* = co-first author, † = corresponding author
HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation
Venkat Giri Magupalli*†, Roberto Negro*†, Yuzi Tian*, Arthur V. Hauenstein*, Giuseppe Di Caprio, Wesley Skillern, Qiufang Deng, Pontus Orning, Hasan B. Alam, Zoltan Maliga, Humayun Sharif, Jun Jacob Hu, Charles L. Evavold, Jonathan C. Kagan, Florian I. Schmidt, Katherine A. Fitzgerald, Tom Kirchhausen, Yongqing Li†, Hao Wu†. Science (2020). PDF
To decode the site and the molecular machinery in inflammasome assembly and activation in macrophages, we aimed to visualize inflammasome assembly by cellular imaging complemented by pharmacological inhibition... Read More
FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation
Hu JJ*, Liu X*†, Xia S, Zhang Z, Zhang Y, Zhao J, Ruan J, Luo X, Lou X, Bai Y, Wang J, Hollingsworth LR, Magupalli VG, Zhao L, Luo HR, Kim J, Lieberman J†, Wu H†. FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation. Nature Immunol (2020). PDF
Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. ... Read More
Gasdermin D activity in inflammation and host defense
The mechanisms underlying the release of interleukin-1 (IL-1) family cytokines from phagocytes have been the subject of intense investigations for more than 30 years. The absence of an amino-terminal secretion signal from members of this family suggests a previously unknown mechanism of protein secretion that transfers cytosolic IL-1 directly across the plasma membrane into the extracellular space. The pore-forming protein gasdermin D (GSDMD) has emerged as the conduit for... Read More
Structural Mechanism for NEK7-licensed activation of NLRP3 inflammasome
The NLRP3 inflammasome can be activated by stimuli that include nigericin, uric acid crystals, amyloid-β fibrils and extracellular ATP. The mitotic kinase NEK7 licenses the assembly and activation of the NLRP3 inflammasome in interphase. Here we report a cryo-electron microscopy structure of inactive human NLRP3 in complex with NEK7, at a resolution of 3.8 Å. Read More
Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signalling
Receptor clustering on the cell membrane is critical in the signaling of many immunoreceptors, and this mechanism has previously been attributed to the extracellular and/or the intracellular interactions. Here, we report an unexpected finding that for death receptor 5 (DR5), a receptor in the tumor necrosis factor receptor superfamily, the transmembrane helix (TMH) alone in the receptor directly... Read More
Molecular mechanism for NLRP6 inflammasome assembly and activation
Inflammasomes are large protein complexes that trigger host defense in cells by activating inflammatory caspases for cytokine maturation and pyroptosis. NLRP6 is a sensor protein in the nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing (NLR) inflammasome family that has been shown to play multiple roles in regulating inflammation and host defenses. Despite the significance of the NLRP6 inflammasome, little is known about the molecular mechanism behind... Read More
Crystal structure of the WD40 domain dimer of LRRK2
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein with both a Ras of complex (ROC) domain and a kinase domain (KD) and, therefore, exhibits both GTPase and kinase activities. Human genetics studies have linked LRRK2 as a major genetic contributor to familial and sporadic Parkinson’s disease (PD), a neurodegenerative movement disorder that inflicts millions worldwide. Read More
Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1-receptor-associated kinase 4 (IRAK4)
Interleukin-1 receptor–associated kinase 4 (IRAK4) is a key player in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is ... Read More
Structures and gating mechanism of human TRPM2
Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain responsible for binding adenosine diphosphate (ADP)–ribose (ADPR), and both ADPR and calcium (Ca2+) are required for TRPM2 activation. Here we report cryo–electron microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca2+. NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM homology... Read More
Cryo-EM structures of ASC and NLRC4 CARD filaments reveal a unified mechanism of nucleation and activation of caspase-1
Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes...Read More
DNA melting initiates the RAG catalytic pathway
The mechanism for initiating DNA cleavage by DDE-family enzymes, including the RAG endonuclease, which initiates V(D)J recombination, is not well understood. Here we report six cryo-EM structures of zebrafish RAG in complex with one or two intact recombination signal sequences (RSSs), at up to 3.9-Å resolution. Unexpectedly, these structures reveal DNA melting at the heptamer of the RSSs, thus resulting in a corkscrew-like rotation of coding-flank DNA and the positioning of the scissile phosphate in the active site. Read More
The Structure of the Necrosome RIPK1-RIPK3 Core,
a Human Hetero-Amyloid Signaling Complex
The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) andVQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. Read More
Cryo-EM structure of the gasdermin A3 membrane pore
Gasdermins mediate inflammatory cell death after cleavage by caspases or other, unknown enzymes. The cleaved N-terminal fragments bind to acidic membrane lipids to form pores, but the mechanism of pore formation remains unresolved. Here we present the cryo-electron microscopy structures of the 27-fold and 28-fold single-ring pores formed by the N-terminal fragment of mouse GSDMA3 (GSDMA3-NT) at 3.8 and 4.2Å resolutions, and of a double-ring pore at 4.6Å resolution. In the 27-fold pore, a 108-stranded... Read More
Assembly mechanism of the CARMA1-BCL10-MALT1-TRAF6 signalosome
The CARMA1–BCL10–MALT1 (CBM) signalosome is a central mediator of T cell receptor and B cell receptor-induced NF-κB signaling that regulates multiple lymphocyte functions. While caspaserecruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1 (CARMA1) nucleates B cell lymphoma 10 (BCL10) filament formation through interactions between CARDs, mucosaassociated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a paracaspase with structural similarity to caspases, Read More
Crystal structure of human IRAK1
Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating innate immune responses against foreign pathogens and other types of dangers through their role in Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) mediated signaling pathways. Upon ligand binding, TLRs and IL-1Rs recruit adaptor proteins, such as myeloid differentiation primary response gene 88 (MyD88), to the membrane, which in turn recruit IRAKs via the death domains... Read More
AID Recognizes Structured DNA for Class Switch Recombination
Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic
hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are... Read More
CIDE domains form functionally important higher-order assemblies for DNA fragmentation
Cell death-inducing DFF45-like effector (CIDE) domains, initially identified in apoptotic nucleases, form a family with diverse functions ranging from cell death to lipid homeostasis. Here we show that the CIDE domains of Drosophila and human apoptotic nucleases Drep2, Drep4, and DFF40 all form head-to-tail helical filaments. Read More
Cryo-EM structure of the DNA-PK holoenzyme
DNA-dependent protein kinase (DNA-PK) is a large protein complex central to the nonhomologous end joining (NHEJ) DNA-repair pathway. It comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer of DNA-binding proteins Ku70 and Ku80. Here, we report the cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs at 4.4-Å resolution and the DNA-PK holoenzyme at 5.8-Å resolution. The DNA-PKcs structure contains three distinct segments: the N-terminal region with an arm and a bridge... Read More
Venkat Giri Magupalli*†, Roberto Negro*†, Yuzi Tian*, Arthur V. Hauenstein*, Giuseppe Di Caprio,Wesley Skillern, Qiufang Deng, Pontus Orning, Hasan B. Alam, Zoltan Maliga, Humayun Sharif, Jun Jacob Hu, Charles L. Evavold, Jonathan C. Kagan, Florian I. Schmidt, Katherine A. Fitzgerald,Tom Kirchhausen, Yongqing Li†, Hao Wu†(2020). HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation. Science. doi: 10.1126/science.aas8995.
Xia S*(2020). Biological mechanisms and therapeutic relevance of the gasdermin family. Mol Aspects Med. doi: 10.1016/j.mam.2020.100890.
Chen H*, Gu L*, Orellana EA, Wang Y, Guo J, Liu Q, Wang L, Shen Z, Wu H, Gregory RI, Xing Y, Shi Y† (2020). METTL4 is an snRNA m6Am methyltransferase that regulates RNA splicing. Cell Res doi: 10.1038/s41422-019-0270-4.
Xia S*, Hollingsworth LR 4th*, Wu H†(2019). Mechanism and Regulation of Gasdermin-Mediated Cell Death. Cold Spring Harb Perspect Biol. pii: a036400. doi: 10.1101/cshperspect.a036400.
Wu H*, Arnold E* (2019). Michael G. Rossmann (1930-2019) Nat Struct Mol Biol. (8):660-662.
Wang L, Wu H† (2019). Keeping the Death Protein in Check. Immunity. 51(1):1-2.
Scott DA*† , Hatcher JM, Liu H, Fu M, Du G, Fontán L, Us I, Casalena G, Qiao Q, Wu H, Melnick A, Gray NS (2019). Quinoline and thiazolopyridine allosteric inhibitors of MALT1. Bioorg Med Chem Lett. 29(14):1694-1698.
Arnold E†* , Wu H*, Johnson JE*. (2019). Michael G. Rossmann (1930-2019), pioneer in macromolecular and virus crystallography: scientist, mentor and friend. Acta Crystallogr D Struct Biol. 75(Pt 6):523-527.
Xia S*, Wang L*, Fu TM†, Wu H† (2019). Mechanism of TRPM2 channel gating revealed by cryo-EM. FEBS J. 286(17):3333-3339.
Xia S, Ruan J†, Wu H†(2019). Monitoring gasdermin pore formation in vitro. Methods Enzymol. 625:95-107
Shen C, Sharif H, Xia S, Wu H† (2019). Structural and mechanistic elucidation of inflammasome signaling by cryo-EM. Curr Opin Struct Biol. 58:18-25
Lee YR, Chen M, Lee JD*, Zhang J*, Lin SY*, Fu TM, Chen H, Ishikawa T, Chiang SY, Katon J, Zhang Y, Shulga YV, Bester AC, Fung J, Monteleone E, Wan L, Shen C, Hsu CH, Papa A, Clohessy JG, Teruya-Feldstein J, Jain S, Wu H, Matesic L, Chen RH, Wei W, Pandolfi PP† (2019). Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway. Science. 364(6441).
Wang L*, Ferrao R*, Li Q, Hatcher JM, Choi HG, Buhrlage SJ, Gray NS, Wu H† (2019). Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor-associated kinase 4 (IRAK4). The Journal of biological chemistry. 294(12):4511-4519.
Choi YJ, Kim S, Choi Y, Nielsen TB, Yan J, Lu A, Ruan J, Lee HR, Wu H, Spellberg B, Jung JU†(2019). SERPINB1-mediated checkpoint of inflammatory caspase activation. Nature immunology. 20(3):276-287.
Mompeán M*, Bozkurt G*, Wu H†.(2019). Mimicry by a viral RHIM. EMBO reports. 20(2).
Ru H, Zhang P, Wu H (2018). Structural gymnastics of RAG-mediated DNA cleavage in V(D)J recombination. Curr Opin Struct Biol. 53:178-186.
Orning P, Weng D1*, Starheim K*, Ratner D*, Best Z, Lee B, Brooks A, Xia S, Wu H, Kelliher MA, Berger SB, Gough PJ, Bertin J, Proulx MM, Goguen JD, Kayagaki N, Fitzgerald KA, Lien E†.(2018). Pathogen blockade of TAK1 triggers caspase-8–dependent cleavage of gasdermin D and cell death Science. 362(6418):1064-1069.
Luong P, Hedl M, Yan J, Zuo T, Fu TM, Jiang X, Thiagarajah JR, Hansen SH, Lesser CF, Wu H, Abraham C†, Lencer WI †(2018) INAVA-ARNO complexes bridge mucosal barrier function with inflammatory signaling. eLife. 2018; 7.
Fontán L*†, Qiao Q, Hatcher JM, Casalena G, Us I, Teater M, Durant M, Du G, Xia M, Bilchuk N, Chennamadhavuni S, Palladino G, Inghirami G, Philippar U, Wu H, Scott DA*, Gray NS, Melnick A*† (2018). Specific covalent inhibition of MALT1 paracaspase suppresses B cell lymphoma growth. The Journal of clinical investigation. 128(10):4397-4412.
Feltham R*, Jamal K*, Tenev T*, Liccardi G, Jaco I, Domingues CM, Morris O, John SW, Annibaldi A, Widya M, Kearney CJ, Clancy D, Elliott PR, Glatter T, Qiao Q, Thompson AJ, Nesvizhskii A, Schmidt A, Komander D, Wu H, Martin S, Meier P† (2018). Mind Bomb Refulates Cell Deth during TNF Signalling by Suppressing RIPK1’s Cytotoxic Potential. Cell Rep. 23(2):470-484.
Annibaldi A †, Wicky John S*, Vanden Berghe T*, Swatek KN*, Ruan J, Liccardi G, Bianchi K, Elliott PR, Choi SM, Van Coillie S, Bertin J, Wu H, Komander D, Vandenabeele P, Silke J, Meier P †(2018). Ubiquitin-Mediated Regulation of RIPK1 Kinase Activity Independent of IKK and MK2. Mol Cell. 69(4):566-580.
Xia S*, Fu TM*, Wu H† (2018). Inflammation NODs to Antagonists of RIP2-XIAP Interaction. Mol Cell. 69(4):535-536.
Fu TM*, Shen C*, Li Q, Zhang P, Wu H† (2018). Mechanism of ubiquitin transfer promoted by TRAF6. Proc Natl Acad Sci U S A. 115(8):1783-1788.
Zhang L, Wu H† (2018). Bad germs are trapped. Cell Res. 28(2):141-142
Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC† (2018). The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages. Immunity. 48(1):35-44
Hauenstein AV, Xu G, Kabaleeswaran V, Wu H† (2017) .Evidence for M1-Linked Polyubiquitin-Mediated Conformational Change in NEMO. J Mol Biol429(24):3793-3800
Kleino A, Ramia NF, Bozkurt G, Shen Y, Nailwal H, Huang J, Napetschnig J, Gangloff M, Chan FK*, Wu H*†, Li J†, Silverman N*†.(2017). Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signalin. Immunity 47(4):635-647
Wang H, Feng Z, Lu A, Jiang Y, Wu H, Xu B† (2017). Instant Hydrogelation Inspired by Inflammasomes. Angew Chem Int Ed Engl.56(26):7579-758
Wang L, Qiao Q, Wu H†(2017). Understanding CARD Tricks in Apoptosomes. Structure. 25(4)575-577
Yi L, Bozkurt G, Li Q, Lo S, Menon AK, Wu H† (2017). Disulfide Bond Formation and N-Glycosylation Modulate Protein-Protein Interactions in GPI-Transamidase (GPIT). Sci Rep. 8:45912
Lamour G, Nassar R*, Chan PH*, Bozkurt G, Li J, Bui JM, Yip CK, Mayor T, Li H, Wu H, Jorg A. Gsponer† (2017). Mapping the Broad Structural and Mechanical Properties of Amyloid Fibrils. Biophys. 112(4):584-594
Katja G. Weinacht*†, Louis-Marie Charbonnier*, Fayhan Alroqi, Ashley Plant, Qi Qiao, Hao Wu, Clement Ma, Troy R. Torgerson, Serogo D. Rosenzweig, Thomas A.Fleisher, Luigi D. Notarangelo, Imelda C. Hanson, Lisa R. Forbes, Talal A. Chatila (2017). Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation. Journal of Allergy and Clinical Immunology. 139(5):1629-1640