The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

<p>Abstract</p> <p>Background</p> <p>Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested <it>in vitro </it>the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also <it>in vivo </it>associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an <it>in vitro </it>co-sedimentation assay was performed.</p> <p>Results</p> <p>The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-helical amino-terminal domain of vimentin <it>in vitro</it>. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated <it>in vivo</it>. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association.</p> <p>Conclusion</p> <p>We hypothesize that Stk33 is involved in the <it>in vivo </it>dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.</p

The mammalian gene function resource: the International Knockout Mouse Consortium.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research

Comparative genomic sequencing analysis of a region in human chromosome 11p15.3, mouse chromosome 7 and analysis of the novel STK33, Stk33 gene

The comparative genomic sequence analysis of a region in human chromosome 11p15.3 and its homologous segment in mouse chromosome 7 between ST5 and LMO1 genes has been performed. 158,201 bases were sequenced in the mouse and compared with the syntenic region in human, partially available in the public databases. The analysed region exhibits the typical eukaryotic genomic structure and compared with the close neighbouring regions, strikingly reflexes the mosaic pattern distribution of (G+C) and repeats content despites its relative short size. Within this region the novel gene STK33 was discovered (Stk33 in the mouse), that codes for a serine/threonine kinase. The finding of this gene constitutes an excellent example of the strength of the comparative sequencing approach. Poor gene-predictions in the mouse genomic sequence were corrected and improved by the comparison with the unordered data from the human genomic sequence publicly available. Phylogenetical analysis suggests that STK33 belongs to the calcium/calmodulin-dependent protein kinases group and seems to be a novelty in the chordate lineage. The gene, as a whole, seems to evolve under purifying selection whereas some regions appear to be under strong positive selection. Both human and mouse versions of serine/threonine kinase 33, consists of seventeen exons highly conserved in the coding regions, particularly in those coding for the core protein kinase domain. Also the exon/intron structure in the coding regions of the gene is conserved between human and mouse. The existence and functionality of the gene is supported by the presence of entries in the EST databases and was in vivo fully confirmed by isolating specific transcripts from human uterus total RNA and from several mouse tissues. Strong evidence for alternative splicing was found, which may result in tissue-specific starting points of transcription and in some extent, different protein N-termini. RT-PCR and hybridisation experiments suggest that STK33/Stk33 is differentially expressed in a few tissues and in relative low levels. STK33 has been shown to be reproducibly down-regulated in tumor tissues, particularly in ovarian tumors. RNA in-situ hybridisation experiments using mouse Stk33-specific probes showed expression in dividing cells from lung and germinal epithelium and possibly also in macrophages from kidney and lungs. Preliminary experimentation with antibodies designed in this work, performed in parallel to the preparation of this manuscript, seems to confirm this expression pattern. The fact that the chromosomal region 11p15 in which STK33 is located may be associated with several human diseases including tumor development, suggest further investigation is necessary to establish the role of STK33 in human health.In der vorliegenden Arbeit wurde eine vergleichende Genomanalyse der humanen Chromosomenregion 11p15.3 mit dem homologen Bereich des murinen Chromosoms 7 zwischen den Genen ST5 und LMO1 durchgeführt. 158.201 Basenpaare der Maus wurden sequenziert und mit dem syntänen Bereich im Mensch-Genom verglichen, der zum Teil selbst sequenziert wurde und zum Teil in den Datenbanken verfügbar war. Die analysierten Bereiche weisen die typischen strukturellen Eigenschaften eines eukaryotischen Genoms auf, z.B. im Hinblick auf (G+C)-Gehalt und repetitive Sequenzen. Innerhalb dieser Region wurde ein für eine Serin/Threonin-Proteinkinase kodierendes neues Gen gefunden, das nach den Regeln des Human Genome Organisation Nomenclature Committee STK33 in Mensch und Stk33 in Maus genannt wurde. Der Nachweis dieses Gens zeigt die Effektivität der vergleichenden Genomanalyse-Strategie. Schwache Genvorhersage-Ergebnisse in der genomischen Sequenz der Maus wurden durch vergleichende Analyse mit der unvollständigen Sequenz des menschlichen Genoms korrigiert. Daraus konnten die vollständigen STK33/Stk33-Transkripte erschlossen werdendie durch Laborexperimente bestätigt wurden. Phylogenetische Analysen zeigen, dass STK33/Stk33 zu der Calcium/Calmodulin-abhängigen Protein-Kinasen Gruppe gehört und eventuell ein Novum der Chordaten ist. Das Gen scheint unter reinigender Selektion, ein Bereich sogar unter starker positiver Selektion zu evolvieren. Beide Versionen der Serin/Threonin-Kinase-33 in Mensch und Maus bestehen aus siebzehn Exonen, die hoch konserviert in den kodierenden Bereichen vorliegen, allen voran die für die Kinase-Domäne. Auch die Exon/Intron-Struktur ist in den kodierenden Bereichen zwischen Mensch und Maus gut konserviert. Der Nachweis der Expression des Gens wird durch Einträge in den EST-Datenbanken unterstützt und wurde in vivo durch Isolierung spezifischer Transkripte aus Gesamt-RNA des menschlichen Uterus und aus mehreren murinen-Geweben komplett bestätigt. Es wurden starke Hinweise auf differentielles Spleißen gefunden, was eventuell für gewebsspezifische Transkriptions-Startpunkte und verschiedene Protein-N-termini sprechen könnte. RT-PCR und Hybridisierungs-Experimente beweisen, dass STK33/Stk33 in nur wenigen Geweben und in relativ geringen Mengen exprimiert wird. STK33 ist in einigen Tumor-Geweben herunterreguliert, insbesondere in Ovarial-Tumoren. RNA in situ Hybridisierungs-Experimente mit Maus-spezifischen Sonden zeigen, dass Stk33 in teilenden Zellen aus Lungengewebe und des Germinalepithels aktiv ist und möglicherweise auch in Nieren- und Lungen-Makrophagen. Erste Tests mit Antikörpern, die in dieser Arbeit entworfen wurden, bestätigen dieses Expressionsmuster. Die Tatsache, dass der chromosomale Bereich 11p15, wo STK33 lokalisiert ist, mit mehreren menschlichen Krankheiten inklusive Tumorentwicklung in Verbindung gebracht wird, zeigt deutlich, dass weitere Untersuchungen nötig sind, um den Zusammenhang von STK33 mit der menschlichen Gesundheit zu klären

Pre- and postexposure efficacy of fully human antibodies against Spike protein in a novel humanized mouse model of MERS-CoV infection

© 2015, National Academy of Sciences. All rights reserved. Traditional approaches to antimicrobial drug development are poorly suited to combatting the emergence of novel pathogens. Additionally, the lack of small animal models for these infections hinders the in vivo testing of potential therapeutics. Here we demonstrate the use of the VelocImmune technology (a mouse that expresses human antibody-variable heavy chains and κ light chains) alongside the VelociGene technology (which allows for rapid engineering of the mouse genome) to quickly develop and evaluate antibodies against an emerging viral disease. Specifically, we show the rapid generation of fully human neutralizing antibodies against the recently emerged Middle East Respiratory Syndrome coronavirus (MERS-CoV) and development of a humanized mouse model for MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of the isolated antibodies. The VelocImmune and VelociGene technologies are powerful platforms that can be used to rapidly respond to emerging epidemics

Serological based monitoring of a cohort of patients with chronic Chagas disease treated with benznidazole in a highly endemic area of northern Argentina

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease