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  • 1
    Publication Date: 2018-05-02
    Description: Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4 + T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.
    Print ISSN: 1535-9476
    Electronic ISSN: 1535-9484
    Topics: Biology , Medicine
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  • 2
    Publication Date: 2011-04-02
    Description: Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKgamma or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1beta was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Gerlach, Bjorn -- Cordier, Stefanie M -- Schmukle, Anna C -- Emmerich, Christoph H -- Rieser, Eva -- Haas, Tobias L -- Webb, Andrew I -- Rickard, James A -- Anderton, Holly -- Wong, Wendy W-L -- Nachbur, Ueli -- Gangoda, Lahiru -- Warnken, Uwe -- Purcell, Anthony W -- Silke, John -- Walczak, Henning -- 10950/Cancer Research UK/United Kingdom -- England -- Nature. 2011 Mar 31;471(7340):591-6. doi: 10.1038/nature09816.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Tumour Immunology Unit, Department of Medicine, Imperial College London, W12 0NN London, UK.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; CD40 Ligand/metabolism ; Carrier Proteins/chemistry/metabolism ; Cell Line ; Humans ; I-kappa B Kinase/metabolism ; Immunity/*immunology ; Inflammation/*metabolism/pathology/prevention & control ; Interleukin-1beta/metabolism ; Mice ; Multiprotein Complexes/chemistry/metabolism ; NF-kappa B/metabolism ; Nerve Tissue Proteins/chemistry/genetics/metabolism ; Phenotype ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; Receptors, Tumor Necrosis Factor/deficiency/genetics/metabolism ; *Signal Transduction ; Skin/cytology/immunology/metabolism/pathology ; Tumor Necrosis Factor-alpha/deficiency/genetics ; Ubiquitin/chemistry/metabolism ; Ubiquitin-Protein Ligase Complexes/chemistry/metabolism ; Ubiquitin-Protein Ligases/chemistry/metabolism ; *Ubiquitination
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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