Deficiency of autophagy in dendritic cells protects against experimental autoimmune encephalomyelitis.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the immune system. DCs present antigens to CD8 and CD4 T cells in the context of class I or II MHC. Recent evidence suggests that autophagy, a conserved intracellular degradation pathway, regulates class II antigen presentation. In vitro studies have shown that deletion of autophagy-related genes reduced antigen presentation by APCs to CD4 T cells. In vivo studies confirmed these findings in the context of infectious diseases. However, the relevance of autophagy-mediated antigen presentation in autoimmunity remains to be elucidated. Here, we report that loss of autophagy-related gene 7 (Atg7) in DCs ameliorated experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-mediated mouse model of multiple sclerosis, by reducing in vivo priming of T cells. In contrast, severity of hapten-induced contact hypersensitivity, in which CD8 T cells and NK cells play major roles, was unaffected. Administration of the autophagy-lysosomal inhibitor chloroquine, before EAE onset, delayed disease progression and, when administered after the onset, reduced disease severity. Our data show that autophagy is required in DCs for induction of EAE and suggest that autophagy might be a potential target for treating CD4 T cell-mediated autoimmune conditions.

Regulation of NF-kappaB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13).

Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated cellular proteins or changes in proteasome expression, suggesting that Rpn13 must have a specialized role in proteasome function. Thus, Rpn13 participation in protein degradation, by recruiting UCH37, is rather selective to specific proteins whose degradation critically depends on UCH37 deubiquitination activity. The specific substrates for the Rpn13/UCH37 complex have not been determined. Because of a previous discovery of an interaction between Rpn13 and inducible nitric oxide synthase (iNOS), we hypothesized that iNOS is one of the substrates for the Rpn13/UCH37 complex. In this study, we show that Rpn13 is involved in iNOS degradation and is required for iNOS interaction with the deubiquitination protein UCH37. Furthermore, we discovered that IkappaB-alpha, a protein whose proteasomal degradation activates the transcription factor NF-kappaB, is also a substrate for the Rpn13/UCH37 complex. Thus, this study defines two substrates, with important roles in inflammation and host defense for the Rpn13/UCH37 pathway.

Src kinase-mediated phosphorylation stabilizes inducible nitric-oxide synthase in normal cells and cancer cells.

Src kinases are key regulators of cellular proliferation, survival, motility, and invasiveness. They play important roles in the regulation of inflammation and cancer. Overexpression or hyperactivity of c-Src has been implicated in the development of various types of cancer, including lung cancer. Src inhibition is currently being investigated as a potential therapy for non-small cell lung cancer in Phase I and II clinical trials. The mechanisms of Src implication in cancer and inflammation are linked to the ability of activated Src to phosphorylate multiple downstream targets that mediate its cellular effector functions. In this study, we reveal that inducible nitric-oxide synthase (iNOS), an enzyme also implicated in cancer and inflammation, is a downstream mediator of activated Src. We elucidate the molecular mechanisms of the association between Src and iNOS in models of inflammation induced by lipopolysaccharide and/or cytokines and in cancer cells and tissues. We identify human iNOS residue Tyr(1055) as a target for Src-mediated phosphorylation. These results are shown in normal cells and cancer cells as well as in vivo in mice. Importantly, such posttranslational modification serves to stabilize iNOS half-life. The data also demonstrate interactions and co-localization of iNOS and activated Src under inflammatory conditions and in cancer cells. This study demonstrates that phosphorylation of iNOS by Src plays an important role in the regulation of iNOS and nitric oxide production and hence could account for some Src-related roles in inflammation and cancer.

The physiologic aggresome mediates cellular inactivation of iNOS.

Nitric Oxide (NO), produced by inducible nitric oxide synthase (iNOS), has been implicated in the pathogenesis of various biological and inflammatory disorders. Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed "physiologic aggresome," is exemplified by aggresome formation of iNOS, an important host defense protein. The functional significance of cellular formation of the iNOS aggresome is hitherto unknown. In this study, we used live cell imaging, fluorescence microscopy, and intracellular fluorescence NO probes to map the subcellular location of iNOS and NO under various conditions. We found that NO production colocalized with cytosolic iNOS but aggresomes containing iNOS were distinctly devoid of NO production. Further, cells expressing iNOS aggresomes produced significantly less NO as compared with cells not expressing aggresomes. Importantly, primary normal human bronchial epithelial cells, stimulated by cytokines to express iNOS, progressively sequestered iNOS to the aggresome, a process that correlated with marked reduction of NO production. These results suggest that bronchial epithelial cells used the physiologic aggresome mechanism for iNOS inactivation. Our studies reveal a novel cellular strategy to terminate NO production via formation of the iNOS aggresome.

A critical role for CHIP in the aggresome pathway.

Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed "physiologic aggresome," is exemplified by aggresome formation of inducible nitric oxide synthase (iNOS), an important host defense protein. CHIP (carboxy terminus of Hsp70-interacting protein) is a highly conserved protein that has been shown to mediate substrate ubiquitination and degradation by the proteasome. In this study, we show that CHIP has a previously unexpected critical role in the aggresome pathway. CHIP interacts with iNOS and promotes its ubiquitination and degradation by the proteasome as well as its sequestration to the aggresome. CHIP-mediated iNOS targeting to the proteasome sequentially precedes CHIP-mediated iNOS sequestration to the aggresome. CHIP is required for iNOS preaggresome structures to form a mature aggresome. Furthermore, CHIP is required for targeting the mutant form of cystic fibrosis transconductance regulator (CFTRDeltaF508) to the aggresome. Importantly, the ubiquitin ligase function of CHIP is required in targeting preaggresomal structures to the aggresome by promoting an iNOS interaction with histone deacetylase 6, which serves as an adaptor between ubiquitinated proteins and the dynein motor. This study reveals a critical role for CHIP in the aggresome pathway.

[Identification of a hrp-associated gene hpaB in the role of pathogenicity of Xanthomonas oryzae pv. oryzae].

Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, is the causal agent of rice bacterial blight disease, which can cause severe yield loss of rice worldwide. To identify genes contributing to virulence and explore the possible mechanism of pathogenicity, transposon mutagenesis was used to isolate nonpathogenic mutants. By screening of a high-quality Tn5-like transposon (EZ: :TN) insertional mutant library of Xoo PXO99 against a host plant (rice cultivar IR24), one virulence-deficient mutant, XOG11, was identified. Genomic fragment flanking the insertion site of the mutant was amplified by thermal asymmetric interlaced polymerase chian reaction ( TAIL-PCR) and sequenced. The result of NCBI blast homologue searching of the fragment shows that the transposon was inserted into a hrp associated gene, hpaB. Xoo hpaB gene is one of the hrp gene cluster members that encode a type [I secretion system (TTSS) and locates at the downstream of hrpE. The product of hpaB in Xoo is a small (Molecular Weight, 17.6kDa), acidic (PI, 4.28) and Leucine-rich (14.4%) protein and shares high homology with corresponding proteins in other Xanthomonas. It suggests that HpaB may play as a TTSS chaperone. Mutant XOGl1 was confirmed both by PCR and Southern blotting: The PCR result by using primers upstream and downstream of hpaB respectively verified Tn5 insertion in hpaB and excluded the rare case of second transfer of the transposon associated with flanking sequence; Southern blot of digested genomic DNA with the probe of Km resistance gene aph proved that XOG11 was inserted by a single-copy transposon, indicating that the loss of pathogenicity in XOG11 was due to the Tn5 insertion in hpaB gene. Genetic complementation by cloning hpaB in the wide host range plasmid pHMI and transferring the recombinant plasmid into XOG11 restored its pathogenicity in IR24. These results suggest that the pathogenicity deficiency of XOG11 is due to the mutation of hpaB gene.

Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris.

Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.