Regulation of NOXO1 Activity through Reversible Interactions with p22phox and NOXA1

Reactive oxygen species (ROS) have been known for a long time to play important roles in host defense against microbial infections. In addition, it has become apparent that they also perform regulatory roles in signal transduction and cell proliferation. The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase homologue that is most abundantly expressed in colon epithelial cells, requires the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX activating protein 1), as well as the flavocytochrome component p22phox for maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is tightly repressed in the resting state, NOX1 produces superoxide constitutively at low levels. These levels can be further increased in a stimulus-dependent manner, yet the molecular details regulating this activity are not fully understood. Here we present the first quantitative characterization of the interactions made between the cytosolic regulators NOXO1 and NOXA1 and membrane-bound p22phox. Using isothermal titration calorimetry we show that the isolated tandem SH3 domains of NOXO1 bind to p22phox with high affinity, most likely adopting a superSH3 domain conformation. In contrast, complex formation is severely inhibited in the presence of the C-terminal tail of NOXO1, suggesting that this region competes for binding to p22phox and thereby contributes to the regulation of superoxide production. Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems. Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions

Structural basis for ubiquitylation by HOIL-1

The linear ubiquitin chain assembly complex synthesises linear Ub chains which constitute a binding and activation platform for components of the TNF signalling pathway. One of the components of LUBAC is the ubiquitin ligase HOIL-1 which has been shown to generate oxyester linkages on several proteins and on linear polysaccharides. We show that HOIL-1 activity requires linear tetra-Ub binding which enables HOIL-1 to mono-ubiquitylate linear Ub chains and polysaccharides. Furthermore, we describe the crystal structure of a C-terminal tandem domain construct of HOIL-1 comprising the IBR and RING2 domains. Interestingly, the structure reveals a unique bi-nuclear Zn-cluster which substitutes the second zinc finger of the canonical RING2 fold. We identify the C-terminal histidine of this bi-nuclear Zn-cluster as the catalytic base required for the ubiquitylation activity of HOIL-1. Our study suggests that the unique zinc-coordinating architecture of RING2 provides a binding platform for ubiquitylation targets

SPATA2-Mediated Binding of CYLD to HOIP Enables CYLD Recruitment to Signaling Complexes

SummaryRecruitment of the deubiquitinase CYLD to signaling complexes is mediated by its interaction with HOIP, the catalytically active component of the linear ubiquitin chain assembly complex (LUBAC). Here, we identify SPATA2 as a constitutive direct binding partner of HOIP that bridges the interaction between CYLD and HOIP. SPATA2 recruitment to TNFR1- and NOD2-signaling complexes is dependent on HOIP, and loss of SPATA2 abolishes CYLD recruitment. Deficiency in SPATA2 exerts limited effects on gene activation pathways but diminishes necroptosis induced by tumor necrosis factor (TNF), resembling loss of CYLD. In summary, we describe SPATA2 as a previously unrecognized factor in LUBAC-dependent signaling pathways that serves as an adaptor between HOIP and CYLD, thereby enabling recruitment of CYLD to signaling complexes

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

National Institute of General Medical Sciences R01 GM0880555T32 GM007270, Francis Crick Institute FCI01, Cancer Research UK, Medical Research Council U117565398, Wellcome Trus

Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases

Abstract RING-between-RING (RBR) ubiquitin ligases work with multiple E2 enzymes and function through an E3-ubiquitin thioester intermediate. The RBR module comprises three domains, RING1, IBR and RING2 that collaborate to transfer ubiquitin from the E2~Ub conjugate, recognised by RING1, onto a catalytic cysteine in RING2 and finally onto the substrate in a multi-step reaction. Recent studies have shown that RING1 domains bind E2~Ub conjugates in an open conformation to supress ubiquitin transfer onto lysine residues and promote formation of the E3 thioester intermediate. However, how the nature of the E2 influences the ubiquitin transfer process is currently unclear. We report here a detailed characterization of the RBR/E2-conjugate recognition step that indicates that this mechanism depends on the nature of the E2 enzyme and differs between UbcH5 and UbcH7. In the case of UbcH5~Ub an interaction with ubiquitin is necessary to stabilize the transfer complex while recognition of UbcH7~Ub is driven primarily by E2-RING1 contacts. Furthermore our analysis suggests that RBRs, in isolation and in complex with ubiquitin-loaded E2s, are dynamic species and that their intrinsic flexibility might be a key aspect of their catalytic mechanism

The Structural Basis for 14-3-3:Phosphopeptide Binding Specificity

AbstractThe 14-3-3 family of proteins mediates signal transduction by binding to phosphoserine-containing proteins. Using phosphoserine-oriented peptide libraries to probe all mammalian and yeast 14-3-3s, we identified two different binding motifs, RSXpSXP and RXY/FXpSXP, present in nearly all known 14-3-3 binding proteins. The crystal structure of 14-3-3ζ complexed with the phosphoserine motif in polyoma middle-T was determined to 2.6 Å resolution. The bound peptide is in an extended conformation, with a tight turn created by the pS +2 Pro in a cis conformation. Sites of peptide–protein interaction in the complex rationalize the peptide library results. Finally, we show that the 14-3-3 dimer binds tightly to single molecules containing tandem repeats of phosphoserine motifs, implicating bidentate association as a signaling mechanism with molecules such as Raf, BAD, and Cbl

Profiling sulfur(VI) fluorides as reactive functionalities for chemical biology tools and expansion of the ligandable proteome

Here, we report a comprehensive profiling study of sulfur(VI) fluorides (SVI-F) in the context of multiple chemical biology applications and illustrate that these motifs present an exciting opportunity to develop tools for a wide scope of protein targets. SVI-Fs are reactive functionalities that offer utility for targeting almost any protein, as they can modify multiple residues including Lys, Tyr, His and Ser. A panel of SVI-F functionalities were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acids. Subsequently, the reactivity of SVI-Fs with CAII and kinase proteins was investigated, in the context of both fragment binders and optimized probes. Finally, the performance of the SVI-F panel in chemoproteomic workflows was analyzed. The studies provided an in-depth understanding of the hydrolytic stability, protein reactivity and chemoproteomic utility of SVI-F functionalities that are suitable for direct incorporation into chemical tools. Such insights offer a valuable guide for the prospective design of SVI-F-containing ligands for various chemical biology workflows and demonstrate the wide range of proteins that SVI-Fs can capture, thus highlighting the opportunity for SVI-Fs to expand the liganded proteome

Interaction between NOD2 and CARD9 involves the NOD2 NACHT and the linker region between the NOD2 CARDs and NACHT domain.

NOD2 activation by muramyl dipeptide causes a proinflammatory immune response in which the adaptor protein CARD9 works synergistically with NOD2 to drive p38 and c-Jun N-terminal kinase (JNK) signalling. To date the nature of the interaction between NOD2 and CARD9 remains undetermined. Here we show that this interaction is not mediated by the CARDs of NOD2 and CARD9 as previously suggested, but that NOD2 possesses two interaction sites for CARD9; one in the CARD-NACHT linker and one in the NACHT itself.This work was funded by a Wellcome Trust Career Development Fellowship (WT085090MA) to TPM and a Medical Research Council grant (U117565398) to KR. RP and JPB were supported by BBSRC Doctoral Training Grants.This is the final published version of the article, which can also be found on the publisher's website at: http://www.sciencedirect.com/science/article/pii/S0014579314004979

Efficient ligand discovery using sulfur(VI) fluoride reactive fragments

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors