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CATERPILLER: A novel gene family important in immunity, cell death, and diseases
Abstract
The newly discovered CATERPILLER (CLR) gene family encodes proteins with a variable but limited number of N-terminal domains, followed by a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). The N-terminal domain consists of transactivation, CARD, Pyrin, or BIR domains, with a minority containing undefined domains. These proteins are remarkably similar in structure to the TIR-NBD-LRR and CC-NBD-LRR disease resistance (R) proteins that mediate immune responses in plants. The NBD-LRR architecture is conserved in plants and vertebrates, but only remnants are found in worms and flies. The CLRs regulate inflammatory and apoptotic responses, and some act as sensors that detect pathogen products. Several CLR genes have been genetically linked to susceptibility to immunologic disorders. We describe prominent family members, including CIITA, CARD4/NOD1, NOD2/CARD15, CIAS1, CARD7/NALP1, and NAIP, in more detail. We also discuss implied roles of these proteins in diversifying immune detection and in providing a check-and-balance during inflammation.
... The consensus design by Mosavi et al., 2002, a design with identical repeats without caps, yields unfolded proteins in inclusion bodies that require refolding [20]. Structure based substitutions of hydrophobic against charged surface residues significantly improve the solubility of the redesigned ankyrins by Mosavi et al. [21]. These molecules are denoted "nANK", where "n" and "ANK" indicate the number of ankyrin repeats, respectively [20], [21]. ...
... Structure based substitutions of hydrophobic against charged surface residues significantly improve the solubility of the redesigned ankyrins by Mosavi et al. [21]. These molecules are denoted "nANK", where "n" and "ANK" indicate the number of ankyrin repeats, respectively [20], [21]. ...
... Consensus sequence alignment with structural features of the ankyrin repeat proteins as published by Binz et al., 2003 [17], Mosavi et al., 2002 [20], Mosavi et al., 2003 [21] and Merz et al., 2008 [23]. Various leucines in the N-Cap (L8R, L20R and L21R) and in the C-Cap (L6R, L15R, L22R) have been replaced by arginines in the TALR sequence [21]. The randomized positions (X), coloured in red, in the consensus sequence designed by Binz et al., 2003 encode for every amino acid except cysteine, glycine and proline; Z within the same amino acid sequence encodes either for histidine asparagine or tyrosine. ...
... Toll-like receptors are transmembrane proteins that are expressed in most cell types, either at the cell surface (TLR1, 2,4,5,6,10) or in endosomes (TLR3, 7,8,9). They can detect a variety of molecules, including proteins, lipopeptides, and nucleic acids (single-stranded RNA, double-stranded RNA, or CpG DNA). ...
... The importance of NLRs in regulating inflammation is highlighted by their evolutionary conservation across vertebrate species and the association of genetic mutations in several NLR genes with autoinflammatory diseases (9). NLRs were previously grouped under the term CATERPILLAR [Caspase-recruitment domain (CARD) transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats] gene family (10). Other research groups have named these proteins NOD-LRR family and NACHT [domain present in NAIP, class II transactivator (CIITA), HET-E, and TP1]-LRR family (8,10). ...
... NLRs were previously grouped under the term CATERPILLAR [Caspase-recruitment domain (CARD) transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats] gene family (10). Other research groups have named these proteins NOD-LRR family and NACHT [domain present in NAIP, class II transactivator (CIITA), HET-E, and TP1]-LRR family (8,10). The study of NLR gene family emerged in the early 2000s following the discovery of their structural similarity to the CIITA, which is the master regulator of MHC class II transcription (11). ...
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) associated with inappropriate activation of lymphocytes, hyperinflammatory responses, demyelination, and neuronal damage. In the past decade, a number of biological immunomodulators have been developed that suppress the peripheral immune responses and slow down the progression of the disease. However, once the inflammation of the CNS has commenced, it can cause serious permanent neuronal damage. Therefore, there is a need for developing novel therapeutic approaches that control and regulate inflammatory responses within the CNS. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are intracellular regulators of inflammation expressed by many cell types within the CNS. They redirect multiple signaling pathways initiated by pathogens and molecules released by injured tissues. NLR family members include positive regulators of inflammation, such as NLRP3 and NLRC4 and anti-inflammatory NLRs, such as NLRX1 and NLRP12. They exert immunomodulatory effect at the level of peripheral immune responses, including antigen recognition and lymphocyte activation and differentiation. Also, NLRs regulate tissue inflammatory responses. Understanding the molecular mechanisms that are placed at the crossroad of innate and adaptive immune responses, such as NLR-dependent pathways, could lead to the discovery of new therapeutic targets. In this review, we provide a summary of the role of NLRs in the pathogenesis of MS. We also summarize how anti-inflammatory NLRs regulate the immune response within the CNS. Finally, we speculate the therapeutic potential of targeting NLRs in MS.
... Computational prediction of phasiRNAs pattern has been a popular method to identify phasiRNAs (Howell et al. 2007;Chen et al. 2007). Many 21-nt pha-siRNAs originating from nucleotide binding (NB) leucinerich repeat (LRR) receptors (NLR) genes, which function in plant defense systems (McHale et al. 2006;Ting and Davis 2005), are identified in Medicago truncatula, tomato and perennial species (Källman et al. 2013;Zhai et al. 2011;Shivaprasad et al. 2012;Fei et al. 2015). The PENTA-TRI-COPEPTIDE REPEAT (PPR) genes (Howell et al. 2007;Xia et al. 2013), MYB transcription factor (TF) genes (Xia et al. 2013(Xia et al. , 2015, AUXIN RESPONSE FACTOR (ARF) genes (Xia et al. 2017) and NAC TF genes (Liu et al. 2017;Ma et al. 2018), among a long list of diverse genes yielding phasiRNAs. ...
... The 21-nt and 24-nt phasiRNAs may be generated from the intergenic non-coding RNAs in maize Recent work identified over 500 loci generating 21-nt pha-siRNA by analyzing 69 small RNA libraries from vegetative and reproductive parts of soybean (Arikit et al. 2014). Results show that 483 loci are overlapped with annotated protein-coding genes including NB-LRR genes, receptorlike kinase-encoding genes, auxin response factor families and MYB/HD-like proteins (McHale et al. 2006;Ting and Davis 2005;Howell et al. 2007;Xia et al. 2013Xia et al. , 2017. Whereas in rice, most phasiRNAs triggered by miR2118 and miR2275 are derived from intergenic clusters (Komiya et al. 2014). ...
Phased secondary small interfering RNAs (phasiRNAs) are generated by double-stranded RNA (dsRNAs), which are cleaved by Dicer as a phase set. Hundreds of 21-nt and 24-nt phasiRNAs have been identified in male reproductive organs of rice and maize. Whereas, the expression profile of phasiRNAs among maize tissues is still exclusive. In this study, to identify 21-nt and 24-nt phasiRNAs among tissues, about 600 million signatures from nine tissues were got including root, seedling, embryo, pollen, anther, immature tassel, immature ear, premature ear and silk. As a result, 269 and 135 21-nt and 24-nt PHAS (phasiRNA precursors) loci were identified, respectively. Interestingly, except male tissues, 21-nt and 24-nt phasiRNAs also were identified in immature ear and silk, respectively, which highly overlapped with those characterized in male reproductive organs. But few phasiRNAs were identified in root, seedling and embryo. 93.4% and 81.3% of 21-nt and 24-nt PHAS loci contained 22-nt motif which matched well with miR2118 and miR2275, respectively. The expression levels of miR2118 and miR2275 in tissues accorded well with that of phasiRNAs. Finally, we found that DCL1 might be the direct Dicer nuclease to promote the maturation of 22-nt miR2118 and miR2275 in maize, because the levels of miR2118 and miR2275 were reduced in seedling and tassel primordia of dcl1 mutants. We provided profiling information of 21-nt and 24-nt phasiRNAs among tissues in maize. It could be helpful to understand the biogenesis of phasiRNAs in maize.
... Of all inflammasomes, NLRP3 with its main constituents NLRP3, ASC, and caspase-1, is the best characterized and studied. Mutations in NLRP3 or its upstream regulators lead to constitutive activation of NLRP3 and are associated with various IL-1β-related autoinflammatory diseases [19][20][21][22][23][24][25]. In addition, hyperactivation of the NLRP3 inflammasome has been linked to harmful inflammation in Alzheimer's disease and metabolic disorders [25]. ...
... Variants in genes encoding constituent proteins of the inflammasome are usually associated with an increased susceptibility to immune disorders in humans. For example, activated variants of NLRP3 and Pyrin are associated with a variety of autoinflammatory diseases that are characterized by excessive production of IL-1β such as familial cold urticaria, Muckle-Wells syndrome, neonatal-onset multisystem inflammatory disease, or familial Mediterranean fever [19][20][21][22]26]. In addition, variants of regulatory proteins of the inflammasome have also been reported. ...
Toll like receptors (TLRs) mediate the recognition of microbial and endogenous insults to orchestrate the inflammatory response. TLRs localize to the plasma membrane or endomembranes, depending on the member, and rely critically on endoplasmic reticulum‐resident chaperones to mature and reach their subcellular destinations. The chaperone canopy FGF signaling regulator 3 (CNPY3) is necessary for the proper trafficking of multiple TLRs including TLR1/2/4/5/9 but not TLR3. However, the exact role of CNPY3 in inflammatory signalling downstream of TLRs has not been studied in detail. Consistent with the reported client specificity, we report here that functional loss of CNPY3 in engineered macrophages impairs downstream signalling by TLR2 but not TLR3. Unexpectedly, CNPY3‐deficient macrophages show reduced interleukin‐1β (IL‐1ß) and IL‐18 processing and production independent of the challenged upstream TLR species, demonstrating a separate, specific role for CNPY3 in inflammasome activation. Mechanistically, we document that CNPY3 regulates caspase‐1 localization to the apoptosis speck and auto‐activation of caspase‐1. Importantly, we were able to recapitulate these findings in macrophages from an early infantile epileptic encephalopathy (EIEE) patient with a novel CNPY3 loss‐of‐function variant. Summarizing, our findings reveal a hitherto unknown, TLR‐independent role of CNPY3 in inflammasome activation, highlighting a more complex and dedicated role of CNPY3 to the inflammatory response than anticipated. This article is protected by copyright. All rights reserved
... There are over 20 identified NLR proteins in humans and over 30 in mice, divided into groups based on structures of the variable N-terminus. The NLRP group of NLRs, for example, contains a pyrin domain at the N-terminus, while the NLRC group contains a caspase activation and recruitment domain (Kanneganti et al., 2007;Ting and Davis, 2005). ...
... As in other PRRs, the defining function of the NLR family is to sense pathogen-or damage-associated molecular patterns (Ting and Davis, 2005;Kanneganti et al., 2007). The NLR family is unique among PRRs in its immense diversity of ligands and downstream effector functions. ...
The gastrointestinal tract harbors a highly complex microbial community, which is referred to as gut microbiota. With increasing evidence suggesting that the imbalance of gut microbiota plays a significant role in the pathogenesis of multiple diseases, interactions between the host immune system and the gut microbiota are now attracting emerging interest. Nucleotide-binding and leucine-rich repeat-containing receptors (NLRs) encompass a large number of innate immune sensors and receptors, which mediate the activation of Caspase-1 and the subsequent release of mature interleukin-1β and interleukin-18. Several family members have been found to restrain rather than activate inflammatory cytokines and immune signaling. NLR family members are central regulators of pathogen recognition, host immunity, and inflammation with utmost importance in human diseases. In this review, we focus on the potential roles played by NLRs in controlling and shaping the microbiota community and discuss how the functional axes interconnecting gut microbiota with NLRs impact the modulation of colitis, inflammatory bowel diseases, and colorectal cancer.
... Three important groups of PAMP receptors are as follows: membrane-bound or secreted C-type lectin-like receptors (CLRs) (McGreal et al., 2004;van Kooyk et al., 2004;Cambi et al., 2005;McGreal et al., 2005), cytoplasmic NACHT-LRR domain receptors (NLRs) Kufer et al., 2005;Martinon and Tschopp, 2005;Ting and Davis, 2005), and transmembrane Toll-like receptors (TLRs) (Dunne and O'Neill, 2005;Hopkins and Sriskandan, 2005;Liew et al., 2005;Takeda and Akira, 2005). NLRs are the members of the so-called CATERPILLER protein family, which can be further subdivided into evolutionarily related neuronal apoptosis-inhibiting proteins (NAIPs), NACHT, LRR, and PYD domain-containing proteins (NALPs, also known as PYPAFs), and nucleotide-binding oligomerization domain-containing proteins (NODs). ...
... They mount an immune response resulting in the removal and destruction of the invading pathogen. Therefore, the impairment of essential signaling cascades by mutant proteins can lead to the dysregulation of human immunity, causing acute or chronic diseases of autoimmunity or immunodeficiency (Beutler, 2004;Ting and Davis, 2005). However, the modification of innate immune responses by therapeutic targeting of the implicated cellular mechanisms may provide new opportunities for clinical treatment of patients Ulevitch, 2004). ...
... NOD1 and NOD2 are located in the cytoplasm and are composed of an LRR ligand-binding domain, an oligomerization domain with NACHT homology, and a caspase recruitment domain (CARD) that transmits the signal. Despite these significant advances and improvements in the understanding of the genetic basis for these receptors, their importance in NCDs and cancer could be underscored by the fact that single-nucleotide polymorphisms (SNPs) occurring in different subpopulations of patients have been barely elucidated [30,31] NOD1/2 and NOD-like receptors (NLRs) belong to the superclass of pattern recognition receptors (i.e., PAMPs, DAMPs), among which are also the innate immune 'Toll-like' receptors (TLRs). Pattern recognition receptors play key roles in determining the function and activity of innate immune effectors. ...
Nucleotide-binding oligomerization domain-like (NOD) receptors rely on the interface between immunity and metabolism. Dietary factors constitute critical players in the activation of innate immunity and modulation of the gut microbiota. The latter have been involved in worsening or improving the control and promotion of diseases such as obesity, type 2 diabetes, metabolic syndrome, diseases known as non-communicable metabolic diseases (NCDs), and the risk of developing cancer. Intracellular NODs play key coordinated actions with innate immune ‘Toll-like’ receptors leading to a diverse array of gene expressions that initiate inflammatory and immune responses. There has been an improvement in the understanding of the molecular and genetic implications of these receptors in, among others, such aspects as resting energy expenditure, insulin resistance, and cell proliferation. Genetic factors and polymorphisms of the receptors are determinants of the risk and severity of NCDs and cancer, and it is conceivable that dietary factors may have significant differential consequences depending on them. Host factors are difficult to influence, while environmental factors are predominant and approachable with a preventive and/or therapeutic intention in obesity, T2D, and cancer. However, beyond the recognition of the activation of NODs by peptidoglycan as its prototypical agonist, the underlying molecular response(s) and its consequences on these diseases remain ill-defined. Metabolic (re)programming is a hallmark of NCDs and cancer in which nutritional strategies might play a key role in preventing the unprecedented expansion of these diseases. A better understanding of the participation and effects of immunonutritional dietary ingredients can boost integrative knowledge fostering interdisciplinary science between nutritional precision and personalized medicine against cancer. This review summarizes the current evidence concerning the relationship(s) and consequences of NODs on immune and metabolic health.
... Caspases-1 and -11 are inflammatory caspases that are recruited into inflammasome SMOCs in response to the cytosolic presence of pathogen-derived signals or molecules (Broz and Dixit, 2016;Lamkanfi and Dixit, 2014). Inflammatory caspases are zymogens that contain a C-terminal enzymatic domain and an N-terminal caspase activation and recruitment domain (CARD), which promotes SMOC formation through homotypic interactions with other CARD-containing adapter proteins as well as homodimerization with each other (Lu et al., 2014;Park et al., 2007;Ting and Davis, 2005). The C-terminal enzymatic domain of caspases is comprised of large (17-20 kDa) and small (10-12 kDa) subunits separated by an interdomain linker (IDL), which contains the self-cleavage site that undergoes autoprocessing following caspase oligomerization (Ross et al., 2022;Walker et al., 1994;Wilson et al., 1994). ...
Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammasomes. Inflammasomes recruit caspases to undergo proximity-induced autoprocessing into an enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high-molecular-weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Expression of fluorescently labeled Casp11 in macrophages revealed that cytosolic LPS induced Casp11 speck formation. Unexpectedly, catalytic activity and autoprocessing were required for Casp11 to form LPS-induced specks in macrophages. Furthermore, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and processing of Casp11 via ectopically expressed TEV protease was sufficient to induce Casp11 speck formation. These data reveal a previously undescribed role for Casp11 catalytic activity and autoprocessing in noncanonical inflammasome assembly, and shed new light on the molecular requirements for noncanonical inflammasome assembly in response to cytosolic LPS.
... The second is C-terminal, which inhibits the function of the NLR protein when leucine-rich repeats (LRRs) are inactivated or in a resting state and adjusts the conformation following the recognition of stimuli to eliminate the inhibitory effect on the NLR protein [19]. The third component is the N-terminal effector domain made up of either pyrin, CARD, or the baculoviral inhibitor of apoptosis protein repeat (BIR) domain before the NACHT domain [20]. Moreover, the NLRs can be further sub-classified into two groups based on the N-terminal domain. ...
In this narrative review, we present the evidence on nucleotide-binding and oligomerization (NOD) domain-like receptor (NLR) family pyrin domain (PYD)-containing 3 (NLRP3) inflammasome activation for its putative roles in the elusive pathomechanism of aging-related cerebral small vessel disease (CSVD). Although NLRP3 inflammasome-interleukin (IL)-1β has been implicated in the pathophysiology of coronary artery disease, its roles in cerebral arteriothrombotic micro-circulation disease such as CSVD remains unexplored. Here, we elaborate on the current manifestations of CSVD and its’ complex pathogenesis and relate the array of activators and aberrant activation involving NLRP3 inflammasome with this condition. These neuroinflammatory insights would expand on our current understanding of CSVD clinical (and subclinical) heterogenous manifestations whilst highlighting plausible NLRP3-linked therapeutic targets.
... In the 1990s, a large family of conserved genes that encode proteins characterized by nucleotide binding domain and leucine rich repeats (NBD-LRRs) was discovered in the animal kingdom. In human, NLR genes were named variously such as CATERPILLER [11,12], NODs [13], NOD-LRR [14,15], NACHT-LRR, [16] and NOD-like receptor [17,18], until the introduction of the standard nomenclature "NLR'' in 2008 after the approval of the Human Genome Organisation (HUGO) Gene Nomenclature Committee and the Mouse Genomic Nomenclature Committee [19]. In teleosts, NLRs were first reported in 2008 by Laing et al. in zebrafish [20]. ...
Nucleotide-binding oligomerization domain-like receptors (NOD-like receptors or NLRs) are key members of the immune system that act as intracellular sentinels. These pathogen recognition receptors are essentially characterized by a central nucleotide binding domain and a C-terminal leucine rich repeat domain responsible for recognition of pathogens. Over the past decade, our understanding of teleosts' NLRs has enhanced significantly although the signaling pathways remain to be elucidated. In this brief review, we have tried to decipher the structural and functional aspects of NLRs in teleost. The review also engages in illustrating the various downstream signaling pathways/molecules reported so far in fishes that enable the NLRs to act as important players in immune responses and defense mechanisms against pathogens. Importantly, we try to explore the lacunae in structural and mechanistic details of NLRs in the teleost that would help in identifying key areas in which research is needed to complete our understanding of NLRs and their structural and functional evolution.
... Interestingly, NOD1 can respond to LPS stimulation similar to the response of plant R protein receptors to pathogenic microbial components, which play key signal transduction roles in immune signalling pathways [56]. Further research revealed that NOD1 can be activated by the PGN fragment DAP (γ-D-glutamyl-meso-diaminopimelic acid) which exists on the cytoplasm of Gram-negative bacteria and certain Gram-positive bacteria, while NOD2 recognizes the Gram-negative PGN fragment MDP (muramyl dipeptide) which exists in both Gram-negative bacteria and Gram-positive bacteria [57][58][59][60]. Oxidative Medicine and Cellular Longevity receptor proteins have been confirmed to assemble into inflammasomes, including the nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (LRR) sequence protein members (NLRP1 [62], NLRP3 [63], NLRC4 [64,65]), AIM2 (melanoma deficiency factor) [66], and pyrin [67,68] (Figure 3). ...
Eukaryotic cells can initiate several distinct self-destruction mechanisms to display essential roles for the homeostasis maintenance, development, and survival of an organism. Pyroptosis, a key response mode in innate immunity, also referred to as caspase-1-dependent proinflammatory programmed necrotic cell death activated by human caspase-1/4/5, or mouse caspase-1/11, plays indispensable roles in response to cytoplasmic insults and immune defense against infectious diseases. These inflammatory caspases are employed by the host to eliminate pathogen infections such as bacteria, viruses, protozoans, and fungi. Gasdermin D requires to be cleaved and activated by these inflammatory caspases to trigger the pyroptosis process. Physiological rupture of cells results in the release of proinflammatory cytokines, the alarmins IL-1β and IL-18, symbolizing the inflammatory potential of pyroptosis. Moreover, long noncoding RNAs play direct or indirect roles in the upstream of the pyroptosis trigger pathway. Here, we review in detail recently acquired insights into the central roles of inflammatory caspases, inflammasomes, and pyroptosis, as well as the crosstalk between pyroptosis and long noncoding RNAs in mediating infection immunity and pathogen clearance.
... Biological studies have shown that the number of NLR receptors expressed in a given species is markedly variable. Because orthologs of NLRs have not been found in Drosophila and C. elegans, it is first thought that the NLR family originated in teleost fish [55,56]. However, analysis of the sea urchin genome sequence revealed that more than 200 NLR-like genes are present in this animal [57][58][59]. ...
Many members of the nucleotide-binding and oligomerization domain (NACHT)- and leucine-rich-repeat-containing protein (NLR) family play crucial roles in pathogen recognition and innate immune response regulation. In our previous work, a unique and Vibrio splendidus -inducible NLRC4 receptor comprising Ig and NACHT domains was identified from the sea cucumber Apostichopus japonicus , and this receptor lacked the CARD and LRR domains that are typical of common cytoplasmic NLRs. To better understand the functional role of AjNLRC4, we confirmed that AjNLRC4 was a bona fide membrane PRR with two transmembrane structures. AjNLRC4 was able to directly bind microbes and polysaccharides via its extracellular Ig domain and agglutinate a variety of microbes in a Ca ²⁺ -dependent manner. Knockdown of AjNLRC4 by RNA interference and blockade of AjNLRC4 by antibodies in coelomocytes both could significantly inhibit the phagocytic activity and elimination of V . splendidus . Conversely, overexpression of AjNLRC4 enhanced the phagocytic activity of V . splendidus , and this effect could be specifically blocked by treatment with the actin-mediated endocytosis inhibitor cytochalasin D but not other endocytosis inhibitors. Moreover, AjNLRC4-mediated phagocytic activity was dependent on the interaction between the intracellular domain of AjNLRC4 and the β-actin protein and further regulated the Arp2/3 complex to mediate the rearrangement of the cytoskeleton and the polymerization of F-actin. V . splendidus was found to be colocalized with lysosomes in coelomocytes, and the bacterial quantities were increased after injection of chloroquine, a lysosome inhibitor. Collectively, these results suggested that AjNLRC4 served as a novel membrane PRR in mediating coelomocyte phagocytosis and further clearing intracellular Vibrio through the AjNLRC4-β-actin-Arp2/3 complex-lysosome pathway.
... [6] Thus, the first one of the plant immune system is transmembrane pattern recognition receptors (PRRs) that react step by step creating microbial or pathogen-associated molecular patterns (MAMPS or PAMPs), like IDA or flg22, and another one works within the cell by considering all things, using the polymorphic NB-LRR protein things scrambled by most R genes. [5,7] ...
Pattern Triggered Immunity (PTI) is distinguished with the activities of pattern recognition receptors (PRRs), which play an essential role in plant defense mechanism. During aggression of microbes, PRRs immediately bind with the PAMPs and recruit co-receptors to initiate the defense signal. Several plant PRRs have been discovered, very few of their functional parameters have been studied. In this study, the crystallographic structures of FLS2-flg22-BAK1 (PDB ID: 4MN8), HAESA-IDA-SERK1 (PDB ID: 5IYX) and PSKR-Phytosulphokine-SERK1 (PDB ID: 4Z64) complexes were simulated for 30ns. Simulated trajectories were analyzed for getting an overview of the immune response of PRRs towards PAMPs with the help of co-receptors. Moreover, MM/PBSA calculation revealed that interaction between FLS2-BAK1 and HAESA-SERK1 show similarity whereas PSKR interacts differently with SERK1. As PRRs play a major role in plant
defense mechanism, it can be hypothesized that binding mechanism with PAMPs and co-receptors will help to understand the interaction pattern of PTI.
... The four best studied families of PRRs are Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), and C-type lectins (CTLs) (6,7). While it was originally thought that many of these PRR families first arose in vertebrates, studies following the advent of next generation sequencing revealed that TLRs, RLRs, NLRs, and CTLs are present in basal metazoans and thus are evolutionarily ancient (8)(9)(10)(11)(12). Studying PRRs in basal taxa informs our understanding of PRR evolution and more broadly immune evolution. ...
Pattern recognition receptors (PRRs) are evolutionarily ancient and crucial components of innate immunity, recognizing danger-associated molecular patterns (DAMPs) and activating host defenses. Basal non-bilaterian animals such as cnidarians must rely solely on innate immunity to defend themselves from pathogens. By investigating cnidarian PRR repertoires we can gain insight into the evolution of innate immunity in these basal animals. Here we utilize the increasing amount of available genomic resources within Cnidaria to survey the PRR repertoires and downstream immune pathway completeness within 15 cnidarian species spanning two major cnidarian clades, Anthozoa and Medusozoa. Overall, we find that anthozoans possess prototypical PRRs, while medusozoans appear to lack these immune proteins. Additionally, anthozoans consistently had higher numbers of PRRs across all four classes relative to medusozoans, a trend largely driven by expansions in NOD-like receptors and C-type lectins. Symbiotic, sessile, and colonial cnidarians also have expanded PRR repertoires relative to their non-symbiotic, mobile, and solitary counterparts. Interestingly, cnidarians seem to lack key components of mammalian innate immune pathways, though similar to PRR numbers, anthozoans possess more complete immune pathways than medusozoans. Together, our data indicate that anthozoans have greater immune specificity than medusozoans, which we hypothesize to be due to life history traits common within Anthozoa. Overall, this investigation reveals important insights into the evolution of innate immune proteins within these basal animals.
... The NACHT domain has ATPase activity and is considered essential for NLRP3 oligomerisation Kelley et al., 2019). c) An N-terminal effector domain consisting of either a CARD, Pyrin or baculoviral inhibitor of apoptosis protein repeat (BIR) domain before the NACHT domain (Ting and Davis, 2005). Furthermore, NLRs can be sub-classified into two groups based on the N-terminal domain: a) NLRP containing pyrin and b) NOD-like receptor subfamily C (NLRC), which contains CARD (Moossavi et al., 2018). ...
Cerebrovascular diseases are pathological conditions involving impaired blood flow in the brain, primarily including ischaemic stroke, intracranial haemorrhage, and subarachnoid haemorrhage. The nucleotide-binding and oligomerisation (NOD) domain-like receptor (NLR) family pyrin domain (PYD)-containing 3 (NLRP3) inflammasome is a protein complex and a vital component of the immune system. Emerging evidence has indicated that the NLRP3 inflammasome plays an important role in cerebrovascular diseases. The function of the NLRP3 inflammasome in the pathogenesis of cerebrovascular diseases remains an interesting field of research. In this review, we first summarised the pathological mechanism of cerebrovascular diseases and the pathological mechanism of the NLRP3 inflammasome in aggravating atherosclerosis and cerebrovascular diseases. Second, we outlined signalling pathways through which the NLRP3 inflammasome participates in aggravating or mitigating cerebrovascular diseases. Reactive oxygen species (ROS)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), ROS/thioredoxin-interacting protein (TXNIP) and purinergic receptor-7 (P2X7R) signalling pathways can activate the NLRP3 inflammasome; activation of the NLRP3 inflammasome can aggravate cerebrovascular diseases by mediating apoptosis and pyroptosis. Autophagy/mitochondrial autophagy, nuclear factor E2-related factor-2 (Nrf2), interferon (IFN)-β, sirtuin (SIRT), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) reportedly alleviate cerebrovascular diseases by inhibiting NLRP3 inflammasome activation. Finally, we explored specific inhibitors of the NLRP3 inflammasome based on the two-step activation of the NLRP3 inflammasome, which can be developed as new drugs to treat cerebrovascular diseases.
... NOD-like receptors (NLRs) are another important group of PRRs and a total of 22 human NLR proteins have been discovered (25), many of which remain to be functionally characterized. NLRs consist of an N-terminal effector domain, a central NACHT domain, and a C-terminal leucine-rich repeat region (LRRs), and can be subcategorized by their effector domains into pyrin-domain (PYD) containing NLRP proteins, CARD-domain containing NLRC proteins, baculovirus inhibitor of apoptosis (BIR)-domain containing NLRB proteins, and CARD-transcriptional activation-domain (CARD-AD) containing NLRA proteins (26). ...
Tight regulation of inflammatory cytokine and interferon (IFN) production in innate immunity is pivotal for optimal control of pathogens and avoidance of immunopathology. The human Nod-like receptor (NLR) NLRP11 has been shown to regulate type I IFN and pro-inflammatory cytokine responses. Here, we identified the ATP-dependent RNA helicase DDX3X as a novel binding partner of NLRP11, using 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 IKKϵ-mediated phosphorylation of DDX3X, resulting in lower type I IFN induction upon viral infection. These effects were dependent on the 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.
... Researchers have speculated that plant and animal NLR genes originated from the same common ancestor (85,171). The NBD is highly conserved across taxa and likely has prokaryotic origins. ...
Nucleotide-binding domain leucine-rich repeat receptors (NLRs) play important roles in the innate immune systems of both plants and animals. Recent breakthroughs in NLR biochemistry and biophysics have revolutionized our understanding of how NLR proteins function in plant immunity. In this review, we summarize the latest findings in plant NLR biology and draw direct comparisons to NLRs of animals. We discuss different mechanisms by which NLRs recognize their ligands in plants and animals. The discovery of plant NLR resistosomes that assemble in a comparable way to animal inflammasomes reinforces the striking similarities between the formation of plant and animal NLR complexes. Furthermore, we discuss the mechanisms by which plant NLRs mediate immune responses and draw comparisons to similar mechanisms identified in animals. Finally, we summarize the current knowledge of the complex genetic architecture formed by NLRs in plants and animals and the roles of NLRs beyond pathogen detection.
Expected final online publication date for the Annual Review of Plant Biology, Volume 72 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... NLRs are characterized by a common tripartite motif, consisting of a central nucleotide binding and oligomerization (NACHT) domain, C-terminal leucine rich repeats (LRRs) and a variable N-terminal effector domain. According to their effector domain, NLRs are categorized into different subgroups: CARD-transcription and activation domain (CARD-AD) containing NLRA, baculovirus inhibitor of apoptosis (BIR) domain carrying NLRB, caspase activation and recruitment domain (CARD) containing NLRC and pyrin domain (PYD) containing NLRP [104]. NLRX1 contains an unconventional N-terminal domain, which shares no homology with the N-terminal domains of the other protein-family members. ...
Type I interferon signaling contributes to the development of innate and adaptive immune responses to either viruses, fungi, or bacteria. However, amplitude and timing of the interferon response is of utmost importance for preventing an underwhelming outcome, or tissue damage. While several pathogens evolved strategies for disturbing the quality of interferon signaling, there is growing evidence that this pathway can be regulated by several members of the Nod-like receptor (NLR) family, although the precise mechanism for most of these remains elusive. NLRs consist of a family of about 20 proteins in mammals, which are capable of sensing microbial products as well as endogenous signals related to tissue injury. Here we provide an overview of our current understanding of the function of those NLRs in type I interferon responses with a focus on viral infections. We discuss how NLR-mediated type I interferon regulation can influence the development of auto-immunity and the immune response to infection.
... CIITA is the founding member of the NLR protein family [7]. The nucleotide-binding domain also has been described as the NACHT (NAIP, CIITA, HET-E, TP1) domain and the protein family as the CATERPILLER (caspase activation and recruitment domain (CARD), transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats) family [8][9][10]. CIITA was, however, for a very long time, the most exceptional member of this protein family in that it was the only transcriptional regulator. ...
In this review, we discuss the major histocompatibility complex (MHC) class II transactivator (CIITA), which is the master regulator of MHC class II gene expression. CIITA is the founding member of the mammalian nucleotide-binding and leucine-rich-repeat (NLR) protein family but stood apart for a long time as the only transcriptional regulator. More recently, it was found that its closest homolog, NLRC5 (NLR protein caspase activation and recruitment domain (CARD)-containing 5), is a regulator of MHC-I gene expression. Both act as non-DNA-binding activators through multiple protein–protein interactions with an MHC enhanceosome complex that binds cooperatively to a highly conserved combinatorial cis-acting module. Thus, the regulation of MHC-II expression is regulated largely through the differential expression of CIITA. In addition to the well-defined role of CIITA in MHC-II GENE regulation, we will discuss several other aspects of CIITA functions, such as its role in cancer, its role as a viral restriction element contributing to intrinsic immunity, and lastly, its very recently discovered role as an inhibitor of Ebola and SARS-Cov-2 virus replication. We will briefly touch upon the recently discovered role of NLRP3 as a transcriptional regulator, which suggests that transcriptional regulation is, after all, not such an unusual feature for NLR proteins.
... [6] Thus, the first one of the plant immune system is transmembrane pattern recognition receptors (PRRs) that react step by step creating microbial or pathogen-associated molecular patterns (MAMPS or PAMPs), like flagellin, and the second one works inside the cell by considering all things, using the polymorphic NB-LRR protein things encoded by most R genes. [5,7] ...
Pattern Triggered Immunity (PTI) is actually identified by the activities of pattern recognition receptors (PRRs), which play the most important role in first layer of plant defense mechanism. During aggression of microbes, PRRs immediately bind with the pathogen-associated molecular patterns (PAMPs) and recruit co-receptor protein(s) to activate the defense signal and begin plant's immunity. Although several plant PRRs have been discovered, very few of them have been fully characterized and their functional parameters studied. In this study, the crystallographic structure of HAESA-IDA-SERk1 (PDB ID: 5IYX) complex was simulated for 30 ns in different stages like there were five different combinations of the main crystallographic structure. For every combination 30ns simulated trajectories were analyzed for getting an overview of the interaction and immune response of HAESA towards IDA with the help of co-receptor SERk1. Analyzing the interaction revealed its remarkable resemblance to the other crystallographic structures, which are also a member of the same LRR-RK subfamily (leucine-rich repeat – receptor kinase subfamily XII). Furthermore, it was observed that Tyr56 and Arg67 of IDA contributed significantly to the formation of the HAESA-IDA complex. It can thus be predicted that any change to the PAMP at these positions can be greatly detrimental to the plant, leading to the PRR losing its ability to recognize the PAMP. Moreover, from MM/PBSA analysis it is found that binding energy between HAESA and IDA is more favourable than HAESA and SERk1. -636.824 kJ/mol binding energy is found when co-receptor SERk1 is present inside the complex and during its absence the binding energy between HAESA and IDA goes higher. Again, a notable binding energy between IDA and SERk1 is obtained from this analysis. There are several prominent residues found from HAESA and SERk1, mutation at any of these residues will affect on PTI of Arabidopsis thaliana. Since HAESA has been shown to play a key role in Arabidopsis thaliana plant defense mechanism, its hypothesized binding mechanism with IDA and co-receptor SERk1 will help paint a better understanding of the inceptive stages of PTI.
... Toll-like receptor (TLR) signaling pathways, especially the TLR4-MyD88-dependent pathway, and nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathways play an essential role in defense against Salmonella infection [8][9][10]. TLR4 is known to sense extracellular lipopolysaccharide (LPS) from Gram-negative bacteria, whereas NLRs are known to sense intracellular bacteria [11,12]. The recognition of pathogen-associated molecular patterns by TLRs or NODs initiates signal transduction pathways that induce the expression of cytokines [13,14]. ...
Salmonellosis is a zoonosis that is not only harmful to the health of poultry but also poses a threat to human health. Although many measures have been put in place to reduce morbidity, they have not provided satisfactory results. Therefore, it is necessary to clarify the immune mechanisms involved in improving the resistance of chickens against Salmonella. BTB (Broad-complex Tramtrack and Bric-a-brac) Speckle-type POZ (poxvirus and zinc finger) protein (SPOP) regulates protein expression by promoting substrate ubiquitination and degradation. The correlation between SPOP expression and the immune response has not been fully described. Therefore, the aim of this study was to clarify this relationship. In vitro, we stimulated chicken macrophage cells (HD11) with lipopolysaccharide, then analyzed the correlation between SPOP and IL1β or IL8 expression using quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, we infected 7-days-old chickens with Salmonella Typhimurium, then analyzed the association between SPOP expression and the immune response, including IL1β and IL8 expression, IgA production, and bacterial loads. We found that SPOP may participate in the regulation of the immune response in macrophage cells. SPOP expression was negatively correlated with IL-1β and IL-8 expression both in vivo and in vitro. SPOP expression was also negatively related to bacterial loads and immunoglobulin (Ig) A production. These results indicate that SPOP may have important functions in the response to Salmonella infection.
... The second mode of defense mostly acts internally using the polymorphic NB-LRR protein products encoded by most R genes (Dangl 2004). As the name indicated they are characterized by the presence of two important domains, nucleotide binding (NB) and leucine rich repeat (LRR) that are broadly related to animal CATERPILLER/NOD/NLR proteins (Ting and Davis 2005). NB-LRR proteins have the capability to recognize pathogen from diverse kingdoms and activate defense responses. ...
Plants are the ultimate producers of this ecosystem and effect human life directly and/or indirectly. However, being sessile organisms, plants are susceptible to several adverse environmental conditions and pathogen ingress resulting in huge losses to yield and productivity. Therefore, plans are continuously evolving complex regulatory networks to respond to these environmental changes. Due to its impact on pre- and post-harvest losses, biotic stress is of great concern to plant scientists. Interpreting the underlying mechanism of plant response to biotic stresses is therefore of great importance. A brief account about these hostile conditions and plants responses towards them is described in this review.
... Therefore, they hold the view that NLRC5 is an indispensable component in the secretion of inflammasome dependent inflammatory cytokine. It has also been suggested that NLRC5 cooperates with NLRP3 to reconstitute inflammasome activity in an ectopic system (Ting and Davis, 2005). Besides, NLRC5 is widely expressed in tissues with bacterial and viral infections, and it can also induce expression of inflammasome-related proteins (Livak and Schmittgen, 2001). ...
Innate immunity activates the corresponding immune response relying on multiple pattern recognition receptors (PRRs) that includes pattern recognition receptors (PRRs), like NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs), which could accurately recognize invasive pathogens. In particular, NLRs belong to a large protein family of pattern recognition receptors in the cytoplasm, where they are highly correlated with activation of inflammatory response system followed by rapid clearance of invasive pathogens. Among the NLRs family, NLRC5, also known as NOD4 or NOD27, accounts for a large proportion and involves in immune responses far and wide. Notably, in the above response case of inflammation, the expression of NLRC5 remarkably increased in immune cells and immune-related tissues. However, the evidence for higher expression of NLRC5 in immune disease still remains controversial. It is noted that the growing evidence further accounts for the participation of NLRC5 in the innate immune response and inflammatory diseases. Moreover, NLRC5 has also been confirmed to exert a critical role in the control of regulatory diverse signaling pathways. Together with its broad participation in the occurrence and development of immune diseases, NLRC5 can be consequently treated as a potential therapeutic target. Nevertheless, the paucity of absolute understanding of intrinsic characteristics and underlying mechanisms of NLRC5 still make it hard to develop targeting drugs. Therefore, current summary about NLRC5 information is indispensable. Herein, current knowledge of NLRC5 is summarized, and research advances in terms of NLRC5 in characteristics, biological function, and regulatory mechanisms are reviewed.
... With regard to immunotherapy, members of the toll-like receptor family (TLRs) of the innate immune system have been extensively investigated in oncogenesis (Braunstein et al. 2018). Additionally, a second protein family has been discovered: The NLR/NOD/Caterpillar family, which is equally attributed to the innate immune system (Ting and Davis 2005). ...
Purpose
NOD1 and NOD2 (nucleotide-binding oligomerization domain)—receptors are intracellular receptors and belong to the family of pattern recognition receptors being present in both human and murine renal tubular cells. Besides, NOD1 has been proved to promote apoptosis, upon its overexpression. Hence, we aimed to investigate NOD1 and NOD2 expression in human clear cell renal cell carcinoma (ccRCC).
Methods
Tumor and corresponding adjacent healthy tissues from 41 patients with histopathological diagnosis of ccRCC as well as primary isolated renal tubular epithelial cells (TECs) and tumor tissue from a murine xenograft model using CAKI-1 ccRCC cells were analyzed.
Results
NOD1 and NOD2 mRNA was constitutively expressed in both tumor and adjacent healthy renal tissue, with NOD1 being significantly lower and in contrast NOD2 significantly higher expressed in tumor tissue compared to healthy tissues. Immunohistochemically, NOD1 was located not only in the cytoplasm, but also in the nucleus in ccRCC tissue whereas NOD2 was solely localized in the cytoplasm in both human ccRCC as well as in the healthy tubular system. Focusing on the vasculature, NOD2 displayed broader expression than NOD1. In primary TECs as well as CAKI-1 cells NOD1 and NOD2 was constitutively expressed and increasable upon LPS stimulation. In the mouse xenograft model, human NOD1 mRNA was significantly higher expressed compared to NOD2. In contrast hereto, we observed a shift towards lower mouse NOD1 compared to NOD2 mRNA expression.
Conclusion
In view of reduced apoptosis-associated NOD1 expression in ccRCC tissue opposed to higher expression of NOD2 in tumor vasculature, inducibility of NOD expression in TECs as well as the detected shift of NOD1 and NOD2 expression in the mouse xenograft model, modulation of NOD receptors might, therefore, provide a molecular therapeutic approach in ccRCC.
... They can recognize intracellular pathogens and dangerous signals to initiate innate immune responses [22,31]. Most members of the NLRs family members contain three distinct functional domains: an N-terminal effect binding domain (EBD), a central nucleotide binding domain (NBD), and a leucinerich repeats (LRRs) [32]. The majority of the NLRs family functions as inflammatory promoters. ...
The nucleotide-oligomerization domain (NOD)-like receptor subfamily C3 (NLRC3) is a newly discovered and incompletely characterized member of the NLR family which negatively regulates inflammatory responses. Inflammation is considered a critical pathogenesis in pulmonary hypertension (PH). This is the first study to hypothesize that NLRC3 is closely correlated with PH. Total of 43 PH patients who were diagnosed by right heart catheterization (RHC) and 20 age-matched healthy control subjects were included. Echocardiographic variables and blood biochemical parameters were tested. Results of World Health Organization functional class (WHOFC), Borg dyspnea score and 6-minute walk tests (6MWT) were recorded. Mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance (PVR) were measured from RHC. Serum NLRC3 concentrations were detected by ELISA. ROC curve analysis was used to evaluate the diagnostic value of NLRC3 concentrations in PH. We found that serum NLRC3 concentration was significantly decreased in PH compared to the healthy control group. Serum NLRC3 concentration correlated negatively with mPAP and PVR. In addition, a negative correlation between serum NLRC3 concentration and WHOFC were detected. We proposed a cut-off value of 2.897ng/mL for serum NLRC3 concentration which was able to predict PH with 88% sensitivity and 85% specificity. In conclusion, NLRC3 concentrations in PH were significantly decreased, suggesting that NLRC3 may potentially be a diagnosis index and represent a prognostic factor for PH patients.
... Plants and animals diverged from a common unicellular ancestor~1.6-1.8 billion years ago [139,140]. Nevertheless, the similarity of the NLRs of metazoans and plant NLR resistance proteins (R-proteins) was noted soon after their discovery, which led to the hypothesis that they evolved from a common ancestor [141,142]. However, this hypothesis was recently rejected, with the suggestion that the domain architecture of NLRs evolved at least twice [143]. ...
... Plants and animals diverged from a common unicellular ancestor ~1.6-1.8 billion years ago [139,140]. Nevertheless, the similarity of the NLRs of metazoans and plant NLR resistance proteins (R-proteins) was noted soon after their discovery, which led to the hypothesis that they evolved from a common ancestor [141,142]. However, this hypothesis was recently rejected, with the suggestion that the domain architecture of NLRs evolved at least twice [143]. ...
NLR (NOD-like receptor) genes belong to one of the largest gene families in plants. Their role in plants’ resistance to pathogens has been clearly described for many members of this gene family, and dysregulation or overexpression of some of these genes has been shown to induce an autoimmunity state that strongly affects plant growth and yield. For this reason, these genes have to be tightly regulated in their expression and activity, and several regulatory mechanisms are described here that tune their gene expression and protein levels. This gene family is subjected to rapid evolution, and to maintain diversity at NLRs, a plethora of genetic mechanisms have been identified as sources of variation. Interestingly, regulation of gene expression and evolution of this gene family are two strictly interconnected aspects. Indeed, some examples have been reported in which mechanisms of gene expression regulation have roles in promotion of the evolution of this gene family. Moreover, co-evolution of the NLR gene family and other gene families devoted to their control has been recently demonstrated, as in the case of miRNAs.
... The corrected paragraph should read: In mammals, NLRs are able to recognize bacterial flagella, lipopolysaccharide, RNA, and muramyl dipeptides in the cytoplasm (Franchi et al., 2009). NLRs can be divided into five subfamilies according to the difference in the N-terminal effector domain (Ting and Davis, 2005). Class II major histocompatibility complex (MHC-II) transactivator (CIITA), discovered in 1985-1986(Accolla et al., 1985, 1986, and contains an acidic transcriptional activation motif in the N-terminal domain, so it belongs to the NLRA subfamily. ...
[This corrects the article on p. 1629 in vol. 8, PMID: 28878764.].
Phased secondary small interfering RNAs (phasiRNAs) are generated by double-stranded RNA (dsRNAs), which are cleaved by Dicer as a phase set. So far, hundreds of 21-nt and 24-nt phasiRNAs have been identified in male reproductive organs of rice and maize. Whereas, the expression profile of phasiRNAs among maize tissues is still exclusive. In this study, to identify 21-nt and 24-nt phasiRNAs among tissues, about 600 million signatures from nine tissues were got including root, seedling, embryo, pollen, anther, immature tassel, immature ear, premature ear and silk. As a result, 269 and 135 21-nt and 24-nt PHAS (phasiRNA precursors) loci were identified, respectively. Interestingly, except male tissues, 21-nt and 24-nt phasiRNAs also were identified in immature ear and silk, respectively, which highly overlapped with those characterized in male reproductive organs. But few phasiRNAs were identified in root, seedling and embryo. 93.4% and 81.3% of 21-nt and 24-nt PHAS loci contained 22-nt motif which matched well with miR2118 and miR2275, respectively. The expression levels of miR2118 and miR2275 in tissues accorded well with that of phasiRNAs. Finally, we found that DCL1 might be the direct Dicer nuclease to promote the maturation of 22-nt miR2118 and miR2275 in maize, because the levels of miR2118 and miR2275 were reduced in seedling and tassel primordia of dcl1 mutants. We provided profiling information of 21-nt and 24-nt phasiRNAs among tissues in maize. It could be helpful to understand the biogenesis of phasiRNAs in maize.
Le sepsis reste une cause importante de mortalité dans le monde. Le système immunitaire joue un rôle central dans la réponse à l'infection. L'état d'inflammation initial, déclenché par la réponse immunitaire innée, peut évoluer vers une auto-amplification incontrôlée et des défaillances d'organes. Parallèlement, afin d'en assurer le contrôle, une réponse négative anti-inflammatoire se met en place pouvant conduire à un état d'immunosuppression, pourvoyeur d'infections secondaires et de mortalité en soins intensifs. Cet état se caractérise par un déficit quantitatif et qualitatif de la réponse immunitaire. L'expression monocytaire de HLA-DR (mHLA-DR), molécule du CMH de classe II présentant l'antigène aux lymphocytes T, est à ce jour un marqueur pertinent de l'état d'immunosuppression induite par le sepsis. Son expression est régulée principalement par le transactivateur de classe II, CIITA, lui-même contrôlé au niveau transcriptionnel par une région hautement conservée située dans le promoteur. La mise en évidence dans cette région promotrice de polymorphismes génétiques, ou SNPs (Single Nucleotide Polymorphism), impliqués dans des maladies inflammatoires, ainsi que des preuves d'une sélection positive dans la région 5' du gène, nous ont permis de cibler le SNP G-286A*rs3087456.Dans la première partie de notre travail, nous avons démontré le rôle du SNP dans la diminution de l'activité du promoteur III de CIITA, la récupération de celle-ci après stimulation par IFN-γ, et son rôle dans la mortalité des patients en choc septique en lien avec la diminution de mHLA-DR.Dans la suite de notre travail, nous avons présenté des données de sécurité et d'efficacité du traitement par IFN-γ chez des patients atteints d'immunosuppression induite par le sepsis. De plus, nous avons validé une technique de mesure par cytométrie de flux de mHLA-DR au lit du malade sur des faibles volumes de sang frais. Nous avons ensuite décrit les phénotypes myéloïdes et leurs rôles dans l'immunosuppression chez une série de cas graves de patients atteints de pneumopathie à COVID-19 se compliquant de sepsis bactérien. Enfin, nous avons réalisé une étude sur les biomarqueurs immunologiques d'infections associées aux soins chez des patients pédiatriques transplantés du foie, dont une partie des expériences et les analyses sont encore en cours.
Background: Generalized vitiligo is a common, multifaceted, polygenic condition in which autoimmune loss of melanocytes results in depigmented skin patches, overlying hair and mucous membranes. NLRP1 has been proposed to be implicated in the susceptibility of a broad variety of autoimmune disorders, including generalized vitiligo (GV). Genetic polymorphisms in the NLRP1 encoding gene (formerly known as NALP1) have previously been found to be linked with GV and there is uncertainty as to their role in the modulation of NLRP1 expression. Oxidative stress is a significant pathogenesis theory for vitiligo. Glutathione S-transferases (GSTs) are enzymes active in the defense of cells against chemical toxicity and stress.This study validates some of the Unani concepts of humors or temperaments (Phenotypes), with regard to Vitiligo, Where vitiligo is regarded as a phlegmatic disease. We selected Vitiligo subjects with Phlegmatic Clinical Phenotype for our study, with an aim to determine its association with the genetic biomarkers- NLRP1, GSTM1 and GSTT1 null genotypes and other biochemical parameters. Methods: The Unani clinicians randomly selected 100 vitiligo patients with a phlegmatic Clinical Phenotype who were attending NRIUMSD for treatment and 100 healthy volunteers belonging to Phlegmatic (Phlegmatic clinical Phenotype). Besides looking at temperaments/ humors as susceptibility factors – we included a genetic factor- NLRP1, GSTM1- and GSTT1-null genotypes to our investigation. We have genotyped the NLRP1, GSTM1- and GSTT1-null genotypes by PCR-RFLP and by Multiplex PCR, GST protein level estimation by ELISA method. Results: NLRP1 rs2670660 polymorphism was shown to be in significant association with GV, with the presence of minor alleles in active GV. We found that the frequencies of GSTM1 null genotype and GSTT1 null genotype in vitiligo patients were significantly high compared to the controls (OR= 1.47, 95% CI=0.765--2.861), (OR = 4.75, 95% CI = 2.131-10.63), respectively. In combination analysis with both genes, the results suggested significant association of vitiligo risk with both GSTM1\GSTT1 null genotypes (OR=4.83, 95% CI=1.523– 15.32).We observed a significant decrease (p<0.001) in GST protein levels. Conclusion: Our findings indicate that NLRP1 rs2670660 polymorphism may be genetic risk factor for susceptibility to GV and the null genotypes of GSTM1 and GSTT1 of both genes increase the risk of the disease. A significant decrease (p<0.001) in GST protein levels appeared to be a key feature in Vitiligo subjects, Therefore, detection of antioxidant enzyme levels can be effective biomarkers for early detection of the disease. We believed that GSTM1-and GSTT1-null genotype polymorphisms were associated with an increased risk of vitiligo. This is the first study of its kind along with Clinical Phenotype as per Unani Philosophy.
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.
NOD-like receptors (NLRs) are a kind of pattern recognition receptors, which are vital for detection on pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and then trigger downstream immune responses in vertebrates. Although many NLR like genes have been identified in invertebrates in recent years, knowledge about their immune functions is still very limited. In the present study, a NLR like gene, designated as LvNLRPL1, was identified in Litopenaeus vannamei. It was widely expressed in multiple tissues and responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvNLRPL1 could accelerate the proliferation of Vibrio in the target tissue hepatopancreas and increase the mortality rate of shrimp after Vibrio infection. Meanwhile, knockdown of LvNLRPL1 also up-regulated the expression of Caspase 2, 3 and 5 in hemocytes, which caused apoptosis of more hemocytes. These results indicated that LvNLRPL1 played important immune functions in shrimp during Vibrio infection through regulating the apoptosis of hemocytes in shrimp. To our knowledge, this is the first time to reveal the immune function of a NLR like gene in crustaceans.
Abstract: Cell death is an essential immunological apparatus of host defense, but dysregulation of
mutually inclusive cell deaths poses severe threats during microbial and parasitic infections leading
to deleterious consequences in the pathological progression of infectious diseases. Nucleotidebinding oligomerization domain (NOD)-Leucine-rich repeats (LRR)-containing receptors (NLRs),
also called nucleotide-binding oligomerization (NOD)-like receptors (NLRs), are major cytosolic
pattern recognition receptors (PRRs), their involvement in the orchestration of innate immunity
and host defense against bacteria, viruses, fungi and parasites, often results in the cleavage of
gasdermin and the release of IL-1β and IL-18, should be tightly regulated. NLRs are functionally
diverse and tissue-specific PRRs expressed by both immune and non-immune cells. Beyond the
inflammasome activation, NLRs are also involved in NF-κB and MAPK activation signaling, the
regulation of type I IFN (IFN-I) production and the inflammatory cell death during microbial
infections. Recent advancements of NLRs biology revealed its possible interplay with pyroptotic cell
death and inflammatory mediators, such as caspase 1, caspase 11, IFN-I and GSDMD. This review
provides the most updated information that caspase 8 skews the NLRP3 inflammasome activation
in PANoptosis during pathogen infection. We also update multidimensional roles of NLRP12 in
regulating innate immunity in a content-dependent manner: novel interference of NLRP12 on TLRs
and NOD derived-signaling cascade, and the recently unveiled regulatory property of NLRP12
in production of type I IFN. Future prospects of exploring NLRs in controlling cell death during
parasitic and microbial infection were highlighted
The NLR (NACHT-LRR) family of proteins have been implicated in the regulation of immune responses and cell death pathways. Some NLR family members can form multiprotein complexes, called inflammasomes, involved in the activation of pro-inflammatory caspases. Mutations in the NALP3/CIAS1/cryopyrin gene, a member of the NLR family, are linked to three auto-inflammatory disorders: Muckle-Wells syndrome, familial cold auto-inflammatory syndrome and neonatal-onset multisystem inflammatory disease. NALP3 along with the adaptor molecule ASC activates caspase-1 in response to a wide variety of stimuli. Here we review recent findings on the biology of NALP3 suggesting that it has functions beyond that of pathogen recognition.
Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. Caspase-1 is a member of inflammatory caspases that play important roles in the innate immune system. However, few studies have been performed in lower vertebrates such as teleosts and implications of extracellular ATP-mediated immune signalling in fish. Here we identified and characterized novel ASC and caspase-1 genes (namely EcASC and EcCaspase-1) from the orange-spotted grouper (Epinephelus coioides). EcASC and EcCaspase-1 encode 204- and 388-aa proteins which shared 55.34% and 72.89% identity with those in Siniperca chuatsi and Perca flavescens, respectively. EcASC contained a PYRIN domain (aa 5-82) and CARD domain (aa 107-201). EcCaspase1 contained a CARD domain (aa 1-88) and a CASc domain (aa 127-376). Both EcASC and EcCaspase-1 were distributed in all tissues tested in the healthy grouper. The expression of EcASC and EcCaspase-1 was significantly upregulated in response to ATP infection. Subcellular localization analysis showed that EcCaspase-1 exhibited a clear distribution in both cytoplasm and nucleus. In contrast, EcASC was observed in the cytoplasm as speck-like structures, which are called "pyroptosomes". EcCaspase-1 co-localized with the spot-like protein (EcASC). Overexpression of EcASC and EcCaspase-1 inhibited NF-κB activation and promoted P53 activation in grouper spleen (GS) cells. Extracellular ATP was an effective signaling molecule that activates the innate immune response, rapidly upregulating the expression of EcASC and EcCaspase1, and enhancing their promotion of proinflammatory cytokine expression in GS cells. Both EcASC and EcCaspase-1 promoted ATP-induced apoptosis. Our results suggested that the interactions of inflammatory EcCaspase-1 with EcASC proteins were associated with extracellular ATP-mediated immune signaling in fish.
SGT1/SUGT1, a co-chaperone of HSP90, is involved in multiple cellular activities including cullin E3 ubiquitin ligase activity. In various species, SGT1 homologs have been identified, indicating that SGT1 genes and proteins are highly conserved. SGT1 functions in multiple distinct biological processes, such as plant and mammal innate immune systems, yeast CBF3 kinetochore assembly, human CENP-A deposition and kinetochore assembly, SCF function and cyclic AMP signaling, centrosome organization and cytokinesis, and brain and heart diseases. Domain-specific functions, interactors, and roles in subcellular localization of SGT1 are described in this chapter. Importantly, SGT1 contributes to cancer development by stabilizing oncoproteins, and the SGT1-HSP90 complex is a potential target for therapies aimed at cancer, brain and heart disease. Recent advances in our understanding of the physiologic role of SGT1 are briefly reviewed in this chapter.
Inflammation occurs as a result of acute trauma, invasion of the host by different pathogens, pathogen-associated molecular patterns (PAMPs) or chronic cellular stress generating damage-associated molecular patterns (DAMPs). Thus inflammation may occur under both sterile inflammatory conditions including certain cancers, autoimmune or autoinflammatory diseases (Rheumatic arthritis (RA)) and infectious diseases including sepsis, pneumonia-associated acute lung inflammation (ALI) or acute respiratory distress syndrome (ARDS). The pathogenesis of inflammation involves dysregulation of an otherwise protective immune response comprising of various innate and adaptive immune cells and humoral (cytokines and chemokines) mediators secreted by these immune cells upon the activation of signaling mechanisms regulated by the activation of different pattern recognition receptors (PRRs). However, the pro-inflammatory and anti-inflammatory action of these immune cells is determined by the metabolic stage of the immune cells. The metabolic process of immune cells is called immunometabolism and its shift determined by inflammatory stimuli is called immunometabolic reprogramming. The article focuses on the involvement of various immune cells generating the inflammation, their interaction, immunometabolic reprogramming, and the therapeutic targeting of the immunometabolism to manage inflammation.
LOR1 is a member of a fifteen member gene family in Arabidopsis termed LOR (LURP-one related). Of the LOR gene family members, LOR1 shows strong constitutive expression based on microarray data against Hyaloperonospora arabidopsidis and Pseudomonas Syringe. LURP1 is another member of LOR family and a member of LURP gene cluster that shows an unusually pronounced transcriptional up-regulation in response to Hyaloperonospora arabidopsidis (Hpa). Mutation in LOR1 revealed a highly significant role of this gene in basal defense against Hpa whereas mutations in LURP1 resulted in reduced immunity mediated by the R-gene RPP5 against Hpa. Based on the resemblance of LOR1 protein to human Phospholipid scramlase1 (PLSCR1) stable CaMV35S:GFP-LOR1 and CaMV35S:GFP-LURP1 expression lines were created to analyze the subcellular localization of these proteins. GFP-LOR1 and GFP-LURP1 complemented loss of resistance in their respective mutant backgrounds. Like PLSCR, both GFP-LOR1 and GFP-LURP1 were found localized at the plasma membrane and in the nucleus but unlike PLSCR1 that is predominantly localized to the nucleus after cytokine induction, both GFP-LOR1 and GFP-LURP1 did not change their localization after infection with Hpa. LOR1 and LURP1 used as bait protein in yeast two hybrid system did not result in the identification of any interacting proteins from a screen of 2.3 × 105 cDNA for each from control and disease sampled plants treated with Hpa isolates.
The article describes the basic principles of the immune systems' function. Its given notions of organs and cells of the immune system, especially native and adaptive immunity. The article presents the evolution of age-related features of the immune response.
Abstract: In order to understand mechanisms of immune evasion in muscle-invasive urothelial bladder cancer (MIBC), we aimed at characterizing the protein expression of T-cells in lymph nodes of MIBC patients to clarify how immune signaling in lymph nodes is impacted by proximity to the tumour. The sentinel node (SN) is key to the tumor microenvironment, where regulatory T-cells (Tregs) play an immunosuppressive role. Proteomic analysis of Tregs and effector T-cells in SN and non-draining lymph nodes found SN-Tregs in MIBC patients to up-regulate growth and immune signaling pathways, the cytokine IL-16 being central in the signaling network. Experimental validation showed that in Tregs, tumoral factors increase IL-16 processing into bioactive forms and increase FOXP3 expression. In conclusion, altered IL-16 processing caused by tumour-released factors stimulate expansion of SN-Tregs in MIBC, creating an immunosuppressive environment and contributing to immune escape.
RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.
The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR. analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.
Systematic analysis of the Arabidopsis genome provides a basis for detailed studies of genome structure and evolution. Members of multigene families were mapped, and random sequence alignment was used to identify regions of extended similarity in the Arabidopsis genome. Detailed analysis showed that the number, order, and orientation of genes were conserved over large regions of the genome, revealing extensive duplication covering the majority of the known genomic sequence. Fine mapping analysis showed much rearrangement, resulting in a patchwork of duplicated regions that indicated deletion, insertion, tandem duplication, inversion, and reciprocal translocation. The implications of these observations for evolution of the Arabidopsis genome as well as their usefulness for analysis and annotation of the genomic sequence and in comparative genomics are discussed.
Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large in the order of ∼1.4 MDa. We now demonstrate that dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M r values of ∼1.4 MDa and ∼700 kDa. Kinetic analysis demonstrates that in vitro the ∼700-kDa complex is produced more rapidly than the ∼1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide orN-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the ∼700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the ∼700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.
Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-kappaB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-kappaB induction. Dominant-negative versions of CARD4 block activation of NF-kappaB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.
Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding
domain, which regulate apoptosis and/or NF-κB activation. Nod2, a third member of the family, was identified. Nod2 is composed
of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1
were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor
κB (NF-κB) activation, which required IKKγ and was inhibited by dominant negative mutants of IκBα, IKKα, IKKβ, and IKKγ. Nod2
interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-κB activity induced
by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK
containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to
activate a NF-κB signaling pathway.
Genetic variation in NOD2 has been associated with susceptibility to Crohn's disease (CD) and specifically with ileal involvement. The reason for the unique association of NOD2 mutations with ileal disease is unclear. To identify a possible link, we tested expression of NOD2 in intestinal tissue of CD patients and controls.
Fifty five specimens of ileum or colon from 21 CD patients, seven ulcerative colitis (UC) patients, and five controls with pathology other than CD or UC were stained for NOD2 using an immunoperoxidase method.
Using a monoclonal antibody against NOD2 developed in our laboratory, we detected uniform expression of NOD2 in terminal ileum Paneth cells from controls and patients as well as in metaplastic Paneth cells in the colon. Mechanical purification showed enriched expression of NOD2 mRNA in ileal crypts. In Paneth cells, NOD2 was located in the cytosol in close proximity to the granules that contain antimicrobial peptides. We detected minimal NOD2 in the villous epithelium of the ileum or in the colonic epithelium from both CD patients and controls.
These results suggest a role for NOD2 in the regulation of Paneth cell mediated responses against intestinal bacteria and a plausible mechanism to explain the selective association of NOD2 mutations with ileal disease. The impaired capacity of CD associated mutations to sense luminal bacteria may result in increased susceptibility to certain gut microbes.
Expression of NOD2/CARD15 in the Paneth cell may be critical in the pathogenesis of Crohn’s disease
The emergence and application of novel molecular techniques over the last decade has provided a needed catalyst to studies of the pathogenesis of the chronic inflammatory bowel diseases (IBD): Crohn’s disease (CD) and ulcerative colitis (UC). Successful development of genetically engineered models of intestinal inflammation has not only provided insight into the dysregulation of the mucosal immune system characteristic of IBD but has also emphasised the critical and complex role of the bacterial flora in establishing and maintaining chronic intestinal inflammation.1 These advances in understanding pathophysiology in turn have already led to novel therapeutic approaches.2,3
However, it is in studies of human genetics that landmark progress has been made, widely recognised not only within gastroenterology but also by investigators in all complex diseases.4 Genome wide scanning led initially to the identification of a number of susceptibility loci, the statistical evidence for which satisfy stringent criteria for definite linkage.5 The subsequent detection of the NOD2/CARD15 gene6–8 within the IBD1 linkage interval and the association of mutations within this gene with susceptibility to CD is widely regarded as the most stringent proof of principle for hypothesis free genome scanning in complex diseases.
In the time that has elapsed since the discovery of NOD2/CARD15, the contribution of this gene in determining susceptibility and disease behaviour in IBD has received detailed examination. It is now clear that NOD2/CARD15 mutations are associated with susceptibility to CD but not UC.6 However, the contribution is subject to considerable ethnic and even regional variation. Whereas mutations may be carried by up to 50% of central Europeans with CD,9 these mutations are not present in Japanese10 or Afro-American11 patients. Even within Europe, …
Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.
Scaffold molecules interact with multiple effectors to elicit specific signal transduction pathways. CIITA, a non-DNA-binding regulator of class II major histocompatibility complex (MHC) gene transcription, may serve as a transcriptional scaffold. Regulation of the class II MHC promoter by CIITA requires strict spatial-helical arrangements of the X and Y promoter elements. The X element binds RFX (RFX5/RFXANK-RFXB/RFXAP) and CREB, while Y binds NF-Y/CBF (NF-YA, NF-YB, and NF-YC). CIITA interacts with all three. In vivo analysis using both N-terminal and C-terminal deletion constructs identified critical domains of CIITA that are required for interaction with NF-YB, NF-YC, RFX5, RFXANK/RFXB, and CREB. We propose that binding of NF-Y/CBF, RFX, and CREB by CIITA results in a macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.
RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.
Plant breeders have used disease resistance genes (R genes) to control plant disease since the turn of the century. Molecular
cloning of R genes that enable plants to resist a diverse range of pathogens has revealed that the proteins encoded by these
genes have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms
for the expression of resistance to a wide range of unrelated pathogens. Characterization of the molecular signals involved
in pathogen recognition and of the molecular events that specify the expression of resistance may lead to novel strategies
for plant disease control.
CIITA activates the expression of multiple genes involved in antigen presentation and it is believed to be required for both constitutive and IFN gamma-inducible expression of these genes. To understand the role of CIITA in vivo, we have used gene targeting to generate mice that lack CIITA. CIITA-deficient (-/-) mice do not express conventional MHC class II molecules on the surface of splenic B cells and dendritic cells. In addition, macrophages resident in the peritoneal cavity do not express MHC class II molecules upon IFN gamma stimulation nor do somatic tissues of mice injected with IFN gamma, in contrast with wild-type mice. The levels of Ii and H-2M gene transcripts are substantially decreased but absent in CIITA (-/-) mice. The transcription of nonconventional MHC class II genes is, however not affected by CIITA deficiency. A subset of thymic epithelial cells express MHC class II molecules. Nonetheless, very few mature CD4 T cells are present in the periphery of CIITA (-/-) mice despite MHC class II expression in the thymus. Consequently, CIITA(-/-) mice are impaired in T-dependent antigen responses and MHC class II-mediated allogeneic responses.
In tomato, resistance to Pseudomonas syringae pv. tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf. Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants. prf mutant alleles (4) were found to carry alterations within the Prf coding sequence. A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion. Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.
Transient expression of the Pseudomonas syringae avirulence gene avrPto in plant cells resulted in a Pto-dependent necrosis. The AvrPto avirulence protein was observed to interact directly with the Pto resistance protein in the yeast two-hybrid system. Mutations in the Pto and avrPto genes which reduce in vivo activity had parallel effects on association in the two-hybrid assay. These data suggest that during infection the pathogen delivers AvrPto into the plant host cell and that resistance is specified by direct interaction of Pto with AvrPto.
Resistance to bacterial speck disease in tomato occurs when the Pto kinase in the plant responds to expression of the avirulence gene avrPto in the Pseudomonas pathogen. Transient expression of an avrPto transgene in plant cells containing Pto elicited a defense response. In the yeast two-hybrid system, the Pto kinase physically interacted with AvrPto. Alterations of AvrPto or Pto that disrupted the interaction in yeast also abolished disease resistance in plants. The physical interaction of AvrPto and Pto provides an explanation of gene-for-gene specificity in bacterial speck disease resistance.
We show here that transient forebrain ischemia selectively elevates levels of neuronal apoptosis inhibitory protein (NAIP) in rat neurons that are resistant to the injurious effects of this treatment. This observation suggests that increasing NAIP levels may confer protection against ischemic cell death. Consistent with this proposal, we demonstrate that two other treatments that increase neuronal NAIP levels, systemic administration of the bacterial alkaloid K252a and intracerebral injection of an adenovirus vector capable of overexpressing NAIP in vivo, reduce ischemic damage in the rat hippocampus. Taken together, these findings suggest that NAIP may play a key role in conferring resistance to ischemic damage and that treatments that elevate neuronal levels of this antiapoptotic protein may have utility in the treatment of stroke.
The following pair of articles, the first by Gil Segal and Howard Shuman, and the second by James Kirby and Ralph Isberg (Trends Microbiol. 6, 256-258), explore the genetics and function of the icm/dot genes of Legionella pneumophila. This gene family is implicated in several aspects of virulence and appears to constitute components of a conjugal transfer system that has been adopted to prevent phagosome-lysosome fusion in the host cell and to mediate host cytotoxicity by pore formation. Whether these functions are natural consequences or operate in parallel remains to be discovered.
The nematode CED-4 protein and its human homolog Apaf-1 play a central role in apoptosis by functioning as direct activators of death-inducing caspases. A novel human CED-4/Apaf-1 family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that mediate disease resistance in plants. CARD4 interacted with the serine-threonine kinase RICK and potently induced NF-kappaB activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-kappaB and apoptotic signaling pathways and may be a component of the host innate immune response.
Paneth cells in mouse small intestinal crypts secrete granules rich in microbicidal peptides when exposed to bacteria or bacterial antigens. The dose-dependent secretion occurs within minutes and α-defensins, or cryptdins, account for 70% of the released bactericidal peptide activity. Gram-negative bacteria, Gram-positive bacteria, lipopolysaccharide, lipoteichoic acid, lipid A and muramyl dipeptide elicit cryptdin secretion. Live fungi and protozoa, however, do not stimulate degranulation. Thus intestinal Paneth cells contribute to innate immunity by sensing bacteria and bacterial antigens, and discharge microbicidal peptides at effective concentrations accordingly.
The PYRIN domain is a recently identified protein-protein interaction domain that is found at the N terminus of several proteins
thought to function in apoptotic and inflammatory signaling pathways. We report here that PYPAF1 (PYRIN-containing Apaf1-like protein 1) is a novel PYRIN-containing signaling protein that belongs to the nucleotide-binding site/leucine-rich repeat
(NBS/LRR) family of signaling proteins. The expression of PYPAF1 is highly restricted to immune cells, and its gene maps to
chromosome 1q44, a locus that is associated with the rare inflammatory diseases Muckle-Wells syndrome and familial cold urticaria.
To identify downstream signaling partners of PYPAF1, we performed a mammalian two-hybrid screen and identified ASC as a PYRIN-containing
protein that interacts selectively with the PYRIN domain of PYPAF1. When expressed in cells, ASC recruits PYPAF1 to distinct
cytoplasmic loci and induces the activation of NF-κB. Furthermore, coexpression of PYPAF1 with ASC results in a potent synergistic
activation of NF-κB. These findings suggest that PYPAF1 and ASC function as upstream activators of NF-κB signaling.
Legionella pneumophila survives within macrophages by evading phagosome–lysosome fusion. To determine whether L. pneumophila resides in an intermediate endosomal compartment or is isolated from the endosomal pathway and to investigate what bacterial factors contribute to establishment of its vacuole, we applied a series of fluorescence microscopy assays. The majority of vacuoles, aged 2.5 min to 4 h containing post-exponential phase (PE) L. pneumophila, appeared to be separate from the endosomal pathway, as judged by the absence of transferrin receptor, LAMP-1, cathepsin D and each of four fluorescent probes used to label the endocytic pathway either before or after infection. In contrast, more than 70% of phagosomes that contained Escherichia coli, polystyrene beads, or exponential phase (E) L. pneumophila matured to phagolysosomes, as judged by co-localization with LAMP-1, cathepsin D and fluorescent endosomal probes. Surprisingly, neither bacterial viability nor the putative Dot/Icm transport complex was absolutely required for vacuole isolation; although phagosomes containing either formalin-killed PE wild-type or live PE dotA or dotB mutant L. pneumophila rapidly accumulated LAMP-1, less than 20% acquired lysosomal cathepsin D or fluorescent endosomal probes. Therefore, a Dot-dependent factor(s) isolates the L. pneumophila phagosome from a LAMP-1-containing compartment, and a formalin-resistant Dot-independent activity inhibits vacuolar accumulation of endocytosed material and delivery to the degradative lysosomes.
Studies of a family with "familial cold urticaria" indicate that the syndrome is not a true urticarial disorder, but appears to be a systemic reaction generated by cooling of the skin. The systemic reaction appears to be mediated by an unidentified humoral substance, probably not histamine. Leukocytosis is a constant accompaniment of the generalized reaction and seems to play a role in the pathogenesis of the skin lesions. The generalized reaction can be prevented by the prior administration of Pseudomonas polysaccharide complex (Piromen).
Type II major histocompatibility complex combined immune deficiency (type II MHC CID or bare lymphocyte syndrome) is a congenital immunodeficiency disease characterized by absent MHC class II expression. Four distinct complementation groups have been identified. Recently, the defective gene in group II type II MHC CID has been isolated and termed CIITA. Here, we demonstrate that CIITA is an MHC class II gene-specific transcription activator. The transcription activation function is provided by the N-terminal acidic domain (amino acids 26-137), which is experimentally exchangeable with a heterologous viral transcription-activating domain. The specificity of CIITA for three major MHC class II genes, DR, DQ and DP, is mediated by its remaining C-terminal residues (amino acids 317-1130). The transactivation of multiple cis elements, especially S and X2, of the DR alpha proximal promoter in group II CID cells is CIITA dependent. Since CIITA overexpression in normal cells did not increase class II expression, we propose that initiation of CIITA expression serves as the on-off switch, while availability of downstream interactor(s) limits transcription.
CIITA, a gene that can complement a transcriptional mutation of the major histocompatibility complex (MHC) class II genes, was tested for its ability to function as a coactivator, CIITA cDNA clones isolated showed alternative RNA splicing, but only one splice site combination was able to restore class II MHC gene expression. DNA-mediated transfection experiments showed that CIITA directs its activity through the X box element; the presence of CIITA leads to the formation of a higher order complex at the X box region; and CIITA contains a potent activation domain. These findings support the hypothesis that CIITA directly interacts with the MHC class II-specific transcription factors and is required for expression.
The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.
Hereditary major histocompatibility complex (MHC) class II deficiency (or bare lymphocyte syndrome) is a form of severe primary immunodeficiency with a total lack of MHC class II expression. It is due to a defect in the regulation of MHC class II genes. A novel gene was isolated by complementation cloning, using an MHC class II-negative mutant cell line. This gene (CIITA) functions as a transactivator of MHC class II gene expression and restores expression of all MHC class II isotypes in mutant cells. In addition, CIITA fully corrects the MHC class II regulatory defect of cells from patients with bare lymphocyte syndrome. In this disease we have identified a splicing mutation that results in a 24 amino acid deletion in CIITA, resulting in loss of function of the transactivator. Hence, the CIITA gene is essential for MHC class II gene expression and has been shown to be responsible for hereditary MHC class II deficiency.
Class II major histocompatibility complex (MHC) genes are regulated in a coordinate manner. A set of conserved upstream elements termed the W/Z/S, X1, X2, and Y boxes are found 5{prime} to class II genes, the R gene, and the HLA-DM genes. These conserved elements are required for tissue-specific and IFN{gamma}-mediated regulation of these genes. The DNA-binding proteins RFX, X2BP, and NFY have been found to specifically interact with the X1, X2, and Y box elements, respectively, as well as with each other. A role for an additional factor was recently demonstrated by the cloning of a gene that could complement the MHC class H gene-specific transcriptional deficiency in the mutant cell line RJ2.2.5 as well as cell lines isolated from patients exhibiting the bare lymphocyte syndrome. This gene was termed the class II transactivator or CIITA. While both genetic and biochemical studies have indicated interactions between the DNA-binding proteins described above, direct interactions with CIITA have not been described. 23 refs., 3 figs.
Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Recently, we isolated a candidate gene, encoding neuronal apoptosis inhibitor protein (NAIP), for SMA. This gene is homologous to two baculovirus inhibitor of apoptosis proteins (Cp-IAP and Op-IAP) and is partly deleted in individuals with type I SMA. A second SMA candidate gene encoding survival motor neuron (SMN), which is contiguous with the NAIP locus on 5q13.1, was also reported. Here we demonstrate a NAIP-mediated inhibition of apoptosis induced by a variety of signals, and have identified three additional human complementary DNAs and a Drosophila melanogaster sequence that are also homologous to the baculovirus IAPs. The four open reading frames (ORFs) possess three baculoviral inhibition of apoptosis protein repeat (BIR) domains and a carboxy-terminal RING zinc-finger. The human iap genes have a distinct but overlapping pattern of expression in fetal and adult tissues. These proteins significantly increase the number of known apoptotic suppressors.
Congenital MHC class II deficiency or bare lymphocyte syndrome (BLS; McKusick 209920) is caused by defects in trans-acting regulatory factors that control MHC class II expression and is therefore a disease of gene regulation. There are at least four complementation groups and the genetic and molecular dissection of this rare disease has contributed considerably to our current understanding of the molecular mechanisms governing MHC class II expression. Identification of the gene that is defective in BLS complementation group A, CIITA (MHC class II transactivator), has led to the discovery that CIITA acts as a master control factor of MHC class II expression. We have identified the CIITA mutations in a second patient from BLS group A. Two novel mutations abolish CIITA function, as shown by transfection experiments. Molecular analysis of these two novel mutations, together with the one described earlier in the first patient, is informative in terms of CIITA structure-function relationships.
The class II transactivator (CIITA) is a master transcription regulator of gene products involved in the exogenous antigen presentation pathway, including major histocompatibility complex (MHC) class II, invariant chain, and DM. An extensive analysis of the putative functional domains of CIITA is undertaken here to explore the action of CIITA. Antibodies to CIITA protein were produced to verify that these mutant proteins are expressed. Both acidic and proline/serine/threonine-rich domains are essential for class II MHC promoter activation. In addition, three guanine nucleotide-binding motifs are essential for CIITA activity. Of these mutants, two exhibited strong transdominant-negative functions. These two mutants provide a plausible approach to manipulate MHC class II expression and immune responses.
The constitutive and cytokine-induced levels of major histocompatibility (MHC) class I expression are tightly controlled at the transcriptional level. In this study, it is shown that the cis-acting regulatory element site alpha of the MHC class I promoter is essential for the IFN gamma-induced transactivation of MHC class I gene expression through the ISRE. Moreover, it was discovered that the class II transactivator (CIITA), which is itself under the control of the IFN gamma induction pathway, strongly transactivates MHC class I gene expression and exerts its activity through site alpha. Therefore, site alpha is a crucial regulatory element, mediating the classic route of IFN gamma induction via the ISRE as well as a novel route of MHC class I transactivation involving CIITA.
Major histocompatibility complex (MHC) class I-deficient cell lines were used to demonstrate that the MHC class II transactivator (CIITA) can induce surface expression of MHC class I molecules. CIITA induces the promoter of MHC class I heavy chain genes. The site alpha DNA element is the target for CIITA-induced transactivation of class I. In addition, interferon-gamma (IFNgamma)-induced MHC class I expression also requires an intact site alpha. The G3A cell line, which is defective in CIITA induction, does not induce MHC class I antigen and promoter in response to IFNgamma. Trans-dominant-negative forms of CIITA reduce class I MHC promoter function and surface antigen expression. Collectively, these data argue that CIITA has a role in class I MHC gene induction.
We report here the purification and cDNA cloning of Apaf-1, a novel 130 kd protein from HeLa cell cytosol that participates in the cytochrome c-dependent activation of caspase-3. The NH2-terminal 85 amino acids of Apaf-1 show 21% identity and 53% similarity to the NH2-terminal prodomain of the Caenorhabditis elegans caspase, CED-3. This is followed by 320 amino acids that show 22% identity and 48% similarity to CED-4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of Apaf-1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3, leading to apoptosis.
Following the recent realization that TCR beta transgenes can severely inhibit the rearrangement of endogenous Vbeta gene segments in the absence of pre-TCR alpha (pT alpha) chains, we tested whether the pre-TCR has an essential role in TCR beta allelic exclusion under more physiological conditions by analyzing TCR rearrangement in immature thymocytes by single-cell PCR. Our results in pT alpha+ mice are consistent with an ordered model of TCR beta rearrangement beginning on one allele and continuing on the other only when the first attempt is unsuccessful. By contrast, a higher proportion of thymocytes from pT alpha-/- mice exhibited two productive TCR beta alleles. Thus, the pre-TCR-independent suppression of rearrangement by TCR beta transgenes represents a transgene artifact, whereas under physiological conditions the pre-TCR is essential for allelic exclusion.
We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.
CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation.
Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major
histocompatibility complex class II and associated genes by interferon-γ, but CIITA does not bind directly to DNA. Expression
of CIITA in G3A cells, which lack endogenous CIITA, followed by in vivo genomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters
of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-γ-inducible cell types,
but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable
and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.
Activation of procaspase-9 by Apaf-1 in the cytochrome c/dATP-dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we designed an in vitro Apaf-1-procaspase-9 activation system using recombinant components. Here, we show that deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively active and capable of processing procaspase-9 independent of cytochrome c an dATP. Apaf-1-mediated processing of procaspase-9 occurs at Asp-315 by an intrinsic autocatalytic activity of procaspase-9 itself. We provide evidence that Apaf-1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6, and -7.
Classical genetic and molecular data show that genes determining disease resistance in plants are frequently clustered in the genome. Genes for resistance (R genes) to diverse pathogens cloned from several species encode proteins that have motifs in common. These motifs indicate that R genes are part of signal-transduction systems. Most of these R genes encode a leucine-rich repeat (LRR) region. Sequences encoding putative solvent-exposed residues in this region are hypervariable and have elevated ratios of nonsynonymous to synonymous substitutions; this suggests that they have evolved to detect variation in pathogen-derived ligands. Generation of new resistance specificities previously had been thought to involve frequent unequal crossing-over and gene conversions. However, comparisons between resistance haplotypes reveal that orthologs are more similar than paralogs implying a low rate of sequence homogenization from unequal crossing-over and gene conversion. We propose a new model adapted and expanded from one proposed for the evolution of vertebrate major histocompatibility complex and immunoglobulin gene families. Our model emphasizes divergent selection acting on arrays of solvent-exposed residues in the LRR resulting in evolution of individual R genes within a haplotype. Intergenic unequal crossing-over and gene conversions are important but are not the primary mechanisms generating variation.
The severe immunodeficiency type II bare lymphocyte syndrome (BLS) lacks class II MHC gene transcription. One defect from a complementation group A type II BLS patient is a 24 aa deletion in the MHC class II transactivator (CIITA). We show here that the molecular defect present in this protein is a failure of CIITA to undergo nuclear translocation. This defect was mapped to a position-dependent, novel nuclear localization sequence that cannot be functionally replaced by a classical NLS. Fusion of this 5 aa motif to an unrelated protein leads to nuclear translocation. Furthermore, this motif is not critical for transactivation function. This is a description of a genetic disease resulting from a novel defect in the subcellular localization of a transcriptional coactivator.
Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-kappaB) and bound RICK, a CARD-containing kinase that also induces NF-kappaB activation. Nod1 mutants inhibited NF-kappaB activity induced by RICK, but not that resulting from tumor necrosis factor-alpha stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-kappaB activation pathways.
Class II transactivator (CIITA) is an unusual transcriptional coactivator in that it contains a functionally important, GTP-binding consensus domain. To assess the functional role of the GTP-binding domain of CIITA in vivo, we have generated knockout mice that bear a mutation in the CIITA gene spanning the GTP-binding domain. Upon analysis, these mice show no detectable CIITA mRNA; hence, they represent mice with deleted CIITA rather than mice with defects in the GTP-binding domain only. In these knockout mice, MHC class II expression is nearly eliminated, although a faint RT-PCR signal is visible in spleen, lymph node, and thymus, suggestive of the presence of CIITA-independent regulation of MHC class II expression. Invariant chain expression is also greatly reduced, but to a lesser extent than MHC class II. Serum IgM is not decreased, but the serum IgG level is greatly reduced, further confirming the absence of MHC class II Ag-dependent Ig class switching. Induction of MHC class II expression by IL-4 or LPS was absent on B cells, and Mac-1+ cells showed no detectable induction of MHC class II by either IL-4, LPS, or IFN-gamma. These findings demonstrate a requirement for CIITA in IFN-gamma-, IL-4-, and endotoxin-induced MHC class II expression as well as the possibility of rare CIITA-independent MHC class II expression.