RESUMO
An important consideration in drug discovery is the prioritization of tractable protein targets that are not only amenable to binding small molecules, but also alter disease biology in response to small molecule binding. Covalent fragment-based drug discovery has emerged as a powerful approach to aid in the identification of such protein targets. The application of irreversible binding mechanisms enables the identification of fragment hits for challenging-to-target proteins, allows proteome-wide screening in a cellular context, and makes it possible to determine functional effects with modestly potent ligands without the requirement for extensive compound optimization. Here, we provide an overview of recent approaches to covalent fragment-based screening and discuss how these have been applied to establish the tractability of unexplored binding sites on protein targets.
Assuntos
Descoberta de Drogas , Proteínas , Bibliotecas de Moléculas Pequenas , Descoberta de Drogas/métodos , Humanos , Ligantes , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas/química , Proteínas/metabolismo , Ligação Proteica , Sítios de LigaçãoRESUMO
The tripartite motif (TRIM) protein family members have been implicated in a multitude of physiologies and pathologies in different tissues. With diverse functions in cellular processes including regulation of signaling pathways, protein degradation, and transcriptional control, the impact of TRIM dysregulation can be multifaceted and complex. Here, we focus on the cellular and molecular roles of TRIMs identified in the brain in the context of a selection of pathologies including cancer and neurodegeneration. By examining each disease in parallel with described roles in brain development, we aim to highlight fundamental common mechanisms employed by TRIM proteins and identify opportunities for therapeutic intervention.
RESUMO
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.
Assuntos
Cisteína , Fluoretos , Cisteína/química , Ligantes , Aminoácidos , EnxofreRESUMO
Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.
Assuntos
Fluoretos , Proteoma , Proteoma/metabolismo , Fluoretos/química , Enxofre/química , Aminoácidos/química , BiologiaRESUMO
Tripartite motif (TRIM) proteins constitute a large family of RING-type E3 ligases that share a conserved domain architecture. TRIM2 and TRIM3 are paralogous class VII TRIM members that are expressed mainly in the brain and regulate different neuronal functions. Here we present a detailed structure-function analysis of TRIM2 and TRIM3, which despite high sequence identity, exhibit markedly different self-association and activity profiles. We show that the isolated RING domain of human TRIM3 is monomeric and inactive, and that this lack of activity is due to a few placental mammal-specific amino acid changes adjacent to the core RING domain that prevent self-association but not E2 recognition. We demonstrate that the activity of human TRIM3 RING can be restored by substitution with the relevant region of human TRIM2 or by hetero-dimerization with human TRIM2, establishing that subtle amino acid changes can profoundly affect TRIM protein activity. Finally, we show that TRIM2 and TRIM3 interact in a cellular context via their filamin and coiled-coil domains, respectively.
Assuntos
Aminoácidos , Proteínas de Transporte , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Transporte/genética , Ubiquitina-Proteína Ligases/genética , Proteínas com Motivo Tripartido/genéticaRESUMO
The ubiquitin system contains a wealth of potential drug targets for many diseases and conditions, including neurodegenerative, immune, metabolic and developmental diseases, as well as multiple cancers. Despite years of research, relatively few clinical inhibitors or specific chemical probes for proteins within the ubiquitin system exist, with many interesting target proteins yet to be explored. Fragment-based drug discovery (FBDD) offers efficient and broad coverage of chemical space with small libraries, using covalent and non-covalent approaches. Coupled with advances in structural biology and proteomics, FBDD now provides a thorough screening platform for inhibitor discovery within the ubiquitin system. In this mini review, we summarise the current scope of FBDD and how it has been applied to ubiquitin-activating (E1), ubiquitin-conjugating (E2), ubiquitin ligase (E3) and deubiquitinating (DUB) enzymes. We also discuss the newest frontiers of FBDD and how they could be applied to enable inhibitor and novel chemical probe discovery and provide functional insight into the ubiquitin system.
RESUMO
Multidomain proteins composed of individual domains connected by flexible linkers pose a challenge for structural studies due to their intrinsic conformational dynamics. Integrated modelling approaches provide a means to characterise protein flexibility by combining experimental measurements with molecular simulations. In this study, we characterise the conformational dynamics of the catalytic RBR domain of the E3 ubiquitin ligase HOIP, which regulates immune and inflammatory signalling pathways. Specifically, we combine small angle X-ray scattering experiments and molecular dynamics simulations to generate weighted conformational ensembles of the HOIP RBR domain using two different approaches based on maximum parsimony and maximum entropy principles. Both methods provide optimised ensembles that are instrumental in rationalising observed differences between SAXS-based solution studies and available crystal structures and highlight the importance of interdomain linker flexibility.
Assuntos
Proteínas , Ubiquitina-Proteína Ligases , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas/química , Espalhamento a Baixo Ângulo , Ubiquitina-Proteína Ligases/metabolismo , Difração de Raios XRESUMO
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.
RESUMO
TRIM proteins form a protein family that is characterized by a conserved tripartite motif domain comprising a RING domain, one or two B-box domains and a coiled-coil region. Members of this large protein family are important regulators of numerous cellular functions including innate immune responses, transcriptional regulation and apoptosis. Key to their cellular role is their E3 ligase activity which is conferred by the RING domain. Self-association is an important characteristic of TRIM protein activity and is mediated by homodimerization via the coiled-coil region, and in some cases higher order association via additional domains of the tripartite motif. In many of the TRIM family proteins studied thus far, RING dimerization is an important prerequisite for E3 ligase enzymatic activity though the propensity of RING domains to dimerize differs significantly between different TRIMs and can be influenced by other regions of the protein.
Assuntos
Apoptose , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Animais , Catálise , Domínio Catalítico , Dimerização , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Transdução de Sinais , Proteínas com Motivo Tripartido/genéticaRESUMO
The Parkin-coregulated gene (PACRG), which encodes a protein of unknown function, shares a bidirectional promoter with Parkin (PRKN), which encodes an E3 ubiquitin ligase. Because PRKN is important in mitochondrial quality control and protection against stress, we tested whether PACRG also affected these pathways in various cultured human cell lines and in mouse embryonic fibroblasts. PACRG did not play a role in mitophagy but did play a role in tumor necrosis factor (TNF) signaling. Similarly to Parkin, PACRG promoted nuclear factor κB (NF-κB) activation in response to TNF. TNF-induced nuclear translocation of the NF-κB subunit p65 and NF-κB-dependent transcription were decreased in PACRG-deficient cells. Defective canonical NF-κB activation in the absence of PACRG was accompanied by a decrease in linear ubiquitylation mediated by the linear ubiquitin chain assembly complex (LUBAC), which is composed of the two E3 ubiquitin ligases HOIP and HOIL-1L and the adaptor protein SHARPIN. Upon TNF stimulation, PACRG was recruited to the activated TNF receptor complex and interacted with LUBAC components. PACRG functionally replaced SHARPIN in this context. In SHARPIN-deficient cells, PACRG prevented LUBAC destabilization, restored HOIP-dependent linear ubiquitylation, and protected cells from TNF-induced apoptosis. This function of PACRG in positively regulating TNF signaling may help to explain the association of PACRG and PRKN polymorphisms with an increased susceptibility to intracellular pathogens.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Chaperonas Moleculares/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Mitofagia/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Protein ubiquitination plays a key role in the regulation of cellular processes, and misregulation of the ubiquitin system is linked to many diseases. So far, development of tool compounds that target enzymes of the ubiquitin system has been slow and only a few specific inhibitors are available. Here, we report the selection of single-domain antibodies (single-dAbs) based on a human scaffold that recognize the catalytic domain of HOIP, a subunit of the multi-component E3 LUBAC and member of the RBR family of E3 ligases. Some of these dAbs affect ligase activity and provide mechanistic insight into the ubiquitin transfer mechanism of different E2-conjugating enzymes. Furthermore, we show that the co-crystal structure of a HOIP RBR/dAb complex serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.
Assuntos
Inibidores Enzimáticos/farmacologia , Chaperonas Moleculares/química , Anticorpos de Domínio Único/química , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Cristalização , Inibidores Enzimáticos/química , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Proteínas com Motivo Tripartido/química , Proteínas com Motivo Tripartido/metabolismo , Sequência de Aminoácidos , Catálise , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , Proteína 28 com Motivo Tripartido/química , Proteína 28 com Motivo Tripartido/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
Assuntos
Inibidores Enzimáticos/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Ligantes , Células MCF-7 , Modelos Moleculares , Estrutura Secundária de Proteína , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/químicaRESUMO
The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.
Assuntos
Escherichia coli/química , Metaloproteases/química , Infecções por Salmonella/genética , Salmonella enterica/química , Sequência de Aminoácidos/genética , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/patogenicidade , Células HeLa , Humanos , Imunidade Celular , Metaloproteases/genética , NF-kappa B/química , Conformação Proteica , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Transdução de Sinais , Relação Estrutura-Atividade , Fator de Transcrição RelA/química , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/genética , Zinco/químicaRESUMO
RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1.
Assuntos
Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/imunologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Células Cultivadas , Proteína DEAD-box 58/imunologia , Células HEK293 , Humanos , Interferons/biossíntese , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Viral/imunologia , Receptores Imunológicos , Transdução de Sinais , Fatores de Transcrição/química , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
RBR ligases are an enigmatic class of E3 ubiquitin ligases that combine properties of RING and HECT-type E3s and undergo multilevel regulation through autoinhibition, post-translational modifications, multimerization and interaction with binding partners. Here, we summarize recent progress in RBR structures and function, which has uncovered commonalities in the mechanisms by which different family members transfer ubiquitin through a multistep process. However, these studies have also highlighted clear differences in the activity of different family members, suggesting that each RBR ligase has evolved specific properties to fit the biological process it regulates.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ativação Enzimática , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Ubiquitina-Proteína Ligases/químicaRESUMO
The Salmonella-secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors.
Assuntos
Glicosiltransferases/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Glicosiltransferases/química , Humanos , Modelos Moleculares , Conformação Proteica , Salmonella typhimurium/química , Alinhamento de SequênciaRESUMO
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.
Assuntos
Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Calorimetria , Catálise , Humanos , Complexos Multiproteicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Termodinâmica , Ubiquitina/química , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2â¼ubiquitin (E2â¼Ub) via their RINGs but canonical RING E3s promote closed E2â¼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2â¼Ub to favor Ub transfer to the E3 active site. This different RING/E2â¼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2â¼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.