P2Y2R Deficiency Attenuates Experimental Autoimmune Uveitis Development

We aimed to study the role of the nucleotide receptor P2Y₂R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y₂^+/+ and P2Y₂^-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y₂^+/+ and P2Y₂^-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4⁺ T lymphocytes from P2Y₂^+/+ and P2Y₂^-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y₂^-/- mice as in P2Y₂^-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y₂^-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y₂^-/- and P2Y₂^+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y₂^-/- T cells proliferate less and secrete less cytokines than the P2Y₂^+/+ one. We further found that antigen-presenting cells of P2Y₂^-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y₂^-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.     

Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms

Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a d_N/d_S-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family.Receptor-like kinases (RLKs) constitute one of the largest gene families in plants and expanded massively in land plants (Embryophyta; Lehti-Shiu et al., 2009, 2012). For plant RLK gene families, the functions of most members are often not known (especially in recently expanded families), but some described functions include innate immunity (Albert et al., 2010), pathogen response (Dodds and Rathjen, 2010), abiotic stress (Yang et al., 2010), development (De Smet et al., 2009), and sometimes multiple functions (Lehti-Shiu et al., 2012). The RLKs usually consist of three domains: an N-terminal extracellular domain, a transmembrane domain, and a C-terminal kinase domain (KD). In plants, the KD usually has a Ser/Thr specificity (Shiu and Bleecker, 2001), but Tyr-specific RLKs were also described (e.g. BRASSINOSTEROID INSENSITIVE1; Oh et al., 2009). Interestingly, it was estimated that approximately 20% of RLKs contain a catalytically inactive KD (e.g. STRUBBELIG and CORYNE; Chevalier et al., 2005; Castells and Casacuberta, 2007; Gish and Clark, 2011). In Arabidopsis (Arabidopsis thaliana), 44 RLK subgroups (SGs) were defined by inferring the phylogenetic relationships between the KDs (Shiu and Bleecker, 2001). Interestingly, different SGs show different duplication/retention rates (Lehti-Shiu et al., 2009). Specifically, RLKs involved in stress responses show a high number of tandemly duplicated genes whereas those involved in development do not (Shiu et al., 2004), which suggests that some RLK genes are important for the responses of land plants to a changing environment (Lehti-Shiu et al., 2012). There seem to be relatively few RLK pseudogenes compared with other large gene families, and copy retention was argued to be driven by both drift and selection (Zou et al., 2009; Lehti-Shiu et al., 2012). As most SGs are relatively old and RLK subfamilies expanded independently in several plant lineages, duplicate retention cannot be explained by drift alone, and natural selection is expected to be an important driving factor in RLK gene family retention (Lehti-Shiu et al., 2009).Leucine-rich repeat-receptor-like kinases (LRR-RLKs), which contain up to 30 leucine-rich repeat (LRRs) in their extracellular domain, constitute the largest RLK family (Shiu and Bleecker, 2001). Based on the KD, 15 LRR-RLK SGs have been established in Arabidopsis (Shiu et al., 2004; Lehti-Shiu et al., 2009). So far, two major functions have been attributed to them: defense against pathogens and development (Tang et al., 2010b). LRR-RLKs involved in defense are predominantly found in lineage-specific expanded (LSE) gene clusters, whereas LRR-RLKs involved in development are mostly found in nonexpanded groups (Tang et al., 2010b). It was also discovered that the LRR domains are significantly less conserved than the remaining domains of the LRR-RLK genes (Tang et al., 2010b). In addition, a study of four plant genomes (Arabidopsis, grape [Vitis vinifera], poplar [Populus trichocarpa], and rice [Oryza sativa]) showed that LRR-RLK genes from LSE gene clusters show significantly more indications of positive selection or relaxed constraint than LRR-RLKs from nonexpanded groups (Tang et al., 2010b).The genomes of flowering plants (angiosperms) have been shown to be highly dynamic compared with most other groups of land plants (Leitch and Leitch, 2012). This dynamic is mostly caused by the frequent multiplication of genetic material, followed by a complex pattern of differential losses (i.e. the fragmentation process) and chromosomal rearrangements (Langham et al., 2004; Leitch and Leitch, 2012). Most angiosperm genomes sequenced so far show evidence for at least one whole-genome multiplication event during their evolution (Jaillon et al., 2007; D’Hont et al., 2012; Tomato Genome Consortium, 2012). At a smaller scale, tandem and segmental duplications are also very common in angiosperms (Arabidopsis Genome Initiative, 2000; International Rice Genome Sequencing Project, 2005; Rizzon et al., 2006). Although the most common fate of duplicated genes is to be progressively lost, in some cases they can be retained in the genome, and adaptive as well as nonadaptive scenarios have been discussed to play a role in this preservation process (for review, see Moore and Purugganan, 2005; Hahn, 2009; Innan, 2009; Innan and Kondrashov, 2010). Whole-genome sequences also revealed that the same gene may undergo several rounds of duplication and retention. These LSE genes were shown to evolve under positive selection more frequently than single-copy genes in angiosperms (Fischer et al., 2014). That study analyzed general trends over whole genomes. Here, we ask if, and to what extent, this trend is observable at LRR-RLK genes. As this gene family is very dynamic and large, and in accordance with the results of Tang et al. (2010b), we expect the effect of positive selection to be even more pronounced than in the whole-genome average.We analyzed 33 Embryophyta genomes to investigate the evolutionary history of the LRR-RLK gene family in a phylogenetic framework. Twenty LRR-RLK SGs were identified, and from this data set, we deciphered the evolutionary dynamics of this family within angiosperms. The expansion/reduction rates were contrasted between SGs and species as well as in ancestral branches of the angiosperm phylogeny. We then focused on genes whose number increased dramatically in an SG- and/or species-specific manner (i.e. LSE genes). Those genes are likely to be involved in species-specific cellular processes or adaptive interactions and were used as a template to infer the potential occurrence of positive selection. This led to the identification of sites at which positive selection likely acted. We discuss our results in the light of angiosperm genome evolution and current knowledge of LRR-RLK functions. Positive selection footprints identified in LSE genes highlight the importance of combining evolutionary analysis and functional knowledge to guide further investigations.     

Liposome-Encapsulated Clodronate Retards the Development of Experimental Autoimmune Uveitis

Abstract

A study was undertaken to determine if the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (C1₂MDP; Clodronat), a treatment known to deplete monocytes, as well as liver and spleen macrophages, would reduce the number of macrophages in the retina of animals with experimental autoimmune uveitis (EAU) and decrease the severity of the disease. EAU was induced in Lewis rats by immunization with S-antigen (S-Ag). Monocytes and macrophages were depleted via an intravenous injection of Cl₂MDP encapsulated in liposomes. Control groups included rats that received no S-Ag (n= 18), S-Ag and no treatment (n=23), S-Ag and free drug (n = 20), or empty liposomes (n=14). Treated animals received injections of the Cl₂MDP-liposomes, free drug, or empty liposomes. Animals were sacrificed at 14, 21 and 28 days post-S-Ag administration. Intravenous, Cl₂MDP-liposomes produced a statistically significant reduction in the severity of the EAU when compared to controls at both days 14 and 21 following S-Ag injection. Immunohistochemical staining with the monoclonal antibody EDI demonstrated that the severity of the ocular inflammatory response correlated with the number of EDI-positive cells in the retina. Following the cessation of treatment, treated animals developed disease that was as severe at day 28 as that of untreated animals at day 21. These results confirm the importance of monocytes and macrophages in EAU by demonstrating the correlation between the presence of EDI-positive cells in the retina and the resultant damage to the retina. Although the dosing regimen employed here did not provide a cure, strategies designed to prevent the local recruitment and/or activation of mononuclear phagocytes may prove to be useful in the treatment of EAU.     

Leucine-Rich Repeat Kinase 2 (LRRK2)-Deficient Rats Exhibit Renal Tubule Injury and Perturbations in Metabolic and Immunological Homeostasis

Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson’s disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV) and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.     

Domain-Specific Positive Selection Contributes to the Evolution of Arabidopsis Leucine-Rich Repeat Receptor-Like Kinase (LRR RLK) Genes

Leucine-rich repeat receptor-like kinases (LRR RLKs) comprise the largest group within the plant receptor-like kinase (RLK) superfamily, and the Arabidopsis genome alone contains over 200 LRR RLK genes. Although there is clear evidence for diverse roles played by individual LRR RLK genes in Arabidopsis growth and development, the evolutionary mechanism for this functional diversification is currently unclear. In this study, we focused on the LRRII RLK subfamily to investigate the molecular mechanisms that might have led to the functional differentiation of Arabidopsis LRR RLK genes. Phylogenetic analysis of 14 genes in this subfamily revealed three well-supported groups (I, II, and III). RT-PCR analysis did not find many qualitative differences in expression among these 14 genes in various Arabidopsis tissues, suggesting that evolution of regulatory sequences did not play a major role in their functional divergence. We analyzed substitution patterns in the predicted ligand-binding regions of these genes to examine if positive selection has acted to produce novel ligand-binding specificities, using the nonsynonymous/synonymous rate ratio (d _N/d _S) as an indicator of selective pressure. Estimates of d _N/d _S ratios from multiple methods indicate that nonsynonymous substitutions accumulated during divergence of the three lineages. Positive selection is likely to have occurred along the lineages ancestral to groups II and III. We suggest that positive selection on the ligand-binding sites of LRRII RLKs promoted diversification of ligand-binding specificities and thus contributed to the functional differentiation of Arabidopsis LRRII RLK genes during evolution. [Reviewing Editor: Dr. Martin Kreitman]     

Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization

Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.     

Identification and Function of Leucine-Rich Repeat Flightless-I-Interacting Protein 2 (LRRFIP2) in Litopenaeus vannamei

Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G) were identified in Litopenaeus vannamei. All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV). Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of Drosophila and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of L. vannamei upon V. parahaemolyticus but not S. aureus and WSSV infections. The expression of L. vannamei AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.     

Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Experimental Autoimmune Anterior Uveitis (EAAU): Induction by Melanin Antigen and Suppression by Various Treatments

The uveitogenicity of melanin has been a controversial subject for a long time, presumably as a result of the use of ill-defined preparations in the experiments. We have developed procedures for the preparation of purified uveitogenic melanins from the retinal pigment epithelium and choroid that are free from pathogenic retinal photoreceptor proteins. The active melano-antigen is located at the surface of the melanin granules and is probably identical in both tissues. It retains its pathogenicity in hot polar detergent and during in vitro proteolysis, but it is inactivated by macrophage phagocytosis and hydrolysis in hot hydrochloric acid. Lewis rats immunized with microgram doses of bovine retinal pigment epithelial or choroidal melanin develop severe experimental autoimmune anterior uveitis (EAAU) about 10 days later. Retinitis and pinealitis are not observed. Skin melanin prepared in a similar way evokes EAAU as well, but it is only weakly pathogenic. EAAU cannot be transferred by serum, and its development can effectively be inhibited by antibodies to the inciting antigen and by cyclosporin. Vitamin E treatment of the animals causes a delay in its onset. The results indicate that cell-mediated immunity plays a dominant role in the pathogenesis of EAAU. This is the first time it has been shown that purified ocular and skin melanins are able to induce an autoimmune disease. The relevance of this finding for the study of melanin-related immunopathology in man is discussed.     

Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa2

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.Resistance (R) proteins play a central role in the recognition-based immune system of plants. Unlike vertebrates, plants lack an adaptive immune system with highly specialized immune cells. Instead, they rely on an innate immune system in which each cell is autonomous. Two types of immune receptors can be distinguished in plants, pathogen-associated molecular patterns recognition receptors that detect conserved molecular patterns in plant pathogens and intracellular R proteins that recognize specific effectors employed by pathogens as modifiers of host metabolism or defense mechanisms (Jones and Dangl, 2006). Effector-triggered activation of R proteins leads to an array of protective responses, often culminating in programmed cell death at the site of infection (Greenberg and Yao, 2004), thereby preventing further ingress of the pathogen. Pathogens have evolved mechanisms to evade recognition by R proteins and to regain their virulence (Dodds and Rathjen, 2010). This continuous coevolutionary process between host and pathogen has resulted in a reservoir of highly diverse R proteins in plants, enabling them to counteract a wide range of pathogens and pests.The most common class of R proteins consists of nucleotide-binding (NB)-leucine-rich repeat (LRR) proteins with a tripartite domain architecture, which roughly corresponds to an N-terminal response domain (a coiled coil [CC] or Toll/Interleukin-1 receptor [TIR] domain) involved in downstream signaling, a central molecular switch domain (the NB domain present in the mammalian apoptosis regulator Apaf1, plant R proteins, and the Caenorhabditis elegans apoptosis regulator CED4 [NB-ARC]), and a C-terminal sensor domain (the LRR domain). The NB-ARC domain is an extended nucleotide-binding domain that plant immune receptors share with metazoan apoptosis regulators and immune receptors such as Apaf1, CED4, and nucleotide-binding oligomerization domain (NOD-like) receptors (NLRs) and belongs to the STAND (signal transduction ATPases with numerous domains) family of nucleoside triphosphatase domains (van der Biezen and Jones, 1998; Leipe et al., 2004; Albrecht and Takken, 2006; Maekawa et al., 2011b). The overall modular architecture of metazoan STAND nucleoside triphosphatase is similar to that of NB-LRR plant immune receptors, but the domains flanking the NB-ARC domain often differ. In NLRs, for example, several N-terminal domains can be found, including caspase-recruiting domains and Pyrin domains (Proell et al., 2008). In the mammalian protein Apaf1, the sensor involved in cytochrome c detection consists of C-terminal WD40 repeats (Zou et al., 1997).In plant NB-LRR resistance proteins, the recognition of a pathogen effector via the LRR domain is thought to switch the conformation of the protein from a closed, autoinhibited “off” state into an open, active “on” state (Lukasik and Takken, 2009). The activation of NB-LRR proteins is most likely a multistep process in which the NB-ARC domain plays a central role. The three subdomains of the NB-ARC, the NB, ARC1, and ARC2, collectively form a nucleotide-binding pocket that adopts different conformations depending on the bound nucleotide. This mechanism seems to be conserved between proteins from organisms as distant as bacteria, metazoans, and plants (Rairdan and Moffett, 2007; Danot et al., 2009; Takken and Tameling, 2009). The conformational change coincides with the exchange of bound ADP for ATP in the NB-ARC, probably stabilizing the active conformation (Tameling et al., 2006; Ade et al., 2007). Hydrolysis of the bound ATP is hypothesized to return the domains to their inactive state. The exact mechanism by which elicitor recognition via the LRR leads to a conformational change of the NB-ARC and the subsequent activation of immune signaling pathways is not clear.Previous studies have shown that the CC/TIR, NB-ARC, and LRR domains in plant immune receptors interact and cooperate with each other in an interdependent manner (Moffett et al., 2002; Leister et al., 2005; Ade et al., 2007; Rairdan et al., 2008). From these data, a picture emerges in which the LRR domain is not only involved in pathogen recognition, but also plays a role in maintaining an autoinhibited resting state in the absence of pathogens via its interactions with the other domains (Bendahmane et al., 2002; Hwang and Williamson, 2003; Ade et al., 2007; Qi et al., 2012). A similar role as regulatory domain has been found for the sensor domains of other NLRs, such as the mammalian Apaf1 (Hu et al., 1998). For the potato (Solanum tuberosum) immune receptor Rx1, a model plant NB-LRR protein, it has been shown that the LRR cooperates with the ARC subdomains in retaining the inactive state of the protein. The deletion of the ARC and LRR domains leads to a constitutive activity of the NB (Bendahmane et al., 2002; Rairdan et al., 2008). In addition, it was demonstrated that the elicitor, the Potato virus X (PVX) coat protein, modifies the interdomain interactions in Rx1 (Moffett et al., 2002; Rairdan et al., 2008). Sequence exchanges between Rx1 and the highly homologous nematode resistance protein Gpa2 (88% amino acid identity) resulted in incompatibilities between the domains that give rise to inappropriate activation of cell death responses (Rairdan and Moffett, 2006), indicating that the cooperation between the sensor and switch domains depends on an interaction fine tuned by intramolecular coevolution. In this light, it is interesting to note that a functional ortholog of Rx1, Rx2 from Solanum acaule, is almost identical to Rx1 in its LRR region but displays a higher similarity to Gpa2 in stretches of its CC-NB-ARC sequence (Bendahmane et al., 2000).The aim of our study was to pinpoint the molecular determinants controlling the switch between the resting and activation state of NB-LRR proteins. The incompatibility between the ARC and LRR domains of Rx1 and Gpa2 was used as a guideline to dissect the molecular and structural determinants involved in the cooperation between the switch (NB-ARC) and sensor (LRR) domain. An extensive exchange of polymorphic residues between these two homologous NB-LRR proteins resulted in the identification of a minimal fragment of 68 amino acid residues in the ARC2 domain and the first LRR repeats as being crucial for proper activation of Gpa2 and Rx1. Within this minimal region, we identified two amino acids that, despite their proximity in the amino acid sequence, differentiate between elicitor-dependent (position 401) and independent activation (position 403). However, structural modeling of the domains shows that the residue at position 403 operates at the interface of the ARC2 and N-terminal part of the LRR domain, while residue 401 mapped at the interface between the ARC2 and NB domain. Furthermore, an acidic loop region in the ARC2 domain and complementary-charged basic patches in the N-terminal half of the LRR domain are shown to be required for the physical interaction between these domains. We demonstrate that the binding between the CC- NB-ARC and LRR domains is disrupted upon elicitor-dependent activation and that the complementary-charged residues are predicted to facilitate reassociation. Two independent docking simulations of the NB-ARC and LRR domain indicate that the LRR domain binds to the NB-ARC domain at the surface formed by the interaction of the ARC2 and NB subdomains. We present a mechanistic model in which the first repeats of the LRR, the ARC2 subdomain, and the NB form a clamp, which governs the shuttling between a closed, autoinhibited “off” state and an open, active “on” state of the resistance protein. Finally, we discuss the consequences of the functional constraints imposed by the interface of the NB, ARC2, and LRR domain for the generation of novel resistance specificities via evolutionary processes and genetic engineering.     

Cathepsin-mediated Necrosis Controls the Adaptive Immune Response by Th2 (T helper type 2)-associated Adjuvants

Immunologic adjuvants are critical components of vaccines, but it remains unclear how prototypical adjuvants enhance the adaptive immune response. Recent studies have shown that necrotic cells could trigger an immune response. Although most adjuvants have been shown to be cytotoxic, this activity has traditionally been considered a side effect. We set out to test the role of adjuvant-mediated cell death in immunity and found that alum, the most commonly used adjuvant worldwide, triggers a novel form of cell death in myeloid leukocytes characterized by cathepsin-dependent lysosome-disruption. We demonstrated that direct lysosome-permeabilization with a soluble peptide, Leu-Leu-OMe, mimics the alum-like form of necrotic cell death in terms of cathepsin dependence and cell-type specificity. Using a combination of a haploid genetic screen and cathepsin-deficient cells, we identified specific cathepsins that control lysosome-mediated necrosis. We identified cathepsin C as critical for Leu-Leu-OMe-induced cell death, whereas cathepsins B and S were required for alum-mediated necrosis. Consistent with a role of necrotic cell death in adjuvant effects, Leu-Leu-OMe replicated an alum-like immune response in vivo, characterized by dendritic cell activation, granulocyte recruitment, and production of Th2-associated antibodies. Strikingly, cathepsin C deficiency not only blocked Leu-Leu-OMe-mediated necrosis but also impaired Leu-Leu-OMe-enhanced immunity. Together our findings suggest that necrotic cell death is a powerful mediator of a Th2-associated immune response.     

富亮氨酸重复超家族新成员LRRC4的IgC2结构域蛋白的原核表达和纯化

富亮氨酸重复超家族新成员LRRC4基因是新克隆的脑瘤相关基因,采用多聚酶链式反应（PCR）方法获得长约500bp含IgC2结构域的DNA序列,扩增产物克隆至pGEX-4T-2质粒中,构建GST融合表达质粒,在大肠杆菌中诱导表达融合蛋白,经包涵体沉淀,溶解,Glutathione-Sepharose亲和层析纯化获得融合蛋白,并以Western blot鉴定证实,通过IgC2结构域蛋白的纯化分离该结构域,为进一步研究该结构域及LRRC4基因的结构和功能奠定了基础。     

The Arabidopsis Malectin-Like Leucine-Rich Repeat Receptor-Like Kinase IOS1 Associates with the Pattern Recognition Receptors FLS2 and EFR and Is Critical for Priming of Pattern-Triggered Immunity

Plasma membrane-localized pattern recognition receptors such as FLAGELLIN SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize microbe-associated molecular patterns (MAMPs) to activate the first layer of plant immunity termed pattern-triggered immunity (PTI). A reverse genetics approach with genes responsive to the priming agent β-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hypersusceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants demonstrated defective PTI responses, notably delayed upregulation of PTI marker genes, lower callose deposition, and mitogen-activated protein kinase activities upon bacterial infection or MAMP treatment. Moreover, Arabidopsis lines overexpressing IOS1 were more resistant to P. syringae and demonstrated a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, coimmunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and FLS2 and EFR. IOS1 also associated with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in a ligand-independent manner and positively regulated FLS2/BAK1 complex formation upon MAMP treatment. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a regulatory protein of FLS2- and EFR-mediated signaling that primes PTI activation upon bacterial elicitation.     

Lack of Salt-Inducible Kinase 2 (SIK2) Prevents the Development of Cardiac Hypertrophy in Response to Chronic High-Salt Intake

Cardiac left ventricle hypertrophy (LVH) constitutes a major risk factor for heart failure. Although LVH is most commonly caused by chronic elevation in arterial blood pressure, reduction of blood pressure to normal levels does not always result in regression of LVH, suggesting that additional factors contribute to the development of this pathology. We tested whether genetic preconditions associated with the imbalance in sodium homeostasis could trigger the development of LVH without concomitant increases in blood pressure. The results showed that the presence of a hypertensive variant of α-adducin gene in Milan rats (before they become hypertensive) resulted in elevated expression of genes associated with LVH, and of salt-inducible kinase 2 (SIK2) in the left ventricle (LV). Moreover, the mRNA expression levels of SIK2, α-adducin, and several markers of cardiac hypertrophy were positively correlated in tissue biopsies obtained from human hearts. In addition, we found in cardiac myocytes that α-adducin regulates the expression of SIK2, which in turn mediates the effects of adducin on hypertrophy markers gene activation. Furthermore, evidence that SIK2 is critical for the development of LVH in response to chronic high salt diet (HS) was obtained in mice with ablation of the sik2 gene. Increases in the expression of genes associated with LVH, as well as increases in LV wall thickness upon HS, occurred only in sik2^+/+ but not in sik2^−/− mice. Thus LVH triggered by HS or the presence of a genetic variant of α-adducin requires SIK2 and is independent of elevated blood pressure. Inhibitors of SIK2 may constitute part of a novel therapeutic regimen aimed at prevention/regression of LVH.     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

The Nucleotide-binding Leucine-rich Repeat (NLR) Family Member NLRX1 Mediates Protection against Experimental Autoimmune Encephalomyelitis and Represses Macrophage/Microglia-induced Inflammation

The nucleotide binding domain and leucine-rich repeat-containing (NLR) family of proteins is known to activate innate immunity, and the inflammasome-associated NLRs are prime examples. In contrast, the concept that NLRs can inhibit innate immunity is still debated, and the impact of such inhibitory NLRs in diseases shaped by adaptive immune responses is entirely unexplored. This study demonstrates that, in contrast to other NLRs that activate immunity, NLRX1 plays a protective role in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. When compared with wild-type controls, Nlrx1^−/− mice have significantly worsened clinical scores and heightened CNS tissue damage during EAE. NLRX1 does not alter the production of encephalitogenic T cells in the peripheral lymphatic tissue, but Nlrx1^−/− mice are more susceptible to adoptively transferred myelin-reactive T cells. Analysis of the macrophage and microglial populations indicates that NLRX1 reduces activation during both active and passive EAE models. This work represents the first case of an NLR that attenuates microglia inflammatory activities and protects against a neurodegenerative disease model caused by autoreactive T cells.     

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by ³²P_i revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.The regulation of protein function by phosphorylation and dephosphorylation is a key mechanism of intracellular signaling pathways in eukaryotic organisms. Protein phosphorylation is catalyzed by protein kinases, which are themselves often regulated by phosphorylation (1). The specificity of protein kinases is essential for their cellular functions. In some groups of protein kinases, the specificity is achieved by means of “docking interactions.” Protein kinase docking interactions involve a recognition site on the kinase or a flanking domain that is different from the active site. The most notable example, MAP kinases, uses a docking interaction to specifically recognize substrates, upstream kinases, and phosphatases. Despite the large amount of data on protein kinase docking interactions, e.g. in the MAP kinase field, there is very little information on how these essential interactions are regulated (2–4).3-Phosphoinositide-dependent protein kinase 1 (PDK1)³ belongs to the AGC family of protein kinases and is the activation loop kinase for several other AGC kinases (5). A key feature of the AGC kinase family members except PDK1 is the presence of a C-terminal extension (CT) to the catalytic core that contains a conserved hydrophobic motif (HM) harboring a phosphorylation site. In many AGC kinases, the HM mediates a docking interaction with PDK1. For example, p90 ribosomal S6 kinase (RSK), p70 S6 kinase (S6K) and serum- and glucocorticoid-induced protein kinase (SGK) interact with PDK1 upon phosphorylation of the HM site (6–9). The phosphorylated HM binds to a HM-binding pocket in the catalytic core of PDK1 that was originally termed the PIF-binding pocket (6, 10).Besides its role in the docking of substrates to PDK1, the HM/PIF-binding pocket was also identified as a ubiquitous and key regulatory site in likely all AGC kinases (7, 11). Thus, in AGC kinases studied up to now, the HM/PIF-binding pocket serves as an intramolecular docking site for the phosphorylated HM. In summary, the HM has a dual function in AGC kinase activation, (i) mediating the intermolecular interaction with PDK1 and (ii) acting as an intramolecular allosteric activator that stabilizes the active conformation of the kinase domain via binding to the HM/PIF-binding pocket.The CT of AGC kinases additionally contains a second regulatory phosphorylation site traditionally termed the “turn motif” (TM), and more recently the zipper (Z) site. The Z/TM phosphate interacts with a binding site within the kinase domain, acting like a zipper which serves to support the intramolecular binding of the phosphorylated HM to the HM/PIF-binding pocket (12). Hence, AGC kinases are synergistically activated by phosphorylation at the activation loop, the HM, and the Z/TM sites.Protein kinase C-related protein kinases (PRKs) (13) (also named PAK for protease-activated kinase (14–16) and PKN for protein kinase N (17)) represent a subfamily of AGC kinases. So far, three PRK isoforms were identified, PRK1, PRK2, and PKN3, which are effectors of the small GTP-binding protein Rho. PRKs, as well as the Rho-associated kinases (ROCKs), are considered to be the protein kinases that mediate the phosphorylation events downstream of Rho activation and both can be inhibited by the highly specific protein kinase inhibitor Y27632 (18). The most notable role described for PRK2 is the control of entry into mitosis and exit from cytokinesis (19). In addition, PRK2 phosphorylates the hepatitis C virus (HCV) RNA polymerase (20). In support of a function in HCV RNA replication, PRK2 inhibitors like Y27632 suppress HCV replication (21).The N-terminal region of PRK2 possesses three Rho effector (HR1) domains (13), a pseudosubstrate region that is thought to have an autoinhibitory function (22) and a C2-like domain, which is a potential binding site for lipid activators. The C-terminal region of PRK2 harbors the HM that mediates the docking interaction with the HM/PIF-binding pocket in its upstream kinase PDK1 (10, 23). Interestingly, PRKs and also atypical protein kinase Cs (PKCs, PKCζ, and PKCι/λ), contain an acidic residue instead of a phosphorylatable amino acid at the site equivalent to the HM phosphorylation site in other AGC kinases. Therefore, the molecular events that regulate the interaction of PRK2 and PKCζ with PDK1 must be different from the mechanism characterized for S6K, SGK, and RSK.In the present work we extended and refined the model of docking interaction between PRK2 and PDK1 and characterized C-terminal regions of PRK2 that participate in the regulation of this interaction. The work sheds light on the common as well as specific mechanisms that operate in the regulation of PDK1 docking interaction with its different substrates.