Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC).APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein -catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADPribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the -catenin destruction complex, placing the APC2/ TNKS interaction at the correct intracellular location to regulate -catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases -catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.The Wnt pathway helps direct a myriad of normal developmental and adult homeostasic processes in metazoans, but is also misregulated in several human diseases such as cancer (1, 2). Wnt signaling is regulated through the activity of a multiprotein "destruction complex" that promotes proteolysis of the transcriptional co-activator -catenin (cat) 3 by stimulating phosphorylation of the cat phosphodegron (3). Core components of the destruction complex include the scaffold protein Axin, the tumor suppressor adenomatous polyposis coli (APC), and the kinases CK1 and GSK3.While Wnt signaling plays essential roles during development, it is inappropriately activated in a number of cancers, most notably colorectal cancer. Truncating mutations in the tumor suppressor adenomatous polyposis coli (APC) are the initiating mutational event in more than 80% of all colon cancer cases, and these mutations hyperactivate cat signaling (4). Thus small molecule inhibitors of the Wnt pathway should provide an effective therapeutic strategy. Among these strategies, inhibition of oncogenic cat activity would appear to be the most direct approach, as studies have demonstrated that the accumulation of cat is what initiates oncogenesis, and that tumors have a continued reliance on oncogenic cat signaling (5). Indeed, recent promising antagonists have been identified that specifically disrupt cat binding to TCF or th...
Dopamine receptors play an integral role in controlling brain physiology. Importantly, subtype selective agonists and antagonists of dopamine receptors with biased signaling properties have been successful in treating psychiatric disorders with a low incidence of side effects. To this end, we recently designed and developed SK609, a dopamine D3 receptor (D3R) selective agonist that has atypical signaling properties. SK609 has shown efficacy in reversing akinesia and reducing L-dopa-induced dyskinesia in a hemiparkinsonian rats. In the current study, we demonstrate that SK609 has high selectivity for D3R with no binding affinity on D2R high- or low-affinity state when tested at a concentration of 10 μM. In addition, SK609 and its analogues do not induce desensitization of D3R as determined by repeated agonist treatment response in phosphorylation of ERK1/2 functional assay. Most significantly, SK609 and its analogues preferentially signal through the G-protein-dependent pathway and do not recruit β-arrestin-2, suggesting a functional bias toward the G-protein-dependent pathway. Structure–activity relationship (SAR) studies using analogues of SK609 demonstrate that the molecules bind at the orthosteric site by maintaining the conserved salt bridge interactions with aspartate 110 on transmembrane 3 and aryl interactions with histidine 349 on transmembrane 6, in addition to several hydrophobic interactions with residues from transmembranes 5 and 6. The compounds follow a strict SAR with reference to the three pharmacophore elements: substituted phenyl ring, length of the linker connecting phenyl ring and amine group, and orientation and hydrophobic branching groups at the amine among SK609 analogues for efficacy and functional selectivity. These features of SK609 and the analogues suggest that biased signaling is an inherent property of this series of molecules.
BackgroundSince Plasmodium falciparum transmission relies exclusively on sexual-stage parasites, several malaria control strategies aim to disrupt this step of the life cycle. Thus, a better understanding of which individuals constitute the primary gametocyte reservoir within an endemic population, and the temporal dynamics of gametocyte carriage, especially in seasonal transmission settings, will not only support the effective implementation of current transmission control programmes, but also inform the design of more targeted strategies.MethodsA 1-year prospective cohort study was initiated in June 2013 with the goal of assessing the longitudinal dynamics of P. falciparum gametocyte carriage in a village in Mali with intense seasonal malaria transmission. A cohort of 500 individuals aged 1–65 years was recruited for this study. Gametocyte prevalence was measured monthly using Pfs25-specific RT-PCR, and analysed for the effects of host age and gender, seasonality, and multiclonality of P. falciparum infection over 1 year.ResultsMost P. falciparum infections (51–89%) in this population were accompanied by gametocytaemia throughout the 1-year period. Gametocyte prevalence among P. falciparum-positive individuals (proportion of gametocyte positive infections) was associated with age (p = 0.003) but not with seasonality (wet vs. dry) or gender. The proportion of gametocyte positive infections were similarly high in children aged 1–17 years (74–82% on median among 5 age groups), while older individuals had relatively lower proportion, and those aged > 35 years (median of 43%) had significantly lower than those aged 1–17 years (p < 0.05). Plasmodium falciparum-positive individuals with gametocytaemia were found to have significantly higher P. falciparum multiclonality than those without gametocytaemia (p < 0.033 in two different analyses).ConclusionsTaken together, these results suggest that a substantial proportion of Pf-positive individuals carries gametocytes throughout the year, and that age is a significant determinant of gametocyte prevalence among these P. falciparum-positive individuals. Furthermore, the presence of multiple P. falciparum genotypes in an infection, a common feature of P. falciparum infections in high transmission areas, is associated with gametocyte prevalence.Electronic supplementary materialThe online version of this article (10.1186/s12936-017-2123-2) contains supplementary material, which is available to authorized users.
Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I interferon. One of several upstream receptor cGAS (cyclic-GMP-AMP synthase) binds to cytosolic DNA and generates di-cyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to Stimulator of Interferon Genes (STING). STING recruits TANK binding kinase 1 (TBK1) which acts as a critical node that allows for efficient activation of interferon regulatory factors (IRFs) to drive the anti-viral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine rich repeat containing protein (NLR) that negatively regulates the type I interferon pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I interferons. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two hybrid screening. Here we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3:STING interaction in the cytosol of human epithelial cells. Furthermore, knock down of IQGAP1 in THP1 and HeLa cells causes significantly more interferon-β production in response to cytosolic nucleic acids. This result phenocopies NLRC3 deficient macrophages and fibroblasts and shRNA knock down of NLRC3 in THP1 cells. Our findings suggest IQGAP1 is a novel regulator of type I interferon production possibly via interacting with NLRC3 in human monocytic and epithelial cells.
Nucleotide binding domain, leucine rich repeat CARD containing protein 3 (NLRC3) is a member of the NLR gene family. Members of this family have been associated with human inflammatory diseases such as Crohn’s disease and cryopyrin-associated periodic syndrome. Although NLRC3 is not associated with human diseases, it is expressed preferentially in the immune system and functions in pathogen detection. NLRC3 is an intracellular protein involved in the sensing of lipopolysaccharide and cytosolic nucleic acids. NLRC3 is hypothesized to act as a negative regulator in response to bacterial and viral infection, suggesting that the vertebrate immune system has evolved specific inhibitors to limit the inflammatory response. We performed an unbiased yeast two-hybrid screen using an amino terminal fragment of NLRC3 to identify putative interacting proteins that might help elucidate the mechanism by which NLRC3 might inhibit inflammatory responses. To this end, we identified several interacting proteins. One protein, in particular, IQGAP1, acts as a scaffold important in regulating the cytoskeleton, cell adhesion and proliferation. Structure function analysis has localized the domains necessary and sufficient for interacting with NLRC3. Confocal microscopy demonstrates that these two proteins co-localize in transformed human epithelial cells. Functionally, in the absence of IQGAP1, human monocytic cells are hyperactive in response to cytosolic nucleotides, phenocopying NLRC3 deficiencies. These data suggest that NLRC3 can interact with novel proteins to facilitate squelching of cellular responses to cytosolic nucleotides.
Nucleotide binding domain, leucine rich repeat CARD containing protein 3 (NLRC3) is a member of the NLR gene family. Members of this family have been associated with human inflammatory diseases such as Crohn’s disease and cryopyrin-associated periodic syndrome. Although NLRC3 (and others) are not associated with human diseases, it is expressed preferentially in the immune system and functions in pathogen recognition. NLRC3 is an intracellular protein involved in the sensing of lipopolysaccharide and cytosolic nucleic acids. NLRC3 is hypothesized to act as a negative regulator in response to bacterial and viral infection, suggesting that the vertebrate immune system has evolved specific inhibitors to limit the inflammatory response. We performed an unbiased yeast two-hybrid screen using an amino terminal fragment of NLRC3 to identify putative interacting proteins that might help elucidate the mechanism by which NLRC3 might inhibit inflammatory responses. To this end, we identified several interacting proteins. One protein in particular acts as a scaffold important in regulating the cytoskeleton, cell adhesion and proliferation. Structure function analysis has localized the domains necessary and sufficient for interacting with NLRC3. Additionally, confocal microscopy demonstrates that these two proteins colocalize in transformed human epithelial cells. This data suggests that NLRC3 interacts specifically with a novel scaffold.
Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I interferon. One of several upstream receptor cGAS (cyclic-GMP-AMP synthase) binds to cytosolic DNA and generates di-cyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to Stimulator of Interferon Genes (STING). STING recruits TANK binding kinase 1 (TBK1) which acts as a critical node that allows for efficient activation of interferon regulatory factors (IRFs) to drive the anti-viral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine rich repeat containing protein (NLR) that negatively regulates the type I interferon pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I interferons. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two hybrid screening. Here we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3:STING interaction in the cytosol of human epithelial cells. Furthermore, knock down of IQGAP1 in THP1 and HeLa cells causes significantly more interferon-b production in response to cytosolic nucleic acids. This result phenocopies NLRC3 deficient macrophages and fibroblasts and shRNA knock down of NLRC3 in THP1 cells. Our findings suggest IQGAP1 is a novel regulator of type I interferon production possibly via interacting with NLRC3 in human monocytic and epithelial cells.
Nucleotide binding and oligomerization domain containing protein 2 (NOD2) is an intracellular protein that is involved in the recognition of bacterial cell wall derived muramyl-dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders including Crohn’s disease and Blau syndrome. NOD2 is a member of the Nucelotide-binding domain and leucine-rich repeat containing protein gene family (NLR). Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by pathogenic NOD2 proteins remains unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl-dipeptide in cellular assay. We demonstrate that nucleotide binding is necessary for wild type and pathogenic NOD2 protein activation of the NF-κB signaling pathway. Mutations of the Walker A motif inhibit binding to RIP2k in wild type NOD2, Crohn’s disease-associated NOD2 and Blau syndrome-associated NOD2. Loss of RIP2k binding correlates with decreased NF-κB-dependent luciferase activity. These data suggest that nucleotide binding is necessary for NOD2 function.
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