Crosstalk between E-Cadherin/β-Catenin and NF-κB Signaling Pathways: The Regulation of Host-Pathogen Interaction during Leptospirosis

Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor β-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/β-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.

Meta-QTLs and candidate genes for stripe rust resistance in wheat

In bread wheat, meta-QTL analysis was conducted using 353 QTLs that were available from earlier studies. When projected onto a dense consensus map comprising 76,753 markers, only 184 QTLs with the required information, could be utilized leading to identification of 61 MQTLs spread over 18 of the 21 chromosomes (barring 5D, 6D and 7D). The range for mean R2 (PVE %) was 1.9% to 48.1%, and that of CI was 0.02 to 11.47 cM; these CIs also carried 37 Yr genes. Using these MQTLs, 385 candidate genes (CGs) were also identified. Out of these CGs, 241 encoded known R proteins and 120 showed differential expression due to stripe rust infection at the seedling stage; the remaining 24 CGs were common in the sense that they encoded R proteins as well as showed differential expression. The proteins encoded by CGs carried the following widely known domains: NBS-LRR domain, WRKY domains, ankyrin repeat domains, sugar transport domains, etc. Thirteen breeders’ MQTLs (PVE > 20%) including four pairs of closely linked MQTLs are recommended for use in wheat molecular breeding, for future studies to understand the molecular mechanism of stripe rust resistance and for gene cloning.

NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly

NLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.

In-Silico Characterization and Expression Analysis of NB-ARC Genes in Response to Erwinia mallotivora in Carica papaya

Disease resistance in plants is commonly associated with resistance (R) genes that encode nucleotide binding site-leucine rich repeat (NBS-LRR) domains that are essential for pathogen recognition and defence signalling. In this study, we identified and analyzed the sequence of putative pathogen-responsive NB-ARC transcripts from Carica papaya transcriptome database, carried out the structural and phylogenetic analysis, and determined the expression profile of the transcripts in C. papaya challenged with Erwinia mallotivora. The findings indicate CpNBS1, the only pathogen-responsive NB-ARC protein identified in this study belongs to the CC-NBS-LRR group. Semi-quantitative PCR showed CpNBS1 was differentially expressed in response to E. mallotivora. Structural analysis of the 4993-Eksotika and 4993-Viorica translated proteins showed striking differences in terms of the number of β-sheets and α-helixes as well their ligand-binding surface, suggesting the role of the LRR domain in determining the specificity of recognition of E. mallotivora effector. Collectively, this study provides new insights into the role of NBS-LRR genes in C. papaya and its implications for enhancing of plant disease resistance through genetic engineering.

Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains

Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.

Inborn Errors in the LRR Domain of Nod2 and Their Potential Consequences on the Function of the Receptor

The innate immune system plays a critical role in the early detection of pathogens, primarily by relying on pattern-recognition receptor (PRR) signaling molecules. Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic receptor that recognizes invading molecules and danger signals inside the cells. Recent studies highlight the importance of NOD2′s function in maintaining the homeostasis of human body microbiota and innate immune responses, including induction of proinflammatory cytokines, regulation of autophagy, modulation of endoplasmic reticulum (ER) stress, etc. In addition, there is extensive cross-talk between NOD2 and the Toll-like receptors that are so important in the induction and tuning of adaptive immunity. Polymorphisms of NOD2′s encoding gene are associated with several pathological conditions, highlighting NOD2′s functional importance. In this study, we summarize NOD2′s role in cellular signaling pathways and take a look at the possible consequences of common NOD2 polymorphisms on the structure and function of this receptor.

DDX3X Links NLRP11 to the Regulation of Type I Interferon Responses and NLRP3 Inflammasome Activation

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.

Structural and Evolutionary Adaptation of NOD-Like Receptors in Birds

NOD-like receptors (NLRs) are intracellular sensors of the innate immune system that recognize intracellular pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Little information exists regarding the incidence of positive selection in the evolution of NLRs of birds or the structural differences between bird and mammal NLRs. Evidence of positive selection was identified in four avian NLRs (NOD1, NLRC3, NLRC5, and NLRP3) using the maximum likelihood approach. These NLRs are under different selection pressures which is indicative of different evolution patterns. Analysis of these NLRs showed a lower percentage of codons under positive selection in the LRR domain than seen in the studies of Toll-like receptors (TLRs), suggesting that the LRR domain evolves differently between NLRs and TLRs. Modeling of human, chicken, mammalian, and avian ancestral NLRs revealed the existence of variable evolution patterns in protein structure that may be adaptively driven.

Controlling the Heterodimerisation of the Phytosulfokine Receptor 1 (PSKR1) via Island Loop Modulation

Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing a pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between a leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor’s island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of the PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel “druggable” binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.

Analysis of intraspecies diversity reveals a subset of highly variable plant immune receptors and predicts their binding sites

Plants have two kinds of pathogen recognition receptors: extracellular receptor like kinases and proteins (RLKs and RLPs) and intracellular Nucleotide-Binding Leucine Rich Repeat (NLR) receptors. NLRs comprise three main domains: a central Nucleotide Binding domain (NB-ARC) that mediates receptor oligomerization upon activation, a C-terminal Leucine Rich Repeat (LRR) domain that defines receptor specificity and an N-terminal domain that mediates immunity. Based on the latter domain, the NLRs are subdivided into three monophyletic groups: RNLs (Resistance to Powdery Mildew8), CNLs (Coiled-Coils) and TNLs (Toll/Interleukin-1 Receptor homology). NLRs can be sensors or signal transducers. As sensors, NLRs can recognize pathogens by directly binding the effectors, by recognizing the effector’s action on other proteins, or by recognition of modifications to a non-canonical NLR domain. Continuous generation of NLR diversity is required to keep up with a range of rapidly evolving pathogens.