Huanhuan Liang

Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway

Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends.

Crystal structure of ISG54 reveals a novel RNA binding structure and potential functional mechanisms

Interferon-stimulated gene 56 (ISG56) family members play important roles in blocking viral replication and regulating cellular functions, however, their underlying molecular mechanisms are largely unclear. Here, we present the crystal structure of ISG54, an ISG56 family protein with a novel RNA-binding structure. The structure shows that ISG54 monomers have 9 tetratricopeptide repeat-like motifs and associate to form domain-swapped dimers. The C-terminal part folds into a super-helical structure and has an extensively positively-charged nucleotide-binding channel on its inner surface. EMSA results show that ISG54 binds specifically to some RNAs, such as adenylate uridylate (AU)-rich RNAs, with or without 5′ triphosphorylation. Mutagenesis and functional studies show that this RNA-binding ability is important to its antiviral activity. Our results suggest a new mechanism underlying the antiviral activity of this interferon-inducible gene 56 family member.

Single Amino Acid Substitutions Confer the Antiviral Activity of the TRAF3 Adaptor Protein onto TRAF5

Two single amino acid changes enable the adaptor protein TRAF5 to promote antiviral responses. TRAF5 Becomes Antiviral The adaptor protein TRAF3 promotes antiviral responses by binding to Cardif, a component of a viral response pathway that triggers the production of interferon. Zhang et al. determined the crystal structure of the TRAF domain of TRAF3 bound to the TRAF3-binding peptide of Cardif, as well as that of the TRAF domain of TRAF5, which does not bind to Cardif and cannot substitute for TRAF3 in antiviral responses. The authors identified two amino acids in TRAF3 that participated in binding to Cardif. Substitution of the corresponding residues in TRAF5 with those in TRAF3 produced TRAF5 mutants that bound to Cardif and partially substituted for TRAF3 in antiviral responses. TRAF3 interacts with many proteins that participate in multiple signaling pathways that promote inflammation, and these results raise the possibility of selectively interfering with a subset of its interactions without interfering with its ability to mediate antiviral responses. The TRAF [tumor necrosis factor receptor–associated factor] family of cytoplasmic adaptor proteins link cell-surface receptors to intracellular signaling pathways that regulate innate and adaptive immune responses. In response to activation of RIG-I (retinoic acid–inducible gene I), a component of a pattern recognition receptor that detects viruses, TRAF3 binds to the adaptor protein Cardif [caspase activation and recruitment domain (CARD) adaptor–inducing interferon-β (IFN-β)], leading to induction of type I IFNs. We report the crystal structures of the TRAF domain of TRAF5 and that of TRAF3 bound to a peptide from the TRAF-interacting motif of Cardif. By comparing these structures, we identified two residues located near the Cardif binding pocket in TRAF3 (Tyr440 and Phe473) that potentially contributed to Cardif recognition. In vitro and cellular experiments showed that forms of TRAF5 with mutation of the corresponding residues to those of TRAF3 had TRAF3-like antiviral activity. Our results provide a structural basis for the critical role of TRAF3 in activating RIG-I–mediated IFN production.

A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks

Abstract Chromatin assembly factor 1 (CAF-1) is a histone H3–H4 chaperone that deposits newly synthesized histone (H3–H4)2 tetramers during replication-coupled nucleosome assembly. However, how CAF-1 functions in this process is not yet well understood. Here, we report the crystal structure of C terminus of Cac1 (Cac1C), a subunit of yeast CAF-1, and the function of this domain in stabilizing CAF-1 at replication forks. We show that Cac1C forms a winged helix domain (WHD) and binds DNA in a sequence-independent manner. Mutations in Cac1C that abolish DNA binding result in defects in transcriptional silencing and increased sensitivity to DNA damaging agents, and these defects are exacerbated when combined with Cac1 mutations deficient in PCNA binding. Similar phenotypes are observed for corresponding mutations in mouse CAF-1. These results reveal a mechanism conserved in eukaryotic cells whereby the ability of CAF-1 to bind DNA is important for its association with the DNA replication forks and subsequent nucleosome assembly.

Crystal structure of the ubiquitin-like domain of human TBK1

TANK-binding kinase 1 (TBK1) is an important enzyme in the regulation of cellular antiviral effects. TBK1 regulates the activity of the interferon regulatory factors IRF3 and IRF7, thereby playing a key role in type I interferon (IFN) signaling pathways. The structure of TBK1 consists of an N-terminal kinase domain, a middle ubiquitin-like domain (ULD), and a C-terminal elongated helical domain. It has been reported that the ULD of TBK1 regulates kinase activity, playing an important role in signaling and mediating interactions with other molecules in the IFN pathway. In this study, we present the crystal structure of the ULD of human TBK1 and identify several conserved residues by multiple sequence alignment. We found that a hydrophobic patch in TBK1, containing residues Leu316, Ile353, and Val382, corresponding to the “Ile44 hydrophobic patch” observed in ubiquitin, was conserved in TBK1, IκB kinase epsilon (IKKɛ/IKKi), IκB kinase alpha (IKKα), and IκB kinase beta (IKKβ). In comparison with the structure of the IKKβ ULD domain of Xenopus laevis, we speculate that the Ile44 hydrophobic patch of TBK1 is present in an intramolecular binding surface between ULD and the C-terminal elongated helices. The varying surface charge distributions in the ULD domains of IKK and IKK-related kinases may be relevant to their specificity for specific partners.

Screen Anti-influenza Lead Compounds That Target the PAC Subunit of H5N1 Viral RNA Polymerase

The avian influenza (H5N1) viral RNA polymerase protein PAC was used as a target to screen nine chlorogenic acid derivatives for their polymerase inhibitor activity. Among them, seven compounds were PAC ligands, and four inhibited influenza RNA polymerase activity. These results aid in the design of anti-influenza agents based on caffeoylquinic acid.

NMI and IFP35 serve as proinflammatory DAMPs during cellular infection and injury

Damage-associated molecular patterns (DAMP) trigger innate immune response and exacerbate inflammation to combat infection and cellular damage. Identifying DAMPs and revealing their functions are thus of crucial importance. Here we report that two molecules, N-myc and STAT interactor (NMI) and interferon-induced protein 35 (IFP35) act as DAMPs and are released by activated macrophages during lipopolysaccharide-induced septic shock or acetaminophen-induced liver injury. We show that extracellular NMI and IFP35 activate macrophages to release proinflammatory cytokines by activating nuclear factor-κB through the Toll-like receptor 4 pathway. In addition, the serum levels of NMI are increased in patients who succumbed to severe inflammation. NMI deficiency reduces inflammatory responses and mortality in mouse models of sepsis and liver injury. We therefore propose that extracellular NMI and IFP35 exacerbate inflammation as DAMPs, making them potential therapeutic targets for clinical intervention.Damage-associated molecular patterns (DAMP) are important mediators of innate immunity. Here the authors show that N-myc and STAT interactor (NMI) and interferon-induced protein 35 (IFP35) act as DAMPs to promote inflammation by activating macrophages via the Toll-like receptor 4 and NF-κB pathways.

The structure and polymerase-recognition mechanism of the crucial adaptor protein AND-1 in the human replisome

DNA replication in eukaryotic cells is performed by a multiprotein complex called the replisome, which consists of helicases, polymerases, and adaptor molecules. Human acidic nucleoplasmic DNA-binding protein 1 (AND-1), also known as WD repeat and high mobility group (HMG)-box DNA-binding protein 1 (WDHD1), is an adaptor molecule crucial for DNA replication. Although structural information for the AND-1 yeast ortholog is available, the mechanistic details for how human AND-1 protein anchors the lagging-strand DNA polymerase α (pol α) to the DNA helicase complex (Cdc45-MCM2–7-GINS, CMG) await elucidation. Here, we report the structures of the N-terminal WD40 and SepB domains of human AND-1, as well as a biochemical analysis of the C-terminal HMG domain. We show that AND-1 exists as a homotrimer mediated by the SepB domain. Mutant study results suggested that a positively charged groove within the SepB domain provides binding sites for pol α. Different from its ortholog protein in budding yeast, human AND-1 is recruited to the CMG complex, mediated by unknown participants other than Go Ichi Ni San. In addition, we show that AND-1 binds to DNA in vitro, using its C-terminal HMG domain. In conclusion, our findings provide important insights into the mechanistic details of human AND-1 function, advancing our understanding of replisome formation during eukaryotic replication.

Gasdermins pore cell membrane to pyroptosis

Cell death programs including apoptosis, pyroptosis, au-tophagy, oncosis and necroptosis were defined dependent on distinct biochemical mechanisms and genetic pathways. Pyroptosis is an inflammatory form of programmed cell death characterized by the activation of inflammatory caspases, cell swelling, pore formation on plasma membrane and rapid cellular rupture which cause the release of cell contents followed by robust inflammatory responses (Cookson and Brennan, 2001; Jorgensen and Miao, 2015). Two pathways, canonical and non-canonical, have been uncovered for the activation of the inflammatory caspases. A type of multimeric protein complexes known as inflam-masome play crucial role in canonical pathway. The in-flammasome comprise sensor proteins (NLRs, ALRs, or pyrin), adaptor molecules usually using ASC, and zymogen pro-caspase-1 (Sharma and Kanneganti, 2016). In response to invading pathogens and endogenous danger signals, the inflammasome is assembled and provides a platform for the activation of caspase-1, which is responsible for the matura-tion and secretion of pro-inflammatory cytokines, interleu-kin (IL)-1β and IL-18 (Keller et al., 2008). In the non-canonical pathway, caspases-11 in mice and its ortholog caspase-4/5 in human are directly activated by cytosolic lipopolysaccharides (LPS) (Shi et al., 2014). It was reported that pores with diameter of 1.1–2.4 nm are produced on plasma membrane of salmonella-induced pyroptotic cells, dependent on the activation of caspase-1 (Fink and Cookson, 2006). The pores were proposed to dissipate cellular ionic gradients, resulting in increased water influx and eventually membrane rupture. However, how these plasma membrane pores are formed and regulated by inflam-matory caspases had been unclear for a decade. To understand the mechanism of pore formation during pyroptosis process, immunologists studied the substrates and downstream signaling pathways of activated inflammatory caspases. Some significant advances have been made recently. A crucial substrate of inflammatory caspases, gasdermin D (GSDMD), was independently identified by several Their results showed that GSDMD is cleaved by the activated caspases-1/4/5/11 between its N-terminal and C-terminal domains. The N terminal domain of GSDMD (GSDMD-N) possesses intrinsic pyroptosis-inducing activity and determines the secretion of IL-1β and IL-18. Most recently, the mechanism for the cytotoxicity of GSDMD-N in pyroptosis was further dissected (Aglietti et al. The researchers found that human 293T cells developed swelling bubbles and became ruptured soon after GSDMD-N was produced in the cells. Therefore, they hypothesized that GSDMD-N killed cells by disrupting the integrity of cell membrane, similar to mixed lineage kinase domain-like protein (MLKL) which oligomerized and associated to the inner leaflet of the plasma membrane …

pH-dependent conformational changes of a Thogoto virus matrix protein reveal mechanisms of viral assembly and uncoating.

Orthomyxoviruses are a family of ssRNA virus, including influenza virus, infectious salmon anaemia virus and Thogoto virus. The matrix proteins of orthomyxoviruses play crucial roles in some essential processes of the viral life cycle. However, the mechanisms of the matrix proteins involved in these processes remain incompletely understood. Currently, only the structure and function of the matrix protein from influenza virus have been studied. Here, we present the crystal structures of the N-terminal domain of matrix protein from Thogoto virus at pH 7.0 and 4.5. By analysing the structures, we identified the conformational changes of monomers and dimers in different pH conditions, mainly caused by two flexible loops, L3 and L5. These structural deviations would reflect the basis of viral capsid assembly or disassembly.