CpG-DNA or its synthetic analog CpG-ODN activates innate immunity through Toll-like receptor 9 (TLR9). However, the mechanism of TLR9 activation by CpG-DNA remains elusive. Here we have identified HMGB1 as a CpG-ODN–binding protein. HMGB1 interacts and preassociates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and hastens TLR9's redistribution to early endosomes in response to CpG-ODN. CpG-ODN stimulates macrophages and dendritic cells to secrete HMGB1; in turn, extracellular HMGB1 accelerates the delivery of CpG-ODNs to its receptor, leading to a TLR9-dependent augmentation of IL-6, IL-12, and TNFα secretion. Loss of HMGB1 leads to a defect in the IL-6, IL-12, TNFα, and iNOS response to CpG-ODN. However, lack of intracellular TLR9-associated HMGB1 can be compensated by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells and regulates inflammatory responses to CpG-DNA.

Discriminating between harmless and pathogenic bacteria is a key challenge faced by the immune system. Ivanov and Roy demonstrate that virulent bacteria disrupt mTOR signaling, which then skews responses towards inflammatory cytokine production. The mammalian immune system has the ability to discriminate between pathogenic microbes and nonpathogenic microbes to control inflammation. Here we investigated the ubiquitination profiles of host proteins after infection of macrophages with a virulent strain of the intracellular bacterium Legionella pneumophila or a nonpathogenic mutant of L. pneumophila. Only infection with pathogenic L. pneumophila resulted in ubiquitination of positive regulators of the metabolic checkpoint kinase mTOR and led to diminished mTOR activity. Detection of pathogen signatures resulted in translational biasing toward proinflammatory cytokines through mTOR-mediated regulation of cap-dependent translation. Thus, there is a pathogen-detection program in macrophages that stimulates protein ubiquitination and the degradation of regulators of mTOR, which suppresses mTOR function and directs a proinflammatory cytokine program.

The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection.

The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase (697–955) followed by a 231-residue-long COOH-terminal tail (C-tail), which contains multiple tyrosine residues. To examine the role of the EGFR C-tail in signal transducer and activator of transcription (STAT) activation, a series of EGFR C-tail truncations were constructed. Transient transfection of 293 cells with EGFR lacking the C-tail, i.e. Y974ΔEGFR or Y992ΔEGFR, led to EGF-independent or constitutive STAT activation, whereas EGF-dependent STAT activation was restored with truncations made COOH-terminal to the next tyrosine residue, i.e.EGFR-Y1045Δ. Transfection with the-truncated form EGFR-Y954Δ resulted in the loss of STAT activation, suggesting that the sequence between Tyr⁹⁷⁴ and Tyr⁹⁵⁴ is essential for STAT activation. Phosphopeptide competition analysis revealed multiple tyrosine residues within the C-tail that can act as the docking sites for both Stat1 and Stat3. A region that negatively regulated STAT activation was also identified, extending from Tyr¹¹¹⁴ to Glu¹¹⁷², consistent with the ability of this region to recruit a suppressor of cytokine signaling factors SOCS1 and SOCS3. When cotransfected with the full-length EGFR, but not Y992ΔEGFR, SOCS1 or SOCS3 inhibited STAT activation by EGF in 293 cells. This suggests that both SOCS1 and SOCS3 can negatively regulate EGFR activation, presumably by inducing ubiquitination-dependent EGFR degradation upon ligand binding. These findings may therefore offer clues to how the EGF receptor C-tail regulates STAT activity.

CpG‐DNA and its related synthetic CpG oligodeoxynucleotides (CpG‐ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG‐DNA‐responsive serine/threonine kinases; however, the target protein of CpG‐DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow‐derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) results in defective phosphorylation and activation of Akt by CpG‐DNA. Unexpectedly, loss of the Toll‐like receptor 9 has a minimal effect on Akt activation in response to CpG‐DNA. Further in vitro analysis using purified DNA‐PK and recombinant Akt proteins reveals that DNA‐PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA‐PKcs associates with Akt upon CpG‐DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA‐PKcs in CpG‐DNA signaling and define a CpG‐DNA/DNA‐PKcs/Akt pathway.

Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly-ubiquitin conjugates to proteins associated with L. pneumophila-containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila-derived molecules resulted in the production of ubiquitin-rich dendritic cell aggresome-like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that Legionella jordanis was unable to suppress DALIS formation after creating a ubiquitin-decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.