LRR-containing Proteins Research Articles

LvCD14L Acts as a Novel Pattern Recognition Receptor and a Regulator of the Toll Signaling Pathway in Shrimp.

Leucine-rich repeat (LRR) is a structural motif has important recognition function in immune receptors, such as Tolls and NOD-like receptors (NLRs). The immune-related LRR proteins can be divided into two categories, LRR-containing proteins and LRR-only proteins. The latter contain LRR motifs while they are without other functional domains. However, the functional mechanisms of the LRR-only proteins were still unclear in invertebrates. Here, we identified a gene encoding a secretory LRR-only protein, which possessed similarity with vertebrate CD14 and was designated as LvCD14L, from the Pacific whiteleg shrimp Litopenaeus vannamei. Its transcripts in shrimp hemocytes were apparently responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvCD14L with dsRNA resulted in significant increase of the viable bacteria in the hepatopancreas of shrimp upon V. parahaemolyticus infection. Further functional studies revealed that LvCD14L could bind to microorganisms' PAMPs, showed interaction with LvToll1 and LvToll2, and regulated the expression of LvDorsal and LvALF2 in hemocytes. These results suggest that LvCD14L functions as a pattern recognition receptor and activates the NF-κB pathway through interaction with LvTolls. The present study reveals a shrimp LvCD14L-Tolls-NF-κB signaling pathway like the CD14/TLR4/NF-κB signaling pathway in mammalians, which enriches the functional mechanism of secretory LRR-only immune receptors during pathogens infection in invertebrates.

An Oomycete-Specific Leucine-Rich Repeat-Containing Protein Is Involved in Zoospore Flagellum Development in Phytophthora sojae

A leucine-rich repeat (LRR) is a widespread structural motif of 20 to 30 amino acids with characteristic repetitive sequences rich in leucine. LRR-containing proteins are critical for ligand recognition and binding, participating in plant development and defense. Like plants, oomycetes also harbor genes encoding LRR-containing proteins, but their functions remain largely unknown. We identified a zoospore-upregulated gene from Phytophthora sojae with LRRs and an extra structural maintenance of chromosomes-like domain. We generated knockout and complemented knockout strains of this LRR protein and found that its deletion resulted in a pronounced reduction in zoospore mobility and chemotaxis, cyst germination, and virulence. Interestingly, micro-examination of zoospores under a scanning electron microscope revealed irregularly shaped zoospores without flagella in these deletion mutants. In addition, the reintroduction of this LRR protein into the knockout mutant reversed all the deficiencies. Our data demonstrate a critical role for the Phytophthora LRR protein in modulating zoospore development, which impairs migration to the host soybean and affects the spread of Phytophthora pathogens.

Hidden in plain sight: Systematic investigation of Leucine-rich repeat containing genes unveil the their regulatory network in response to Fusarium wilt in tung tree

Leucine-rich repeat-containing genes (LRRs) have been reported to play important roles in responses to diseases. However, we poorly understood the response of LRRs to Fusarium wilt infection in tung tree (Vernicia fordii), which is an important economic tree. Here, 437 LRR-containing proteins containing nine types of LRR domains were identified in V. fordii genome. Phylogenetic analysis suggested that nine types of LRR domains could not be divided into separate classes, implying that these LRR domains had a common origin. Totally, 27 LRRs were related to possible resistance to Fusarium wilt after 2, 8, and 13 days post-inoculation. We further found that Vf06G1605 was up-regulate under Fusarium wilt infection after these three time points, Vf10G1602 and Vf02G1413 were up-regulated at 8, and 13 dpi, while Vf07G2320 was down-regulated at these three time points. The WGCNA and promoter elements suggested that WRKY possibly regulate the responses of LRRs to Fusarium wilt infection. This study highlighted the phylogeny and function of LRRs in V. fordii and provided a systematic analysis of these genes in the V. fordii genome. Our results presented here might clearly illustrate physiological mechanisms of resistance to Fusarium wilt infection and the target of marker-assisted breeding in V. fordii.

Identification and functional study of an LRR domain containing membrane protein in Litopenaeus vannamei

Leucine-rich repeat (LRR) is a vital structure in some pattern recognition receptors such as TLRs, NLRs and newly reported LRR-containing proteins. Apart from some limited reported LRR-containing proteins, most of LRR proteins, especially immune-related proteins, remain uncharacterized functionally. In the present study, a transmembrane protein containing several LRR motifs, designated as LvLRRm, was identified from the shrimp Litopenaeus vannamei. LvLRRm contained a long signal peptide, one LRRNT region, 12 LRR motifs, one LRRCT region and a transmembrane region. The transcripts of LvLRRm were widely distributed in all tested tissues of shrimp and they were responsive to Vibrio parahaemolyticus infection in several immune-related tissues including Oka, intestine, gill and hemocytes. Knockdown of LvLRRm by dsRNA interference led to a decreased survival rate of shrimp infected by Vibrio parahaemolyticus and an increased in vivo Vibrio propagation. Meanwhile, knockdown of LvLRRm also down-regulated the expression levels of genes involved in antibacterial immune signaling pathways, including the transcription factors LvDorsal and LvRelish, and several antimicrobial peptides. These data suggested that LvLRRm played important roles in shrimp against Vibrio infection, which was probably functioning through activation of antibacterial immune signaling pathways. The present study provided new evidence to elucidate the immune function of LRR-containing proteins in invertebrates.

Transcriptional Profile of Aedes aegypti Leucine-Rich Repeat Proteins in Response to Zika and Chikungunya Viruses.

Aedes aegypti (L.) is the primary vector of chikungunya, dengue, yellow fever, and Zika viruses. The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in invertebrates and vertebrates, as well as plants. We focused on the AaeLRIM1 and AaeAPL1 gene expressions in response to Zika virus (ZIKV) and chikungunya virus (CHIKV) infection using a time course study, as well as the developmental expressions in the eggs, larvae, pupae, and adults. RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in Ae. aegypti in response to Zika virus (Accession number: GSE118858, accessed on: August 22, 2018, GEO DataSets). RNA-seq analysis data showed that AaeLRIM1 (AAEL012086-RA) and AaeAPL1 (AAEL009520-RA) were significantly upregulated 2.5 and 3-fold during infection by ZIKV 7-days post infection (dpi) of an Ae. aegypti Key West strain compared to an Orlando strain. The qPCR data showed that LRR-containing proteins related genes, AaeLRIM1 and AaeAPL1, and five paralogues were expressed 100-fold lower than other nuclear genes, such as defensin, during all developmental stages examined. Together, these data provide insights into the transcription profiles of LRR proteins of Ae. aegypti during its development and in response to infection with emergent arboviruses.

Force-Induced Unfolding of Leucine-Rich Repeats of Glycoprotein Ibα Strengthens Ligand Interaction

Leucine-rich repeat (LRR) is a versatile motif widely present in adhesive proteins and signal-transducing receptors. The concave structure formed by a group of LRRs is thought to facilitate binding to globular protein domains with increased affinities. However, little is known about the conformational dynamics of LRRs in such a structure, e.g., whether and how force induces conformational changes in LRRs to regulate protein binding and signal transduction. Here we investigated the platelet glycoprotein Ibα (GPIbα), a demonstrated mechanoreceptor with known crystal structures for the N-terminal domain (GPIbαN), as a model for LRR-containing proteins using a combined method of steered molecular dynamics simulations and single-molecule force spectroscopy with a biomembrane force probe. We found that force-induced unfolding of GPIbαN starts with LRR2–4 and propagates to other LRRs. Importantly, force-dependent lifetimes of individual VWF-A1 bonds with GPIbα are prolonged after LRR unfolding. Enhancement of protein-protein interactions by force-induced LRR unfolding may be a phenomenon of interest in biology.

New role identified for LRR-containing proteins in T cell development.

![Figure][1] Insight from Juan-Carlos Zuniga-Pflucker There are thousands of leucine-rich repeat (LRR)–containing proteins, which share a common structural horseshoe-shaped motif but have distinct and unrelated functions. Among the LRR-containing proteins involved in the immune system

Leucine-Rich Repeat (LRR) Domains Containing Intervening Motifs in Plants.

LRRs (leucine rich repeats) are present in over 14,000 proteins. Non-LRR, island regions (IRs) interrupting LRRs are widely distributed. The present article reviews 19 families of LRR proteins having non-LRR IRs (LRR@IR proteins) from various plant species. The LRR@IR proteins are LRR-containing receptor-like kinases (LRR-RLKs), LRR-containing receptor-like proteins (LRR-RLPs), TONSOKU/BRUSHY1, and MJK13.7; the LRR-RLKs are homologs of TMK1/Rhg4, BRI1, PSKR, PSYR1, Arabidopsis At1g74360, and RPK2, while the LRR-RLPs are those of Cf-9/Cf-4, Cf-2/Cf-5, Ve, HcrVf, RPP27, EIX1, clavata 2, fascinated ear2, RLP2, rice Os10g0479700, and putative soybean disease resistance protein. The LRRs are intersected by single, non-LRR IRs; only the RPK2 homologs have two IRs. In most of the LRR-RLKs and LRR-RLPs, the number of repeat units in the preceding LRR block (N1) is greater than the number of the following block (N2); N1 » N2 in which N1 is variable in the homologs of individual families, while N2 is highly conserved. The five families of the LRR-RLKs except for the RPK2 family show N1 = 8 − 18 and N2 = 3 − 5. The nine families of the LRR-RLPs show N1 = 12 − 33 and N2 = 4; while N1 = 6 and N2 = 4 for the rice Os10g0479700 family and the N1 = 4 − 28 and N2 = 4 for the soybean protein family. The rule of N1 » N2 might play a common, significant role in ligand interaction, dimerization, and/or signal transduction of the LRR-RLKs and the LRR-RLPs. The structure and evolution of the LRR domains with non-LRR IRs and their proteins are also discussed.

Inhibition of Protein-protein Interactions in Plasmodium falciparum: Future Drug Targets

The rapid development by malaria parasites of resistance to almost all the chemotherapeutic agents so far used for their control means that constant efforts to develop new drugs are necessary. In this review, we propose that the exploration of protein-protein interactions as a new strategy to identify antimalarial drug targets is an attractive and a promising area of research. Nevertheless, one of the most important criteria is that the targeted gene should encode an essential protein within a complex that is able to affect parasite survival. Recently, our research on the biology of Plasmodium falciparum allowed us to identify the interaction of Protein Phosphatase type 1 and actin with two essential partners, PfLRR1 and PfLRR7 respectively, both of which belong to the Leucine Rich Repeat (LRR) protein family. LRR-containing proteins are composed of several consensus LRR motifs LXLXXNXL (where X is any amino acid) that provide sites for the assembly of protein interactions. The LRR combines structural versatility, adaptability and more importantly a high degree of interaction specificity. In addition, it has been shown that a single mutation in a particular LRR motif abolishes the protein-protein interaction and contributes to the expression of severe pathology in humans. This clearly infers that blocking the interaction related to 'hot spots' of LRR motifs can be considered as good targets to block parasite growth and development. Thus, the inhibition of protein-protein interactions by peptides, peptidomimetics or small-molecule inhibitors that interfere with binding domains can contribute to defining new potential drug targets.

LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains

Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains.

Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

Comparative structural analysis of the binding domain of follicle stimulating hormone receptor

Proteins with leucine-rich repeats (LRRs) specialize in mediating protein-protein interactions. The hormone binding portion of the receptor for follicle stimulating hormone (FSH) is an LRR protein by sequence, and the crystal structure of this domain from human FSH receptor in a complex with FSH shows that it does indeed have an LRR structure. It differs from other LRR domains, however, in being an all-beta protein composed of highly irregular repeats and having only slight overall curvature. Despite these distinctions and a superficial resemblance to beta-helical proteins, the binding domain of FSH receptor clearly is an LRR protein. The structure does consist of two parts with distinctively different curvatures. Comparison with the structures of other LRR-containing proteins shows a correlation between curvature and main-chain hydrogen bonding pattern of the parallel beta-sheet. The hormone-binding site is located at the concave surface of the receptor structure, a feature common to proteins with LRR motifs. Analysis of the ligand-binding site of LRR-containing proteins reveals that they generally utilize extensive interface area and a large number of charged residues to facilitate high-affinity protein-protein interactions.

Cell Signalling: The Merry Lives of BAK1

Plant receptor-like kinases characterised by leucine-rich repeats have been shown to play dual roles in seemingly unrelated biological processes, inviting comparison with TOLL-like receptors of animals.

Census, molecular characterization and developmental expression of Leucine-Rich-Repeat proteins in Plasmodium falciparum

The capacity of Leucine-Rich Repeat or LRR proteins to interact with many ligands enables them to contribute to important cellular functions ranging from the regulation of the cell cycle to protein trafficking and signal transduction. In Plasmodium falciparum, little is known about the expression of these LRR proteins. Here, we identified the PfLRR genes and determined their transcriptional expression during the intraerythrocytic parasite life cycle. Exhaustive analysis of the P. falciparum genome revealed 14 potential genes encoding LRR-containing proteins, designated from PfLRR1 to PfLRR14. Molecular cloning and sequencing of the corresponding cDNA indicated that all PfLRRs contain 4–10 LRR motifs. Real-time quantitative PCR revealed that most of genes are highly expressed in late intraerythrocytic stages, including late trophozoites and schizonts. The ability of P. falciparum to express LRR-containing proteins will enable further investigations into the parasite interactome and create opportunities for discovering candidate drug targets.

Whole-Genome Sequence ofListeria welshimeriReveals Common Steps in Genome Reduction withListeria innocuaas Compared toListeria monocytogenes

We present the complete genome sequence of Listeria welshimeri, a nonpathogenic member of the genus Listeria. Listeria welshimeri harbors a circular chromosome of 2,814,130 bp with 2,780 open reading frames. Comparative genomic analysis of chromosomal regions between L. welshimeri, Listeria innocua, and Listeria monocytogenes shows strong overall conservation of synteny, with the exception of the translocation of an F(o)F(1) ATP synthase. The smaller size of the L. welshimeri genome is the result of deletions in all of the genes involved in virulence and of "fitness" genes required for intracellular survival, transcription factors, and LPXTG- and LRR-containing proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri relative to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri, including horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria spp.

High expression of a novel leucine-rich repeat protein in hemocytes and the lymphoid organ of the black tiger shrimp Penaeus monodon

A novel leucine-rich repeat (LRR) cDNA has been cloned from hemocytes of the black tiger shrimp Penaeus monodon by 5′ rapid amplification of cDNA ends. The full-length of P. monodon LRR ( PmLRR) consisted of 2604 bp with a 1686-bp open reading frame, encoding 561 amino acids. The deduced protein contained a high proportion of leucine residues (17%) and had significant homology to LRR-containing proteins from bacteria to humans. Sixteen tandem LRR motifs of 23–24 amino acids in length occurred in the primary sequence. The computed 3D structure revealed a horseshoe shape consisting of alternately repeated strand and helical domains. Such structures are generally considered to mediate protein–protein interactions and to our knowledge, this is the first report of an LRR protein from a crustacean. PmLRR expression was tissue-specific (i.e. highest in hemocytes, intestine and lymphoid organ) suggesting that it may play some roles in shrimp defense against pathogens. A preliminary test suggested that PmLRR may be down-regulated after viral injection.

THE EVOLUTION OF ADAPTIVE IMMUNITY

Approximately 500 mya two types of recombinatorial adaptive immune systems appeared in vertebrates. Jawed vertebrates generate a diverse repertoire of B and T cell antigen receptors through the rearrangement of immunoglobulin V, D, and J gene fragments, whereas jawless fish assemble their variable lymphocyte receptors through recombinatorial usage of leucine-rich repeat (LRR) modular units. Invariant germ line-encoded, LRR-containing proteins are pivotal mediators of microbial recognition throughout the plant and animal kingdoms. Whereas the genomes of plants and deuterostome and chordate invertebrates harbor large arsenals of recognition receptors primarily encoding LRR-containing proteins, relatively few innate pattern recognition receptors suffice for survival of pathogen-infected nematodes, insects, and vertebrates. The appearance of a lymphocyte-based recombinatorial system of anticipatory immunity in the vertebrates may have been driven by a need to facilitate developmental and morphological plasticity in addition to the advantage conferred by the ability to recognize a larger portion of the antigenic world.

Expression and interaction analysis of Arabidopsis Skp1-related genes.

Specific protein degradation has been observed in several aspects of development and differentiation in many organisms. One example of such proteolysis is regulated by protein polyubiquitination that is promoted by the SCF complex consisting of Skp1, cullin, and an F-box protein. We examined the activities of the Arabidopsis Skp1-related proteins (ASKs). Among 19 annotated ASK genes, we isolated 16 of the corresponding cDNAs (ASK1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19), and examined their gene products for interactions with 24 representatives of F-box proteins carrying various classes of the C-terminal domains using the yeast two-hybrid system. As a result, we found diverse binding specificities: ASK1, ASK2, ASK11 and ASK12 interacted well with COI1, FKF1, UFO-like protein, LRR-containing F-box proteins, and other F-box proteins with unknown C-terminal motifs. We also observed specific interaction between F-box proteins and ASK3, ASK9, ASK13, ASK14, ASK16 and ASK18. In contrast, we detected no interaction between any of the 12 ASK proteins and F-box proteins containing CRFA, CRFB or CRFC domains. Both histochemical and RT-PCR analysis of eight ASK genes expression revealed unique expression patterns for the respective genes.

Structural principles of leucine-rich repeat (LRR) proteins.

LRR-containing proteins are present in over 2000 proteins from viruses to eukaryotes. Most LRRs are 20-30 amino acids long, and the repeat number ranges from 2 to 42. The known structures of 14 LRR proteins, each containing 4-17 repeats, have revealed that the LRR domains fold into a horseshoe (or arc) shape with a parallel beta-sheet on the concave face and with various secondary structures, including alpha-helix, 3(10)-helix, and pII helix on the convex face. We developed simple methods to charactere quantitatively the arc shape of LRR and then applied them to all known LRR proteins. A quantity of 2Rsin(phi/2), in which R and phi are the radii of the LRR arc and the rotation angle about the central axis per repeating unit, respectively, is highly conserved in all the LRR proteins regardless of a large variety of repeat number and the radius of the LRR arc. The radii of the LRR arc with beta-alpha structural units are smaller than those with beta-3(10) or beta-pII units. The concave face of the LRR beta-sheet forms a surface analogous to a part of a Möbius strip.

Basolateral targeting by leucine-rich repeat domains in epithelial cells.

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure-function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.