proteins as described above for SWAP and SEA, the antigens were aliquoted and stored below −30°C.…”

Section: Cloning and Expression Of Recombinant Smtr Proteinsmentioning

confidence: 99%

Tandem repeat recombinant proteins as potential antigens for the sero-diagnosis of Schistosoma mansoni infection

Kalenda

Kato

Goto

et al. 2015

Parasitology International

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The diagnosis of schistosome infection, followed by effective treatment and/or mass drug administration, is crucial to reduce the disease burden. Suitable diagnostic tests and field-applicable tools are required to sustain schistosomiasis control programs. We therefore assessed the potential of tandem repeat (TR) proteins for serodiagnosis of Schistosoma mansoni infection using an experimental mouse model. TR genes in the genome of S. mansoni were searched in silico and 7 candidates, named SmTR1, 3, 8, 9, 10, 11 and 15, were selected. Total RNA was extracted from S. mansoni adult worms and eggs. Target TR genes were amplified, cloned, and the proteins were expressed in Escherichia coli competent cells. Female BALB/c mice were infected with 100 S. mansoni cercariae and sera were collected each week post-infection for 18 weeks. The levels of IgG antibodies to SmTR antigens were compared to those to soluble egg antigen (SEA) and to soluble worm antigen preparation (SWAP). Sera of infected mice reacted to all the antigens whereas those of naïve mice did not. IgG responses to SmTR1, 3, 9 and 10 were detected at the early stage of infection. Interestingly, antibodies reacting to SmTR3, 9, 10 and 15 dramatically decreased 4 weeks after treatment with praziquantel, while those against SEA and SWAP remained elevated. Our study suggests that TR proteins, especially SmTR10, may be suitable antigens for sero-diagnosis of infection by S. mansoni and are potential markers for monitoring and surveillance of schistosomiasis, including re-infection after treatment with praziquantel.

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Development of a rapid and comprehensive proteomics-based arboviruses detection system

Cited by 12 publications

References 26 publications

Serine-arginine protein kinase 1 regulates Ebola virus transcription

Serine-arginine protein kinase 1 regulates Ebola virus transcription

Immunoproteomics Identification of Major IgE and IgG4 Reactive Schistosoma japonicum Adult Worm Antigens Using Chronically Infected Human Plasma

Tandem repeat recombinant proteins as potential antigens for the sero-diagnosis of Schistosoma mansoni infection

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