{"id":30140,"date":"2024-08-22T15:16:54","date_gmt":"2024-08-22T13:16:54","guid":{"rendered":"https:\/\/fgu.antstudio.dev\/?post_type=vyzkumny-projekt&p=30140"},"modified":"2024-09-02T08:24:20","modified_gmt":"2024-09-02T06:24:20","slug":"structural-basis-of-the-14-3-3-protein-dependent-activation-of-ubiquitin-ligase-nedd4-2","status":"publish","type":"vyzkumny-projekt","link":"https:\/\/fgu.cas.cz\/en\/research-project\/structural-basis-of-the-14-3-3-protein-dependent-activation-of-ubiquitin-ligase-nedd4-2\/","title":{"rendered":"Structural basis of the 14-3-3 protein-dependent activation of ubiquitin ligase Nedd4-2"},"content":{"rendered":"
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    Our results show the the structural glimpse on the 14-3-3-dependent regulation of human ubiquitin ligase Nedd4-2. Our data could be important for understanding of the regulation process of Nedd4-2 as well as of the role of 14-3-3 proteins in the regulation of other ubiquitin ligases.<\/b><\/p>\n<\/div>\n

    Our research team has been studying the 14-3-3 proteins which are highly conserved regulatory molecules found in all eukaryotes. First they have been isolated from the bovine brain and their unusual name \u201c14-3-3\u201d, originates from their elution and migration pattern on two-dimensional DEAE-cellulose chromatography and starch gel electrophoresis. 14-3-3 proteins have the ability of binding the functionally different signal proteins, including kinases, fosfatases and transmembrane receptors by changing their function. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many processes, including cell cycle regulation, metabolism control, apoptosis,and control of gene transcription.<\/li>\n<\/ol>\n<\/div>\n

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    Unraveling the mechanism of inhibition of human ubiquitin ligase Nedd4-2 by 14-3-3 protein<\/h2>\n

    The neural precursor cell expressed developmentally downregulated\u00a04 (Nedd4-2) is a member of the HECT E3 ubiquitin\u00a0ligase family.\u00a0As such, this enzyme targets proteins\u00a0for ubiquitination in mammalian programmed cell death.\u00a0Nedd4-2 has modular multidomain\u00a0architecture, consisting of an N-terminal C2\u00a0domain, four WW domains, which contain two conserved\u00a0tryptophan residues and a proline residue, and a C-terminal\u00a0catalytic HECT domain.\u00a0Nedd4-2 is also regulated by phosphorylation\u00a0on\u00a0three sites (Ser342, Thr367 and Ser448) recognized by 14-3-3 protein.\u00a0SAXS-based\u00a0structural analysis combined with chemical cross-linking and\u00a0fluorescence spectroscopy provided the first glimpse into the 14-3-3-mediated inhibition of Nedd4-2, showing that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by\u00a0altering interactions between its structured domains (Pohl et. al 2021)<\/a>.<\/em>\u00a0Furthermore, the formation of the complex causes both steric hindrance of the WW3 and WW4 domains and a conformational change of the catalytic HECT domain (Joshi et al., 2022<\/a>)<\/em>.\u00a0Because WW domains presumably mediate Nedd4-2 binding to its substrates, such occlusions combined with conformational changes in the catalytic domain likely affect substrate ubiquitination, thus\u00a0explaining the 14-3-3-mediated modulation of the ubiquitination of some Nedd4-2 substrates.<\/p>\n

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    Structural analysis of the complex between Nedd4-2 and 14-3-3 protein by integrative structural biology approach. a)+b) Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements with the estimation of the apparent dissociation constant of the complex. c) SAXS-based structural modeling with the crystal structures of the 14-3-3 binding sites of Nedd4-2 (Pohl et. al (2021) Communications Biology<\/a>).<\/em>\u00a0<\/em><\/p>\n

    14-3-3 proteins and ubiquitin ligase Nedd4-2<\/strong><\/p>\n

    In 2021, using a series of biochemical and biophysical methods, we elucidated that the key sites for interaction with the 14-3-3 protein are the pSer342 and pSer448 sites, we elucidated the principle of this interaction by solving the crystal structure of 7NMZ, and we found that binding of the 14-3-3 protein blocks WW3 in the central channel of the 14-3-3 protein (Pohl et. al 2021<\/a><\/em>).<\/p>\n

    In 2022, using fluorescence spectroscopy methods, we elucidated that the 14-3-3 protein reduces both mobility and solvent accessibility of the WW2, WW3 and WW4 domains of Nedd4-2. Conversely, binding of the 14-3-3 protien has the opposite effect on the active site of the HECT domain, which is more exposed to solvent and more mobile in the 14-3-3:Nedd4-2 complex\u00a0(Joshi et al., 2022<\/a>).<\/em><\/p>\n

    Current projects involve:<\/strong><\/p>\n

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    1. The role of calcium ions in Nedd4-2 regulation and structure changes induced by calcium binding<\/li>\n
    2. CryoEM of the 14-3-3:Nedd4-2 complex<\/li>\n
    3. Searching for new E3 ubiquitin ligases regulated by 14-3-3 proteins<\/li>\n<\/ol>\n

       <\/p>\n","protected":false},"excerpt":{"rendered":"

      Our results show the the structural glimpse on the 14-3-3-dependent regulation of human ubiquitin ligase Nedd4-2. Our data could be important for understanding of the regulation process of Nedd4-2 as well as of the role of 14-3-3 proteins in the regulation of other ubiquitin ligases. Our research team has been studying the 14-3-3 proteins which […]<\/p>\n","protected":false},"author":1,"template":"","meta":{"_acf_changed":false,"inline_featured_image":false,"footnotes":""},"oddeleni":[181],"poskytovatel":[],"stav-projektu":[209],"class_list":["post-30140","vyzkumny-projekt","type-vyzkumny-projekt","status-publish","hentry","oddeleni-structural-biology-of-signaling-proteins","stav-projektu-current-projects"],"acf":[],"_links":{"self":[{"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/vyzkumny-projekt\/30140","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/vyzkumny-projekt"}],"about":[{"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/types\/vyzkumny-projekt"}],"author":[{"embeddable":true,"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/users\/1"}],"version-history":[{"count":0,"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/vyzkumny-projekt\/30140\/revisions"}],"wp:attachment":[{"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/media?parent=30140"}],"wp:term":[{"taxonomy":"oddeleni","embeddable":true,"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/oddeleni?post=30140"},{"taxonomy":"poskytovatel","embeddable":true,"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/poskytovatel?post=30140"},{"taxonomy":"stav-projektu","embeddable":true,"href":"https:\/\/fgu.cas.cz\/en\/wp-json\/wp\/v2\/stav-projektu?post=30140"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}