Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Vadas, Oscar; Dbouk, Hashem A.; Shymanets, Aliaksei; Perišić, Olga; Burke, John E.; Saab, Widian F. Abi; Khalil, Bassem D.; Harteneck, Christian; Bresnick, Anne R.; Nürnberg, Bernd; Backer, Jonathan M.; Williams, Roger L.

doi:10.1073/pnas.1304801110

Cited by 121 publications

(203 citation statements)

References 55 publications

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“…Rab5 was previously implicated as an effector for class IA PI3Kb 44 , although the nature of this interaction was not characterized. At a molecular level, the PI3K catalytic subunit p110g is traditionally recruited by binding directly to Ras, along with a class IB regulatory subunit, and the Gbg subunit of an activated GPCR complex 45 . We now confirm that Rab8a and PI3Kg bind directly to each other, using homology modelling and mutagenesis of critical residues.…”

Section: Discussionmentioning

confidence: 99%

Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling

et al. 2014

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Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro-and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kg) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kg function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kg do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.

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Section: Discussionmentioning

confidence: 99%

Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling

et al. 2014

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“…Hydrogen-deuterium exchange was essentially as described in ref. 30. For details of these and other biophysical methods, see SI Methods.…”

Section: Methodsmentioning

confidence: 99%

“…Exchange is stopped at various times by reducing the pH, the protein digested with pepsin, and the deuterium content of peptides determined by mass spectrometry (29). Because the rate of exchange depends strongly on tertiary structure and solvent accessibility, such studies can give information about binding or protein conformation, which are revealed by changes in the kinetics of deuterium incorporation into specific peptides (30,31). Fig.…”

Section: Significancementioning

confidence: 99%

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Mund

Lewis

Maslen

2014

Proc. Natl. Acad. Sci. U.S.A.

125

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The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/ Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.inhibitor | ubiquitin | HECT ligase | phage display U biquitin ligases are involved in almost every important function of cells, including the removal of misfolded proteins, the regulation of signaling pathways, DNA repair, the cell cycle, and apoptosis (1-4). This broad range of functions is mediated by a large number of E3 ubiquitin ligases, which recognize a similarly large range of substrates. Ubiquitination begins with the ATP-dependent formation of a ubiquitin (Ub)-E2 enzyme thioester intermediate by the E1 enzyme. The E3 enzymes then transfer ubiquitin from this intermediate to a lysine residue on the substrate (3). There are two main classes of E3: the RING domain ligases and their relatives, of which there are several hundred in humans, and the HECT domain ligases, which number around 28 (5-7). Because of their diversity and numerous functions and the fact that some ligases are frequently overexpressed in cancer cells, ubiquitin ligases have attracted much attention as possible targets for therapeutic intervention (2,5,6,8).Despite this interest, it has proven challenging to make small molecule inhibitors of ubiquitin ligases. The action of these enzymes involves primarily protein-protein interactions among them, the Ub-E2 conjugate, and the substrate. For the RING enzymes, this is simultaneous, and the Ub moiety is transferred directly from E2 to substrate; for the HECT ligases, the interaction is sequential, with a thioester-linked Ub-E3 intermediate forming transiently. Such protein-protein interactions are considered difficult to block with small molecules. Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrat...

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“…HDX‐MS has also been used as a powerful tool to map out protein‐protein interfaces both in solution and on membrane surfaces 15, 16, 17, 18, 19, 20. Herein we describe an experimental approach to use HDX‐MS to define dynamic regions within a protein complex, in this case the type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11, and use this information to generate optimized constructs for X‐ray crystallography.…”

Section: Introductionmentioning

confidence: 99%

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

et al. 2016

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The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.

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Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Cited by 121 publications

References 55 publications

Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling

Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

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