Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains

Joshi, Rohit; Pohl, Pavel; Strachotová, Dita; Heřman, Petr; Obšil, Tomáš; Obšilová, Veronika

doi:10.1016/j.bpj.2022.02.025

Cited by 7 publications

(5 citation statements)

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“…SAXS-based structural analysis combined with chemical cross-linking and fluorescence 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 altering interactions between its structured domains (Figures 3E,F) (Pohl et al, 2021). Furthermore, 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). Because 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 explaining the 14-3-3-mediated modulation of the ubiquitination of some Nedd4-2 substrates.…”

Section: E3 Ligase Nedd4-2 Regulationmentioning

confidence: 99%

Structural insights into the functional roles of 14-3-3 proteins

Obšilová

Obšil

2022

Front. Mol. Biosci.

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Signal transduction cascades efficiently transmit chemical and/or physical signals from the extracellular environment to intracellular compartments, thereby eliciting an appropriate cellular response. Most often, these signaling processes are mediated by specific protein-protein interactions involving hundreds of different receptors, enzymes, transcription factors, and signaling, adaptor and scaffolding proteins. Among them, 14-3-3 proteins are a family of highly conserved scaffolding molecules expressed in all eukaryotes, where they modulate the function of other proteins, primarily in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins participate in key cellular processes, such as cell-cycle control, apoptosis, signal transduction, energy metabolism, and protein trafficking. To date, several hundreds of 14-3-3 binding partners have been identified, including protein kinases, phosphatases, receptors and transcription factors, which have been implicated in the onset of various diseases. As such, 14-3-3 proteins are promising targets for pharmaceutical interventions. However, despite intensive research into their protein-protein interactions, our understanding of the molecular mechanisms whereby 14-3-3 proteins regulate the functions of their binding partners remains insufficient. This review article provides an overview of the current state of the art of the molecular mechanisms whereby 14-3-3 proteins regulate their binding partners, focusing on recent structural studies of 14-3-3 protein complexes.

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Section: E3 Ligase Nedd4-2 Regulationmentioning

confidence: 99%

Structural insights into the functional roles of 14-3-3 proteins

Obšilová

Obšil

2022

Front. Mol. Biosci.

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“…In isolation, each of the techniques FP, FRET, ITC, and SPR act as a unique lens through which to study 14-3-3 PPIs, but these methods alone do not provide a full picture. Other approaches not discussed here (e.g., microscale thermophoresis (Centorrino et al, 2022;Kozeleková et al, 2022), differential scanning fluorimetry (Cossar et al, 2021;Waløen et al, 2021;Joshi et al, 2022), analytical ultracentrifugation (Horvath et al, 2021;Leysen et al, 2021;Neves et al, 2021;Pohl et al, 2021;Srdanović et al, 2022), single angle x-ray scattering (Kast and Dominguez, 2019;Leysen et al, 2021) also contribute new understanding and support hypotheses. NMR (Kuusk et al, 2019;Neves et al, 2021) and native mass spectrometry (Bellamy-Carter et al, 2021) are also important techniques that shows great promise for studying 14-3-3 PPIs.…”

Section: Discussionmentioning

confidence: 93%

“…Synthetic peptides that mimic the binding motifs of the phosphorylated partner protein to 14-3-3 have a sufficiently small size relative to 14-3-3 and can be modified to incorporate a fluorescent label. Their use in place of the natural protein partner makes FP a particularly versatile technique for studying In this review of recent 14-3-3 literature, we have identified 15 studies that used FP as part of the analytical workflow (Soini et al, 2021a;Cossar et al, 2021;Falcicchio et al, 2021;Gogl et al, 2021;Guillory et al, 2021;Horvath et al, 2021;Ruks et al, 2021;Sijbesma et al, 2021;Centorrino et al, 2022;Hazegh Nikroo et al, 2022;Joshi et al, 2022;Srdanovic et al, 2022;Srdanović et al, 2022;Stevers et al, 2022). Because it is such a well-established technique, here we focus on select examples that demonstrate how FP can be used to address three key challenges in current 14-3-3 research: (i) Quantifying the cooperative nature of 14-3-3 molecular glues; (ii) Understanding 14-3-3 isoform specificity and partner protein binding mechanisms; (iii) Studying the kinetic characteristics of 14-3-3 PPIs.…”

Section: Fluorescence Polarisationmentioning

confidence: 99%

“…It is possible that FP kinetic data collected on much shorter timescales could shed more new light on the dynamics of 14-3-3 PPIs and molecular glues. In relation to this, experiments that measure fluorescence anisotropy decay on the nanosecond timescale can also provide fascinating insight into the environment of the fluorophore under different conditions ( Horvath et al, 2021 ; Joshi et al, 2022 ).…”

Section: Fluorescence Polarisationmentioning

confidence: 99%

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Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions

Thurairajah

Hudson

Doveston

2022

Front. Mol. Biosci.

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14-3-3 proteins are a family of regulatory hubs that function through a vast network of protein-protein interactions. Their dysfunction or dysregulation is implicated in a wide range of diseases, and thus they are attractive drug targets, especially for molecular glues that promote protein-protein interactions for therapeutic intervention. However, an incomplete understanding of the molecular mechanisms that underpin 14-3-3 function hampers progress in drug design and development. Biophysical methodologies are an essential element of the 14-3-3 analytical toolbox, but in many cases have not been fully exploited. Here, we present a contemporary review of the predominant biophysical techniques used to study 14-3-3 protein-protein interactions, with a focus on examples that address key questions and challenges in the 14-3-3 field.

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“…Specifically, 14-3-3 binds Nedd4-2 at pSer and pThr sites, blocking its binding to the epithelial sodium channel ENaC, thus suppressing Nedd4-2-dependent ubiquitination and degradation of ENaC. Moreover, recent biophysical/crystallography analysis of the Nedd4-2:14-3-3 interaction revealed that 14-3-3 proteins prevent intramolecular interactions between Nedd4-2 WW3/WW4 domains and the HECT domain, potentially affecting catalytic activity in addition to regulating binding to ENaC 34 , 35 .…”

Section: Discussionmentioning

confidence: 99%

Primate-specific isoform of Nedd4-1 regulates substrate binding via Ser/Thr phosphorylation and 14-3-3 binding

Kefalas,

Rotin

2023

Sci Rep

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Nedd4 (Nedd4-1) is an E3 ubiquitin ligase involved in crucial biological processes such as growth factor receptor signaling. While canonical Nedd4-1 comprises a C2-WW(4)-HECT domain architecture, alternative splicing produces non-canonical isoforms that are poorly characterized. Here we characterized Nedd4-1(NE), a primate-specific isoform of Nedd4-1 that contains a large N-terminal Extension (NE) that replaces most of the C2 domain. We show that Nedd4-1(NE) mRNA is ubiquitously expressed in human tissues and cell lines. Moreover, we found that Nedd4-1(NE) is more active than the canonical Nedd4-1 isoform, likely due to the absence of a C2 domain-mediated autoinhibitory mechanism. Additionally, we identified two Thr/Ser phosphoresidues in the NE region that act as binding sites for 14-3-3 proteins, and show that phosphorylation on these sites reduces substrate binding. Finally, we show that the NE region can act as a binding site for the RPB2 subunit of RNA polymerase II, a unique substrate of Nedd4-1(NE) but not the canonical Nedd4-1. Taken together, our results demonstrate that alternative splicing of the ubiquitin ligase Nedd4-1 can produce isoforms that differ in their catalytic activity, binding partners and substrates, and mechanisms of regulation.

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Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains

Cited by 7 publications

References 62 publications

Structural insights into the functional roles of 14-3-3 proteins

Structural insights into the functional roles of 14-3-3 proteins

Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions

Primate-specific isoform of Nedd4-1 regulates substrate binding via Ser/Thr phosphorylation and 14-3-3 binding

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