The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

Zhang, Qi; Wang, Zhongduo; Hou, Feng; Harding, Rachel; Huang, Xinyi; Dong, Aiping; Walker, John R.; Tong, Yufeng

doi:10.1016/j.bbagen.2016.10.019

Cited by 20 publications

(23 citation statements)

References 88 publications

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“…Avr2 also shows structural homology to Tumor necrosis factor Receptor Associated Factor (TRAF) domains although, as for ToxA, pairwise sequence identities are typically < 5%. Despite this, Avr2 overlays on the TRAF‐domains of Speckle‐type POZ (SPOP) protein (PDB: 4O1V; Li et al ., ), with TRAF6 (PDB: 1LB5 (Ye et al ., )) and with SIAH1 (PDB: 4X3G; Zhang et al ., ), with r.m.s.d.s of 3.5 Å over 97 residues, 3.2 Å over 95 residues and 2.8 Å over 86 residues, respectively (Figs S2 and S3). TRAFs are cytoplasmic adaptor proteins involved in cell signaling cascades in mammals, including roles in PRR‐ and NLR‐mediated signaling (Xie, ).…”

Section: Resultsmentioning

confidence: 98%

Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition

Cao

Hughes

et al. 2017

New Phytologist

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Summary Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I‐2 carrying tomato (Solanum lycopersicum). Fol strains that evade I‐2 recognition carry point mutations in Avr2 (e.g. Avr2R45H), but retain full virulence.Here we investigate the virulence function of Avr2 and determine its crystal structure. Transgenic tomato and Arabidopsis expressing either wild‐type ΔspAvr2 (deleted signal‐peptide) or the ΔspAvr2 R45H variant become hypersusceptible to fungal, and even bacterial infections, suggesting that Avr2 targets a conserved defense mechanism. Indeed, Avr2 transgenic plants are attenuated in immunity‐related readouts, including flg22‐induced growth inhibition, ROS production and callose deposition.The crystal structure of Avr2 reveals that the protein shares intriguing structural similarity to ToxA from the wheat pathogen Pyrenophora tritici‐repentis and to TRAF proteins. The I‐2 resistance‐breaking Avr2V41M, Avr2R45H and Avr2R46P variants cluster on a surface‐presented loop. Structure‐guided mutagenesis enabled uncoupling of virulence from I‐2‐mediated recognition.We conclude that I‐2‐mediated recognition is not based on monitoring Avr2 virulence activity, which includes suppression of immune responses via an evolutionarily conserved effector target, but by recognition of a distinct epitope.

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Section: Resultsmentioning

confidence: 98%

Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition

Cao

Hughes

et al. 2017

New Phytologist

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“…Secondly, X-ray structural and biochemical analysis of mouse SIAH1a and human SIAH1 utilized the N-terminal truncated forms of SIAH1, missing ~80-90 amino acids to delete the entire catalytic RING-domain (missing nearly one third of SIAH1). Such a large N-terminal-truncated mutant SIAH1 might have unknown and unpredictable structural consequences on correct protein folding and thus may yield a possibly incorrect SIAH1 3D structure inappropriate and thus premature for conducting large-scale small molecule inhibitor screens [55][56][57][58][59]. Thirdly, several published anti-SIAH inhibition strategies were designed using such truncated SIAH proteins, and several newly identified SIAH small molecule inhibitors were chemically screened and identified by targeting these RING-domaindeleted SIAH mutant proteins [60][61][62][63][64].…”

Section: Discussionmentioning

confidence: 99%

The phylogenetic functional conservation ofDrosophilaSeven-In-Absentia (SINA) E3 ligase and its two human paralogs, SIAH1 and SIAH2, inDrosophilaeye development

Sciver

Cao

Tang

2020

Preprint

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Key WordsSINA E3 ligase; ubiquitin-mediated proteolysis; RAS signaling transduction, Drosophila eye development, photoreceptor cell fate determination, and peripheral neuronal system development Abbreviations DmSINA PD -Drosophila SINA PD , DPP -decapentaplegic, EGFR -epidermal growth factor receptor, EMS -ethyl methanesulfonate, ERK -extracellular signal-regulated kinases, FLPrecombinase that recognizes FRT site, GAL4 -yeast DNA-binding transcriptional activator, GFP -green fluorescent protein, GMR -Glass multiple reporter, hSIAH1 PD -human SIAH1 PD , hSIAH2 PD -human SIAH2 PD , ORF -open reading frame, PNS -peripheral nervous system, PD -proteolysis deficient, RING -really interesting new gene, SEM -scanning electron microscopy, SEV -sevenless, SIAH -human homologs of SINA, SINA -seven in absentia, SINA/SIAH inhibitor -SINA PD /SIAH PD , UAS -yeast upstream activator sequence, WT -wild type Co-1 st authors Abstract Seven-IN-Absentia (SINA) is the most downstream signaling gatekeeper identified thus far in the RAS/EGFR pathway that controls photoreceptor cell fate determination in Drosophila.Underscoring the central importance of SINA is its phylogenetic conservation in metazoans, with over 83% amino acid identities shared between Drosophila SINA and human SINA homologs (SIAHs). SIAH is a major tumor vulnerability in multidrug-resistant and incurable cancer. SIAH inhibition is an effective strategy to shut down the tumor-driving K-RAS/EGFR/HER2 pathway activation that promotes malignant tumor growth and metastatic dissemination. To further delineate the SINA function in the RAS/EGFR pathway, a genetic modifier screen was conducted, and 28 new sina mutant alleles were isolated via ethyl methanesulfonate (EMS) and X-ray mutagenesis. Among them, 26 of the new sina mutants are embryonic, larval, or pupal lethal, and stronger than the five published sina mutants (sina 1 , sina 2 , sina 3 , sina 4 , and sina 5 ) which are early adult lethal. By sequencing the SINA-coding region of sina ES10 , sina ES26 , sina ES79 , and sina ES473 homozygous mutant animals, we identified three invariable amino acid residues in SINA's RING-domain whose single point mutation ablates SINA function. To demonstrate the functional conservation of this medically important family of RING domain E3 ligases in Drosophila, we established a collection of transgenic lines, expressing either wild type (WT) or proteolysis-deficient (PD) SINA/SIAH inhibitors of Drosophila SINA WT/PD and human SIAH1 WT/PD /2 WT/PD under tissue-specific GAL4-drivers in Drosophila eye, wing, and salary gland.Our results showed that Drosophila SINA and human SIAH1/2 are functionally conserved. Our bioengineered SINA PD /SIAH PD inhibitors are effective in blocking the RAS-dependent neuronal cell fate determination in Drosophila.

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“…Drosophila SINA and two of its human homologs, SIAH1 and SIAH2, belong to a highly evolutionarily conserved family of RING domain E3 ligases [ 20 , 21 , 23 ]. SINA and SIAH function as homo- and hetero-dimers [ 24 – 27 ].…”

Section: Introductionmentioning

confidence: 99%

“…SINA and SIAH function as homo- and hetero-dimers [ 24 – 27 ]. The family of the SINA/SIAH1/SIAH2 E3 ligases has four highly conserved and distinct functional domains: (1) the Really Interesting New Gene (RING) domain is the catalytic active site for the E3 ligase activity, (2) the SIAH-type zinc finger (SZF) domain contains a dual zinc-finger motif, (3) the substrate-binding site (SBS) recognizes substrates, and (4) the dimerization (DIMER) domain allows for homo- and heterodimer formation between SINA/SIAH proteins [ 23 – 31 ]. The substrate-binding domain (SBD), composed of SZF, SBS and DIMER domains, is responsible for substrate recognition, targeting, interaction, and degradation [ 23 , 25 – 31 ].…”

Section: Introductionmentioning

confidence: 99%

“…The family of the SINA/SIAH1/SIAH2 E3 ligases has four highly conserved and distinct functional domains: (1) the Really Interesting New Gene (RING) domain is the catalytic active site for the E3 ligase activity, (2) the SIAH-type zinc finger (SZF) domain contains a dual zinc-finger motif, (3) the substrate-binding site (SBS) recognizes substrates, and (4) the dimerization (DIMER) domain allows for homo- and heterodimer formation between SINA/SIAH proteins [ 23 – 31 ]. The substrate-binding domain (SBD), composed of SZF, SBS and DIMER domains, is responsible for substrate recognition, targeting, interaction, and degradation [ 23 , 25 – 31 ]. As an E3 ligase, SINA/SIAH is known to interact, modify, and target a multitude of substrates/partners/regulators to orchestrate ubiquitin-mediated proteolysis and regulate protein stability, protein complex assembly, protein subcellular localization, and other cellular functions in normal development and human diseases [ 10 , 32 , 33 ].…”

Section: Introductionmentioning

confidence: 99%

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Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa

2017

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BackgroundThe RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling “gatekeeper” whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway.ResultsVertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines.ConclusionThrough phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future effort to design logical, potent, and durable anti-SIAH-based anticancer strategies against oncogenic K-RAS-driven metastatic human cancers. Thus, this method of evolutionary study should be of interest in cancer biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-1024-x) contains supplementary material, which is available to authorized users.

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The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

Abstract: The crystallographic models provide structural insights into the substrate binding of the SIAH family E3 ubiquitin ligases that are critically involved in regulating cancer-related pathways. Our results suggest caution should be taken when using menadione as a specific SIAH2 inhibitor.

Cited by 20 publications

References 88 publications

Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition

Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition

The phylogenetic functional conservation ofDrosophilaSeven-In-Absentia (SINA) E3 ligase and its two human paralogs, SIAH1 and SIAH2, inDrosophilaeye development

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa

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