Shan Li

Glutamine Deamidation and Dysfunction of Ubiquitin/NEDD8 Induced by a Bacterial Effector Family

Gee-Up, NEDD8 Diverse bacterial pathogens, such as Burkholderia pseudomallei and enteropathogenic Escherichia coli (EPEC), inject effector proteins into eukaryotic host cells to subvert host cell biology. Cui et al. (p. 1215; published online 5 August) show that certain bacterial effector proteins promoted the glutamine deamidation of ubiquitin and the ubiquitin-related protein, NEDD8. The modification of ubiquitin inhibited the formation of polyubiquitin chains while, unexpectedly, the same chemical alteration on NEDD8 interfered with the cell cycle, offering insights not only into the cytopathic effects of bacterial infection but also into mechanisms that may operate during viral infection and cancer. Pathogenic bacterial proteins interfere with eukaryotic ubiquitination pathways to induce cytopathic effects. A family of bacterial effectors including Cif homolog from Burkholderia pseudomallei (CHBP) and Cif from Enteropathogenic Escherichia coli (EPEC) adopt a functionally important papain-like hydrolytic fold. We show here that CHBP was a potent inhibitor of the eukaryotic ubiquitination pathway. CHBP acted as a deamidase that specifically and efficiently deamidated Gln40 in ubiquitin and ubiquitin-like protein NEDD8 both in vitro and during Burkholderia infection. Deamidated ubiquitin was impaired in supporting ubiquitin-chain synthesis. Cif selectively deamidated NEDD8, which abolished the activity of neddylated Cullin-RING ubiquitin ligases (CRLs). Ubiquitination and ubiquitin-dependent degradation of multiple CRL substrates were impaired by Cif in EPEC-infected cells. Mutations of substrate-contacting residues in Cif abolished or attenuated EPEC-induced cytopathic phenotypes of cell cycle arrest and actin stress fiber formation.

Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains

The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors. Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv

The small RNA transcriptomes of bread wheat and its emerging model Brachypodium distachyon were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNAs) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of expressed sequence tags, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5′ rapid amplification of complementary DNA ends experiments demonstrated their capability to cleave predicted target genes including three disease-resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in B. distachyon.

A Legionella type IV effector activates the NF-κB pathway by phosphorylating the IκB family of inhibitors

NF-κB is critical in innate immune defense responses against invading microbial pathogens. Legionella pneumophila infection of lung macrophages causes Legionnaires disease with pneumonia symptoms. A set of NF-κB-controlled genes involved in inflammation and anti-apoptosis are up-regulated in macrophages upon L. pneumophila infection in a Legionella Dot/Icm type IV secretion system-dependent manner. Among ≈100 Dot/Icm substrates screened, we identified LegK1 as the sole Legionella protein that harbors a highly potent NF-κB-stimulating activity. LegK1 does not affect MAPK and IFN pathways. Activation of the NF-κB pathway by LegK1 requires its eukaryotic-like Ser/Thr kinase activity and is independent of upstream components in the NF-κB pathway, including TRAFs, NIK, MEKK3, and TAK1. Cell-free reconstitution revealed that LegK1 stimulated NF-κB activation in the absence of IKKα and IKKβ, and LegK1 efficiently phosphorylated IκBα on Ser-32 and Ser-36 both in vitro and in cells. LegK1 seems to mimic the host IKK as LegK1 also directly phosphorylated other IκB family of inhibitors including p100 in the noncanonical NF-κB pathway. Phosphorylation of p100 by LegK1 led to its maturation into p52. Thus, LegK1 is a bacterial effector that directly activates the host NF-κB signaling and likely plays important roles in modulating macrophage defense or inflammatory responses during L. pneumophila infection.

Chemical probing reveals insights into the signaling mechanism of inflammasome activation.

Caspase-1-mediated IL-1β production is generally controlled by two pathways. Toll-like receptors (TLRs) recognize pathogen-derived products and induce NF-κB-dependent pro-IL-1β transcription; NOD-like receptors (NLRs) assemble caspase-1-activating inflammasome complexes that sense bacterial products/danger signals. Through a targeted chemical screen, we identify bromoxone, a marine natural product, as a specific and potent inhibitor of the caspase-1 pathway. Bromoxone is effective over diverse inflammatory stimuli including TLR ligands plus ATP/nigericin, cytosolic DNA, flagellin and Bacillus anthracis lethal toxin. Bromoxone also efficiently suppresses caspase-1 activation triggered by several types of bacterial infection. Bromoxone acts upstream or at the level of the inflammasome in a transcription-independent manner. Bromoxone also inhibits pro-IL-1β expression by targeting components upstream of IKK in the TLR-NF-κB pathway. The unique dual activities of bromoxone are shared by the known TAK1 inhibitor that specifically blocks Nalp3 inflammasome activation. Hinted from the mechanistic and pharmacological properties of bromoxone, we further discover that several known NF-κB inhibitors that act upstream of IKK, but not those targeting IKK or IKK downstream, are potent blockers of different NLRs-mediated caspase-1 activation. Our study uncovers a possible non-transcriptional molecular link between the NLR (Nalp3)-mediated inflammasome pathway and TLR-NF-κB signaling, and suggests a potential strategy to develop new anti-inflammatory drugs.

Synthesis of and Specific Antibody Generation for Glycopeptides with Arginine N-GlcNAcylation†

As a unique and unappreciated protein posttranslational modification, arginine N-glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N-glycosylation, we report the first general strategy for a rapid and cost-effective synthesis of glycopeptides carrying single or multiple arginine N-GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N-GlcNAcylated peptides or proteins in a sequence-independent manner.

Hijacking of death receptor signaling by bacterial pathogen effectors

Death receptors such as Tumor necrosis factor receptor 1, FAS and TNF-associated apoptosis-inducing ligand-R1/2 play a major role in counteracting with bacterial pathogen infection through regulation of inflammation and programmed cell death. The highly regulated death receptor signaling is frequently targeted by gram-negative bacterial pathogens such as Salmonella, Shigella, enteropathogenic Escherichia coli and enterohamorrhagic Escherichia coli, which harbor a conserved type III secretion system that delivers a repertoire of effector proteins to manipulate host signal transductions for their own benefit. This review focuses on how bacterial gut pathogens hijack death receptor signaling to inhibit host NF-κB and programmed cell death pathways.

Total Synthesis of the G2/M DNA Damage Checkpoint Inhibitor Psilostachyin C

A concise total synthesis of the G2/M DNA damage checkpoint inhibitor psilostachyin C is reported using a 1,4-addition-aldol condensation-ring-closing metathesis (RCM) strategy. Initial biological studies indicate that psilostachyin C could enhance the sensitivity of the HeLa cell toward camptothecin (CPT) treatment via the activation of the caspase-3 mediated apoptosis pathway.