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Refinement of the endogenous epitope tagging technology allows the identification of a novel NRAS binding partner in melanoma
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Michal Alon Rafi Emmanuel Nouar Qutob Anna Bakhman Victoria Peshti Alexandra Brodezki David Bassan Mickey Kosloff Yardena Samuels 《Pigment cell & melanoma research》2018,31(5):641-648
The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor‐intensive. To this end, we present a polyadenylation signal‐trap construct for N’‐tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c‐CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants. 相似文献
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Qutob N Balloux F Raj T Liu H Marion de Procé S Trowsdale J Manica A 《Immunogenetics》2012,64(3):165-175
The extreme polymorphism of MHC class I has been argued to be driven by balancing selection from pathogens, with the prediction that populations exposed
to a wider variety of diseases should have higher diversity. We assembled a global database of allotype frequencies for MHC class I genes and investigated possible drivers of genetic diversity, measured in different ways. We first looked for a decline
in diversity with distance from Africa (a consequence of drift during human expansions) and then investigated the link with
pathogen richness once the effect of drift had been corrected for. Using heterozygosity, we recovered a clear decline in diversity
from Africa and confirmed the positive relationship between genetic diversity and pathogen richness for all three classical
MHC class I genes. However, when we considered a sequence-based measure of genetic diversity, the correlation with geographic
distance from Africa vanished for HLA-C, and the correlations with pathogen richness for the three MHC class I genes were much weaker. HLA-C is known to consist of two functional classes of allotypes (classified with respect
to the 80th residue), which interact with different KIR receptors. While this separation provided some improvement in the
fit between genetic diversity and distance from Africa for one class, much clearer and consistent patterns were recovered
when we used the 90th residue to separate HLA-C allotypes into two new classes. This suggests that this residue, which is
also involved in the binding of KIR, might have had an important evolutionary role that has been overlooked. 相似文献
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Roqiya Nouar François Devred Gilles Breuzard Vincent Peyrot 《Biology of the cell / under the auspices of the European Cell Biology Organization》2013,105(4):149-161
Microtubules (MTs) are involved in many crucial processes such as cell morphogenesis, mitosis and motility. These dynamic structures resulting from the complex assembly of tubulin are tightly regulated by stabilising MT‐associated proteins (MAPs) such as tau and destabilising proteins, notably stathmin. Because of their key role, these MAPs and their interactions have been extensively studied using biochemical and biophysical approaches, particularly in vitro. Nevertheless, numerous questions remain unanswered and the mechanisms of interaction between MT and these proteins are still unclear in cells. Techniques coupling cell imaging and fluorescence methods, such as Förster resonance energy transfer and fluorescence recovery after photobleaching, are excellent tools to study these interactions in situ. After describing these methods, we will present emblematic data from the literature and unpublished experimental results from our laboratory concerning the interactions between MTs, tau and stathmin in cells. 相似文献
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