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author:("Li, pinghui")
1.  Single Nucleotide Polymorphisms of Human STING Can Affect Innate Immune Response to Cyclic Dinucleotides 
PLoS ONE  2013;8(10):e77846.
The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3’5’-3’5’ cyclic GMP-AMP (3’3’ cGAMP) produced by Vibrio cholerae and metazoan second messenger 2’5’-3’5’ Cyclic GMP-AMP (2’3’ cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3’3’ cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.
doi:10.1371/journal.pone.0077846
PMCID: PMC3804601  PMID: 24204993
2.  Crystallographic characterization of mouse AIM2 HIN-200 domain bound to a 15 bp and an 18 bp double-stranded DNA 
AIM2 is an innate immune sensor of microbial double-stranded DNA. The HIN-200 domain of mouse AIM2 bound to a 15 bp and an 18 bp dsDNA were crystallized and diffract to about 4.0 Å.
AIM2 (absent in melanoma 2) is an innate immune receptor for cytosolic double-stranded DNA (dsDNA). The engagement of dsDNA by AIM2 activates the AIM2 inflammasome, resulting in the cleavage of pro-interleukin-1β by caspase-1. The DNA-binding HIN-200 domain of mouse AIM2 bound to a 15 bp dsDNA and to an 18 bp dsDNA was purified and crystallized. The AIM2 HIN-200 domain in complex with the 15 bp DNA crystallized in the cubic space group I23 or I213, with unit-cell parameter a = 235.60 Å. The complex of the AIM2 HIN-200 domain and the 18 bp DNA crystallized in a similar unit cell. Diffraction data for the two complexes were collected to about 4.0 Å resolution. Mutagenesis and DNA-binding studies suggest that mouse AIM2 uses a similar surface to human AIM2 to recognize DNA.
doi:10.1107/S174430911203103X
PMCID: PMC3433203  PMID: 22949200
mouse AIM2; HIN-200 domain; DNA binding
3.  Structure of STING bound to c-di-GMP Reveals the Mechanism of Cyclic Dinucleotide Recognition by the Immune System 
STING, stimulator of interferon genes, is an innate immune sensor of cyclic dinucleotides that regulates the induction of type I interferons. STING C-terminal domain forms a V-shaped dimer and binds a c-di-GMP molecule at the dimer interface through direct and solvent-mediated hydrogen bonds. The guanine bases of c-di-GMP stack against the phenolic rings of a conserved tyrosine residue. Mutations at the c-di-GMP binding surface reduce nucleotide binding and affect signaling.
doi:10.1038/nsmb.2331
PMCID: PMC3392545  PMID: 22728658
4.  Peli1 negatively regulates T-cell activation and prevents autoimmunity 
Nature immunology  2011;12(10):1002-1009.
T-cell activation is subject to tight regulation to avoid inappropriate responses against self-antigens. Here we show that genetic deficiency in an ubiquitin ligase, Peli1, causes hyper activation of T cells and renders T cells refractory to suppression by T regulatory cells and transforming growth factor (TGF)-β. As a result, Peli1 knockout mice spontaneously develop autoimmunity, characterized by multiorgan inflammation and autoantibody production. Peli1 deficiency results in accumulation of nuclear c-Rel, a member of the NF-κB family of transcription factors with pivotal roles in T-cell activation. Peli1 negatively regulates c-Rel by mediating its K48 ubiquitination. These results identify Peli1 as a critical factor in the maintenance of peripheral T-cell tolerance and reveal a novel mechanism of c-Rel regulation.
doi:10.1038/ni.2090
PMCID: PMC3178748  PMID: 21874024
5.  New monoclonal anti-mouse DC-SIGN antibodies reactive with acetone-fixed cells 
Journal of immunological methods  2010;360(1-2):66-75.
Mouse DC-SIGN CD209a is a type II transmembrane protein, one of a family of C-type lectin genes syntenic and homologous to human DC-SIGN. Current anti-mouse DC-SIGN monoclonal antibodies (MAbs) are unable to react with DC-SIGN in acetone fixed cells, limiting the chance to visualize DC-SIGN in tissue sections. We first produced rabbit polyclonal PAb-DSCYT14 against a 14-aa peptide in the cytosolic domain of mouse DC-SIGN, and it specifically detected DC-SIGN and not the related lectins, SIGN-R1 and SIGN-R3 expressed in transfected CHO cells. MAbs were generated by immunizing rats and DC-SIGN knockout mice with the extracellular region of mouse DC-SIGN.. Five rat IgG2a or IgM MAbs, named BMD10, 11, 24, 25, and 30, were selected and each MAb specifically detected DC-SIGN by FACS and Western blots, although BMD25 was cross-reactive to SIGN-R1. Two mouse IgG2c MAbs MMD2 and MMD3 interestingly bound mouse DC-SIGN but at 10 fold higher levels than the rat MAbs. When the binding epitopes of the new BMD and two other commercial rat anti-DC-SIGN MAbs, 5H10 and LWC06, were examined by competition assays, the epitopes of BMD11, 24, and LWC06 were identical or closely overlapping while BMD10, 30, and 5H10 were shown to bind different epitopes. MMD2 and MMD3 epitopes were on a 3rd noncompeting region of mouse DC-SIGN. DC-SIGN expressed on the cell surface was sensitive to collagenase treatment, as monitored by polyclonal and MAb. These new reagents should be helpful to probe the biology of DC-SIGN in vivo.
doi:10.1016/j.jim.2010.06.006
PMCID: PMC2924951  PMID: 20558171
Monoclonal Antibody; Polyclonal Antibody; DC-SIGN; CD209a; Dendritic Cells
6.  The Structural Basis of 5′ Triphosphate Double-stranded RNA Recognition by RIG-I C-terminal Domain 
Structure (London, England : 1993)  2010;18(8):1032-1043.
SUMMARY
RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5′ triphosphate (5′ ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here we show that RIG-I CTD binds 5′ ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5′ ppp dsRNA. Crystal structures of RIG-I CTD bound to 5′ ppp dsRNA with GC- and AU- rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5′ triphosphate through extensive electrostatic interactions. Mutagenesis and RNA binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA. Mutations of key residues at the RNA binding surface affected RIG-I signaling in cells.
doi:10.1016/j.str.2010.05.007
PMCID: PMC2919622  PMID: 20637642
7.  Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5′ triphosphate 
Nucleic Acids Research  2010;39(4):1565-1575.
RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5′ triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5′-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5′ ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5′ ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.
doi:10.1093/nar/gkq974
PMCID: PMC3045611  PMID: 20961956

Results 1-7 (7)