Browsing by Subject "NLRC5"

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Characterization of the role of the NLR proteins NLRC5 and NLRP11 in the immune response
(2021) Kienes, Ioannis; Kufer, Thomas
Recognition of conserved molecular patterns by pattern recognition receptors (PRRs) is crucial for the initiation of an innate immune response. Within PRRs, the NOD-like receptor (NLR) family is, in humans, a group of 22 cytosolic proteins, which function as PRRs of the innate immune system and as regulators of adaptive immune responses. However, it has become evident, that several NLR proteins also function as regulators of innate immune responses. In this thesis the function of human NLR proteins NLRC5 and NLRP11 in immune responses was further characterized. The first part of this thesis was focused on the NOD-like receptor NLRC5. NLRC5 and major histocompatibility complex (MHC) class II transcriptional activator (CIITA) are the master regulators of MHC class I and II transcription, respectively. Both proteins can translocate into the nucleus, where they induce transcription of MHC class I and class II, respectively. As NLRC5 and CIITA do not possess intrinsic DNA binding capacities, they exert their function by binding to a common multiprotein complex, termed MHC enhanceosome and through recruitment of further transcriptional regulators. Although MHC enhanceosome components are, as known thus far, identical, NLRC5 and CIITA are specific for their respective transcriptional targets. In this work we employed several techniques to identify novel interaction partners of NLRC5 to understand the mechanisms behind this specificity. As the N-terminal domain death-domain like fold (DD) of NLRC5 has previously been shown to be involved in conferring specificity, we adapted a protocol for proximal ligation by fusion of the NLRC5 DD to biotin ligase from Aquifex aeolicus (BioID2) to unravel the interactome of this NLRC5 domain. By enrichment of biotinylated proteins through streptavidin-biotin precipitation and analysis of the proteins by LC-MS/MS, we aimed to identify novel putative interactors with functions in transcriptional regulation. Additionally, we used yeast 2 hybrid screening of the NLRC5 DD against a library of human CD4+ and CD8+ T cells for the identification of novel interaction partners. This led to the identification of the paired amphipathic helix protein Sin3A (Sin3A) and the negative elongation factor B (NELFB) as interactors of NLRC5 DD. Characterization of their role in transcriptional regulation of MHC class I revealed an inhibitory role of both proteins. However, as we also observed repression of CIITA-mediated MHC class II transcription, both proteins are likely not involved in determination of target specificity of NLRC5. Translocation of NLRC5 into the nucleus is essential for the induction of MHC class I transcription. It has however previously been shown, that forced nuclear localization of NLRC5 strongly diminishes its transcriptional activity. We therefore employed co-immunoprecipitation of differentially localized NLRC5 constructs to identify interaction partners which might be involved in post translational regulation of NLRC5. Further, to advance our understanding of the NLRC5 DD, we aimed to elucidate its crystal structure. For this, we established a protocol for large-scale recombinant expression and purification of the NLRC5 DD for subsequent crystallization of the recombinant protein. The second part of this thesis was focused on NLRP11. Tight regulation of inflammatory cytokine and interferon (IFN) production in innate immunity is pivotal for optimal control of pathogens and avoidance of immunopathology. NLRP11 has previously been shown to regulate type I IFN and other pro-inflammatory responses. To gain a deeper understanding of the underlying mechanism, we aimed to identify novel NLRP11 interactors, through which the inhibition is conferred. Here we generated cell lines stably expressing NLRP11-eGFP as novel tools to elucidate the functions of NLRP11. We identified the ATP-dependent RNA helicase DDX3X as a novel binding partner of NLRP11 by co immunoprecipitation and LC-MS/MS. DDX3X is known to enhance type I IFN responses and NLRP3 inflammasome activation. We demonstrate that NLRP11 can abolish IKKe-mediated phosphorylation of DDX3X, resulting in lower type I IFN induction upon viral infection. These effects were dependent on the leucine-rich repeat (LRR) domain of NLRP11 that we mapped as the interaction domain for DDX3X. In addition, NLRP11 also suppressed NLRP3-mediated caspase-1 activation in an LRR domain-dependent manner, suggesting, that NLRP11 might sequester DDX3X and prevent it from promoting NLRP3-induced inflammasome activation. Taken together, our data revealed DDX3X as a central target of NLRP11, which can mediate the effects of NLRP11 on type I IFN induction, as well as NLRP3 inflammasome activation. This expands our knowledge of the molecular mechanisms underlying NLRP11 function in innate immunity and suggests that both NLRP11 and DDX3X might be promising targets for modulation of innate immune responses.
NLRC5-CIITA fusion protein as an effective inducer of MHC-I expression and antitumor immunity
(2023) Santharam, Madanraj Appiya; Shukla, Akhil; Levesque, Dominique; Kufer, Thomas A.; Boisvert, François-Michel; Ramanathan, Sheela; Ilangumaran, Subburaj
Aggressive tumors evade cytotoxic T lymphocytes by suppressing MHC class-I (MHC-I) expression that also compromises tumor responsiveness to immunotherapy. MHC-I defects strongly correlate to defective expression of NLRC5, the transcriptional activator of MHC-I and antigen processing genes. In poorly immunogenic B16 melanoma cells, restoring NLRC5 expression induces MHC-I and elicits antitumor immunity, raising the possibility of using NLRC5 for tumor immunotherapy. As the clinical application of NLRC5 is constrained by its large size, we examined whether a smaller NLRC5-CIITA fusion protein, dubbed NLRC5-superactivator (NLRC5-SA) as it retains the ability to induce MHC-I, could be used for tumor growth control. We show that stable NLRC5-SA expression in mouse and human cancer cells upregulates MHC-I expression. B16 melanoma and EL4 lymphoma tumors expressing NLRC5-SA are controlled as efficiently as those expressing full-length NLRC5 (NLRC5-FL). Comparison of MHC-I-associated peptides (MAPs) eluted from EL4 cells expressing NLRC5-FL or NLRC5-SA and analyzed by mass spectrometry revealed that both NLRC5 constructs expanded the MAP repertoire, which showed considerable overlap but also included a substantial proportion of distinct peptides. Thus, we propose that NLRC5-SA, with its ability to increase tumor immunogenicity and promote tumor growth control, could overcome the limitations of NLRC5-FL for translational immunotherapy applications.
NOD-like receptors - emerging links to obesity and associated morbidities
(2023) Bauer, Sarah; Hezinger, Lucy; Rexhepi, Fjolla; Ramanathan, Sheela; Kufer, Thomas A.
Obesity and its associated metabolic morbidities have been and still are on the rise, posing a major challenge to health care systems worldwide. It has become evident over the last decades that a low-grade inflammatory response, primarily proceeding from the adipose tissue (AT), essentially contributes to adiposity-associated comorbidities, most prominently insulin resistance (IR), atherosclerosis and liver diseases. In mouse models, the release of pro-inflammatory cytokines such as TNF-alpha (TNF-α) and interleukin (IL)-1β and the imprinting of immune cells to a pro-inflammatory phenotype in AT play an important role. However, the underlying genetic and molecular determinants are not yet understood in detail. Recent evidence demonstrates that nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family proteins, a group of cytosolic pattern recognition receptors (PRR), contribute to the development and control of obesity and obesity-associated inflammatory responses. In this article, we review the current state of research on the role of NLR proteins in obesity and discuss the possible mechanisms leading to and the outcomes of NLR activation in the obesity-associated morbidities IR, type 2 diabetes mellitus (T2DM), atherosclerosis and non-alcoholic fatty liver disease (NAFLD) and discuss emerging ideas about possibilities for NLR-based therapeutic interventions of metabolic diseases.
The role of NLRC5 in obesity
(2024) Bauer, Sarah Katharina; Kufer, Thomas
Obesity and its associated morbidities are major global health problems. It has become evident in the last decades that the state of obesity is intimately linked with our immune system. Pattern recognition receptors (PRRs), the main sensor molecules of the innate immune system, were shown to play an essential role in the pathology of obesity and its associated morbidities. Among others, members of the nucleotide-binding and oligomerization domain (NOD) -like receptors (NLRs), a family of cytosolic PRRs, were associated with the obesity-accompanying low-grade inflammatory response contributing to obesity-associated morbidities. NLRC5 is a NLR protein functioning as key transcriptional regulator of major histocompatibility complex (MHC) class I genes responsible for antigen presentation. Recent observations now suggest novel roles of NLRC5 in metabolic trades, but so far, no confirmation of these singular observations is available, and the underlying mechanisms remain elusive. The aim of this thesis was to characterize the role of the NLR protein NLRC5 in obesity. To this end, two Nlrc5 deficient mouse lines (Nlrc5 deltaExon4-7 and Nlrc5 deltaExon4) were subjected to high-fat diet (HFD) feeding and phenotypic, morphological, and biochemical analyses were performed. Female Nlrc5 deltaExon4-7 mice presented with higher body and adipose tissue (AT) weight gain and larger adipocytes compared to wildtype (WT) animals. This phenotype, however, could not be recapitulated in the Nlrc5 deltaExon4 mouse line. Microbiome analysis revealed subtle alterations of the faecal microbiome by diet:genotype interactions. To further characterize the effect of NLRC5 deficiency on adipocyte differentiation, the CRISPR/Cas9 gene editing system was used to modify Nlrc5 expression in the 3T3-L1 preadipocyte cell line. Using inducible HeLa cell lines with stable GFP-NLRC5 expression we showed NLRC5 to interact with the master regulator of adipogenesis peroxisome proliferator-activated receptor y (PPARy) and to enhance the expression of PPARy target genes. In addition, a contribution of NLRC5 to PPARy’s anti-inflammatory actions was revealed using NLRC5 deficient THP-1 macrophage-like cells and bone marrow-derived macrophages from Nlrc5 deltaExon4-7 mice. To elucidate the mechanism behind the synergy between NLRC5 and PPARy, reporter gene and chromatin immunoprecipitation (ChIP) assays were performed. Lastly, the expression of multiple NLR family members was correlated with body mass index (BMI) in obese human patients and investigated in the adipose tissue and liver of HFD-fed mice, the latter revealing Nlrp10 to be highly upregulated by HFD feeding. Taken together, this thesis provides a comprehensive characterization of Nlrc5 deficient mice on HFD and reveals a function of NLRC5 as transcriptional co-regulator of PPARy targets and its anti-inflammatory properties. In addition, this work provides first insights into the potential mechanism behind the synergistic transcriptional regulation by NLRC5 and PPARy and extends the knowledge on the regulation of NLR expression by HFD feeding.