Chong-Shan Shi

Activation of autophagy by inflammatory signals limits IL-1β production by targeting ubiquitinated inflammasomes for destruction

Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.

MyD88 and Trif Target Beclin 1 to Trigger Autophagy in Macrophages

The Toll-like receptors (TLR) play an instructive role in innate and adaptive immunity by recognizing specific molecular patterns from pathogens. Autophagy removes intracellular pathogens and participates in antigen presentation. Here, we demonstrate that not only TLR4, but also other TLR family members induce autophagy in macrophages, which is inhibited by MyD88, Trif, or Beclin 1 shRNA expression. MyD88 and Trif co-immunoprecipitate with Beclin 1, a key factor in autophagosome formation. TLR signaling enhances the interaction of MyD88 and Trif with Beclin 1, and reduces the binding of Beclin 1 to Bcl-2. These findings indicate TLR signaling via its adaptor proteins reduces the binding of Beclin 1 to Bcl-2 by recruiting Beclin 1 into the TLR-signaling complex leading to autophagy.

TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy.

Regulation of the ubiquitination of Beclin-1 may be a general mechanism that controls autophagy. Controlling the Inner Pac-Man Autophagy is a process by which cellular components can be recycled to provide much-needed raw materials to help cells survive conditions of acute stress. But if a cell has to rely on this process for too long, the cell will die. TLR4 is a receptor that responds to a bacterial component called LPS to activate the transcription factor NF-κB, which drives the expression of pro-inflammatory genes in a process that requires a modifying enzyme called TRAF6 to switch on the pathway and an opposing enzyme called A20 to switch it off. Shi and Kehrl now show that these two enzymes play an analogous role in regulating TLR4-induced autophagy: TRAF6 modifies (ubiquitinates) a protein called Beclin-1, which initiates autophagy, whereas A20 counters the effects of TRAF6 and shuts the process down. The authors’ examination of other pro-autophagy pathways suggests that regulation of the ubiquitination state of Beclin-1 may be a general mechanism for the induction of autophagy by pro-inflammatory stimuli. Autophagy delivers cytoplasmic constituents to autophagolysosomes and is linked to both innate and adaptive immunity. Toll-like receptor 4 (TLR4) signaling induces autophagy and recruits Beclin-1, the mammalian homolog of yeast Atg6, to the receptor complex. We found that tumor necrosis factor receptor (TNFR)–associated factor 6 (TRAF6)–mediated, Lys63 (K63)–linked ubiquitination of Beclin-1 is critical for TLR4-triggered autophagy in macrophages. Two TRAF6-binding motifs in Beclin-1 facilitated the binding of TRAF6 and the ubiquitination of Beclin-1. Lys117, which is strategically located in the Bcl-2 homology 3 (BH3) domain of Beclin-1, was a major site for K63-linked ubiquitination. The deubiquitinating enzyme A20 reduced the extent of K63-linked ubiquitination of Beclin-1 and limited the induction of autophagy in response to TLR signaling. Treatment of macrophages with either interferon-γ or interleukin-1 also triggered the K63-linked ubiquitination of Beclin-1 and the formation of autophagosomes. These results indicate that the status of K63-linked ubiquitination of Beclin-1 plays a key role in regulating autophagy during inflammatory responses.

Activation of Stress-activated Protein Kinase/c-Jun N-terminal Kinase, but Not NF-κB, by the Tumor Necrosis Factor (TNF) Receptor 1 through a TNF Receptor-associated Factor 2- and Germinal Center Kinase Related-dependent Pathway

A key step by which tumor necrosis factor (TNF) signals the activation of nuclear factor-κB (NF-κB) and the stress-activated protein kinase (SAPK, also called c-Jun N-terminal kinase or JNK) is the recruitment to the TNF receptor of TNF receptor-associated factor 2 (TRAF2). However, the subsequent steps in TRAF2-induced SAPK and NF-κB activation remain unresolved. Here we report the identification of a TNF-responsive serine/threonine protein kinase termed GCK related (GCKR) that likely signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1) to activate the SAPK pathway. TNF, TRAF2, and ultraviolet (UV) light, which in part uses the TNF receptor signaling pathway, all increased GCKR activity. A TRAF2 mutant, which inhibits both TRAF2-induced NF-κB and SAPK activation, blocked TNF-induced GCKR activation. Finally, interference with GCKR expression impeded TRAF2- and TNF-induced SAPK activation but not that of NF-κB. This suggests a divergence in the TNF signaling pathway that leads to SAPK and NF-κB activation, which is located downstream of TRAF2 but upstream of GCKR.

Regulator of G Protein Signaling 1 (RGS1) Markedly Impairs Giα Signaling Responses of B Lymphocytes

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-α. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various Gα subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Giα and Gqα GTPase-activating protein and a potential G12α effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12α. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.

Traf6 and A20 differentially regulate TLR4-Induced autophagy by affecting the ubiquitination of Beclin 1

Toll-like receptor 4 (TLR4) signaling triggers autophagy, which has been linked to both adaptive and innate immunity. Engagement of TLR4 recruits to the receptor complex Beclin 1, a key component of a class III phosphatidylinositol 3-kinase complex (PI3KC3) that initiates autophagosome formation. Recently, we found that tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-mediates Lys63 (K63)-linked ubiquitination of Beclin 1 is crucial for TLR4-triggered autophagy in macrophages. We identified two TRAF6-binding motifs in Beclin 1 that facilitate the binding of TRAF6 and the ubiquitination of Beclin 1. A lysine located in the Bcl-2 homology 3 (BH3) domain of Beclin 1 serves as a major site for K63-linked ubiquitination. Opposing TRAF6, the deubiquitinating enzyme A20 reduces the extent of K63-linked ubiquitination of Beclin 1 and limits the induction of autophagy in response to TLR4 signaling. Furthermore, treatment of macrophages with either interferon- or interleukin-1 triggers the K63-linked ubiquitination of Beclin 1 and the formation of autophagosomes. These results indicate that the status of K63-linked ubiquitination of Beclin 1 plays a key role in regulating autophagy during inflammatory responses.

SARS-Coronavirus Open Reading Frame-9b Suppresses Innate Immunity by Targeting Mitochondria and the MAVS/TRAF3/TRAF6 Signalosome

Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002–2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b–mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.

The Mitogen-Activated Protein Kinase Kinase Kinase Kinase GCKR Positively Regulates Canonical and Noncanonical Wnt Signaling in B Lymphocytes

ABSTRACT Wnt ligands bind receptors of the Frizzled (Fz) family to control cell fate, proliferation, and polarity. Canonical Wnt/Fz signaling stabilizes β-catenin by inactivating GSK3β, leading to the translocation of β-catenin to the nucleus and the activation of Wnt target genes. Noncanonical Wnt/Fz signaling activates RhoA and Rac, and the latter triggers the activation of c-Jun N-terminal kinase (JNK). Here, we show that exposure of B-lymphocytes to Wnt3a-conditioned media activates JNK and raises cytosolic β-catenin levels. Both the Rac guanine nucleotide exchange factor Asef and the mitogen-activated protein kinase kinase kinase kinase germinal center kinase-related enzyme (GCKR) are required for Wnt-mediated JNK activation in B cells. In addition, we show that GCKR positively affects the β-catenin pathway in B cells. Reduction of GCKR expression inhibits Wnt3a-induced phosphorylation of GSK3β at serine 9 and decreases the accumulation of cytosolic β-catenin. Furthermore, Wnt signaling induces an interaction between GCKR and GSK3β. Our findings demonstrate that GCKR facilitates both canonical and noncanonical Wnt signaling in B lymphocytes.

Canonical and Noncanonical G-Protein Signaling Helps Coordinate Actin Dynamics To Promote Macrophage Phagocytosis of Zymosan

ABSTRACT Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gαi nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gαi proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca2+ that was partially sensitive to Gαi nucleotide exchange inhibition and expression of GTP-bound Gαi recruited Elmo1 to the plasma membrane. Reducing GDP-Gαi nucleotide exchange, decreasing Gαi expression, pharmacologically interrupting Gβγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gαi remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.

Normal Autophagic Activity in Macrophages from Mice Lacking Gαi3, AGS3, or RGS19

In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3-/-, Gpsm1-/-, or Rgs19-/- mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3-/-, and Gpsm1-/- macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.