NLRP10 Affects the Stability of Abin-1 To Control Inflammatory Responses.

NOD-like receptors (NLR) are critical regulators of innate immune signaling. The NLR family consists of 22 human proteins with a conserved structure containing a central oligomerization NACHT domain, an N-terminal interaction domain, and a variable number of C-terminal leucine-rich repeats. Most NLR proteins function as cytosolic pattern recognition receptors with activation of downstream inflammasome signaling, NF-κB, or MAPK activation. Although NLRP10 is the only NLR protein lacking the leucine rich repeats, it has been implicated in multiple immune pathways, including the regulation of inflammatory responses toward Leishmania major and Shigella flexneri infection. In this study, we identify Abin-1, a negative regulator of NF-κB, as an interaction partner of NLRP10 that binds to the NACHT domain of NLRP10. Using S. flexneri as an infection model in human epithelial cells, our work reveals a novel function of NLRP10 in destabilizing Abin-1, resulting in enhanced proinflammatory signaling. Our data give insight into the molecular mechanism underlying the function of NLRP10 in innate immune responses.

The cofilin phosphatase slingshot homolog 1 (SSH1) links NOD1 signaling to actin remodeling.

NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-κB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-κB activation and innate immune responses.

NLRP10 enhances Shigella-induced pro-inflammatory responses.

Members of the NLR family evolved as intracellular sensors for bacterial and viral infection. However, our knowledge on the implication of most of the human NLR proteins in innate immune responses still remains fragmentary. Here we characterized the role of human NLRP10 in bacterial infection. Our data revealed that NLRP10 is a cytoplasmic localized protein that positively contributes to innate immune responses induced by the invasive bacterial pathogen Shigella flexneri. SiRNA-mediated knock-down studies showed that NLRP10 contributes to pro-inflammatory cytokine release triggered by Shigella in epithelial cells and primary dermal fibroblasts, by influencing p38 and NF-κB activation. This effect is dependent on the ATPase activity of NLRP10 and its PYD domain. Mechanistically, NLRP10 interacts with NOD1, a NLR that is pivotally involved in sensing of invasive microbes, and both proteins are recruited to the bacterial entry point at the plasma membrane. Moreover, NLRP10 physically interacts with downstream components of the NOD1 signalling pathway, such as RIP2, TAK1 and NEMO. Taken together, our data revealed a novel role of NLRP10 in innate immune responses towards bacterial infection and suggest that NLRP10 functions as a scaffold for the formation of the NOD1-Nodosome.

A role for the human nucleotide-binding domain, leucine-rich repeat-containing family member NLRC5 in antiviral responses.

Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I.C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-kappaB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I.C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses.

Derlin-1 and p97/valosin-containing protein mediate the endoplasmic reticulum-associated degradation of human V2 vasopressin receptors.

The endoplasmic reticulum-associated degradation (ERAD), the main quality control pathway of the cell, is crucial for the elimination of unfolded or misfolded proteins. Several diseases are associated with the retention of misfolded proteins in the early secretory pathway. Among them is X-linked nephrogenic diabetes insipidus, caused by mutations in the gene encoding the V2 vasopressin receptor (V2R). We studied the degradation pathways of three intracellularly retained V2R mutants with different misfolded domains in human embryonic kidney 293 cells. At steady state, the wild-type V2R and the complex-glycosylated mutant G201D were partially located in lysosomes, whereas core-glycosylated mutants L62P and V226E were excluded from this compartment. In pulse-chase experiments, proteasomal inhibition stabilized the nonglycosylated and core-glycosylated forms of all studied receptors. In addition, all mutants and the wild-type receptor were found to be polyubiquitinylated. Nonglycosylated and core-glycosylated receptor forms were located in cytosolic and membrane fractions, respectively, confirming the deglycosylation and retrotranslocation of ERAD substrates to the cytosol. Distinct Derlin-1-dependent and -independent ERAD pathways have been proposed for proteins with different misfolded domains (cytosolic, extracellular, and membrane) in yeast. Here, we show for the first time that V2R mutants with different misfolded domains are able to coprecipitate the ERAD components p97/valosin-containing protein, Derlin-1 and the 26S proteasome regulatory subunit 7. Our results demonstrate the presence of a Derlin-1-mediated ERAD pathway degrading wild-type and disease-causing V2R mutants with different misfolded domains in a mammalian system.

Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens.

Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-κB and subsequent activity of the JAK/STAT signaling pathway. While NLRP12 deficiency renders septic mice highly susceptible towards MDP, a sustained sensing of MDP through NOD2 is observed among monocytes lacking NLRP12. This loss of tolerance in monocytes results in greater colonization resistance towards Citrobacter rodentium. Our data show that this is a consequence of NOD2-dependent accumulation of inflammatory mononuclear cells that correlates with induction of interferon-stimulated genes. Our study unveils a relevant process of tolerance towards the gut microbiota that is exploited by an attaching/effacing enteric pathogen.

Roles of NLRP10 in innate and adaptive immunity.

Mammalian nucleotide-binding domain leucine-rich repeat containing proteins (NLRs) are important pattern-recognition receptors, still the function of many NLRs remains poorly defined. Here we review first insights into the molecular function of NLRP10 highlighting the role of this specific NLR in innate and adaptive immune responses.