Alterations in nucleotide-binding oligomerization domain-2 expression, pathway activation, and cytokine production in Yao syndrome.

Yao syndrome (YAOS) is a systemic autoinflammatory disease (SAID), formerly termed nucleotide-binding oligomerization domain-2 (NOD2)-associated autoinflammatory disease. Due to the recent identification of YAOS, the molecular mechanisms underlying its disease pathogenesis are unclear. With specific NOD2 variants as characteristic genotypic features of YAOS, our study examined NOD2 expression, transcript splicing, signaling pathway activation, and cytokine profiles in peripheral blood mononuclear cells (PBMCs) from 10 YAOS patients and six healthy individuals. All participants were genotyped for NOD2 variants; all YAOS patients were heterozygous for the NOD2 IVS8+158 variant (IVS8+158) and four patients also carried a concurrent NOD2 R702W variant (IVS8+158/R702W haplotype). Resembling other SAIDs, plasma levels of TNFα, IL-1ß, IL-6, IFNγ, and S100A12 were unaltered in YAOS patients. Intron-8 splicing of NOD2 transcripts was unaffected by carriage of NOD2 IVS8+158. However, NOD2 transcript level and basal p38 mitogen-activated protein kinase (MAPK) activity were significantly elevated in PBMCs from IVS8+158 YAOS patients. Moreover, these patients' cells had elevated basal IL-6 secretion that was enhanced by muramyl dipeptide (MDP) stimulation. Tocilizumab treatment of a YAOS IVS8+158 patient resulted in marked clinical improvement. In contrast, MDP-stimulated NF-κB activity was uniquely suppressed in haplotype IVS8+158/R702W patients, as was TNFα secretion. Our study demonstrates for the first time that NOD2 expression and pathway activation are aberrant in YAOS, and specific NOD2 genotypes result in distinct NOD2 expression and cytokine profiles. These findings may also help select therapeutic strategies in the future.

A dual role for receptor-interacting protein kinase 2 (RIP2) kinase activity in nucleotide-binding oligomerization domain 2 (NOD2)-dependent autophagy.

Autophagy is triggered by the intracellular bacterial sensor NOD2 (nucleotide-binding, oligomerization domain 2) as an anti-bacterial response. Defects in autophagy have been implicated in Crohn's disease susceptibility. The molecular mechanisms of activation and regulation of this process by NOD2 are not well understood, with recent studies reporting conflicting requirements for RIP2 (receptor-interacting protein kinase 2) in autophagy induction. We examined the requirement of NOD2 signaling mediated by RIP2 for anti-bacterial autophagy induction and clearance of Salmonella typhimurium in the intestinal epithelial cell line HCT116. Our data demonstrate that NOD2 stimulates autophagy in a process dependent on RIP2 tyrosine kinase activity. Autophagy induction requires the activity of the mitogen-activated protein kinases MEKK4 and p38 but is independent of NFκB signaling. Activation of autophagy was inhibited by a PP2A phosphatase complex, which interacts with both NOD2 and RIP2. PP2A phosphatase activity inhibited NOD2-dependent autophagy but not activation of NFκB or p38. Upon stimulation of NOD2, the phosphatase activity of the PP2A complex is inhibited through tyrosine phosphorylation of the catalytic subunit in a process dependent on RIP2 activity. These findings demonstrate that RIP2 tyrosine kinase activity is not only required for NOD2-dependent autophagy but plays a dual role in this process. RIP2 both sends a positive autophagy signal through activation of p38 MAPK and relieves repression of autophagy mediated by the phosphatase PP2A.

The nucleotide synthesis enzyme CAD inhibits NOD2 antibacterial function in human intestinal epithelial cells.

BACKGROUND & AIMS: Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn's disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS: Carbamoyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS: CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS: The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD.

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity.

How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by posttranslational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification [1] that is enriched in axonemes. The enzymes responsible for this posttranslational modification, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) [2]. We show that in cilia of Tetrahymena, TTLL6 E-ligases generate glutamylation mainly on the B-tubule of outer doublet microtubules, the site of force production by ciliary dynein. Deletion of two TTLL6 paralogs caused severe deficiency in ciliary motility associated with abnormal waveform and reduced beat frequency. In isolated axonemes with a normal dynein arm composition, TTLL6 deficiency did not affect the rate of ATP-induced doublet microtubule sliding. Unexpectedly, the same TTLL6 deficiency increased the velocity of microtubule sliding in axonemes that also lack outer dynein arms, in which forces are generated by inner dynein arms. We conclude that tubulin glutamylation on the B-tubule inhibits the net force imposed on sliding doublet microtubules by inner dynein arms.