Structure of the ROC domain from the Parkinson's disease-associated leucine-rich repeat kinase 2 reveals a dimeric GTPase

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity. The structure of ROC and the mechanism(s) by which it regulates kinase activity are not known. Here, we report the crystal structure of the LRRK2 ROC domain in complex with GDP-Mg2+ at 2.0-Å resolution. The structure displays a dimeric fold generated by extensive domain-swapping, resulting in a pair of active sites constructed with essential functional groups contributed from both monomers. Two PD-associated pathogenic residues, R1441 and I1371, are located at the interface of two monomers and provide exquisite interactions to stabilize the ROC dimer. The structure demonstrates that loss of stabilizing forces in the ROC dimer is likely related to decreased GTPase activity resulting from mutations at these sites. Our data suggest that the ROC domain may regulate LRRK2 kinase activity as a dimer, possibly via the C-terminal of ROC (COR) domain as a molecular hinge. The structure of the LRRK2 ROC domain also represents a signature from a previously undescribed class of GTPases from complex proteins and results may provide a unique molecular target for therapeutics in PD.

soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans.

The leucine-rich repeat as a protein recognition motif

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna 1 p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3 Delta 977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3 Delta 977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Gene clustering of the small leucine -rich repeat gene family

The small leucine-rich repeat proteoglycans (or SLRPs) are a group of extracellular proteins (ECM) that belong to the leucine-rich repeat (LRR) superfamily of proteins. The LRR is a protein folding motif composed of 20–30 amino acids with leucines in conserved positions. LRR-containing proteins are present in a broad spectrum of organisms and possess diverse cellular functions and localization. In mammals, the SLRPs are abundant in connective tissues, such as bones, cartilage, tendons, skin, and blood vessels. We have discovered a new member of the class I small leucine rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N-terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9g21.3 where asporin is part of a SLRP gene cluster that includes ECM2, osteoadherin, and osteoglycin. This gene cluster of four LRR-encoding genes is embedded in a 238 kilobase intron of another novel gene named Tes9orf that is expressed primarily in the testes of the adult mouse. The SLRP genes are not present in Drosophila or C. elegans , but reside in three separate gene clusters in the puffer fish, mice and humans. Targeted disruption of individual mouse SLRP genes display minor connective tissue defects such as skin fragility, tendon laxity, minor growth plate defects, and mild osteoporosis. However, double and triple knockouts of SLRP genes exacerbate these phenotypes. Both the double epiphycan/biglycan and the triple PRELP/fibromodulin/biglycan knockout mice exhibit premature osteoarthritis. ^

Caractérisation d'une famille de récepteurs kinases impliqués dans le développement gamétophytique chez Arabidopsis thaliana

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11.

Suppression of Arabidopsis protophloem differentiation and root meristem growth by CLE45 requires the receptor-like kinase BAM3.

Peptide signaling presumably occupies a central role in plant development, yet only few concrete examples of receptor-ligand pairs that act in the context of specific differentiation processes have been described. Here we report that second-site null mutations in the Arabidopsis leucine-rich repeat receptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic root meristem growth defect and the associated perturbed protophloem development of the brevis radix (brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA3/ENDOSPERM SURROUNDING REGION 45 (CLE45) peptide ligand. WT plants transformed with a construct for ectopic overexpression of CLE45 could not be recovered, with the exception of a single severely dwarfed and sterile plant that eventually died. By contrast, we obtained numerous transgenic bam3 mutants transformed with the same construct. These transgenic plants displayed a WT phenotype, however, supporting the notion that CLE45 is the likely BAM3 ligand. The results correlate with the observation that external CLE45 application represses protophloem differentiation in WT, but not in bam3 mutants. BAM3, BRX, and CLE45 are expressed in a similar spatiotemporal trend along the developing protophloem, up to the end of the transition zone. Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brx root meristems, and laser ablation experiments suggest that intertwined regulatory activity of BRX, BAM3, and CLE45 could be involved in the proper transition of protophloem cells from proliferation to differentiation, thereby impinging on postembryonic growth capacity of the root meristem.

Role of an LRR receptor-like kinase in the establishment of a functional root endodermal barrier

The endodermis is a highly conserved cell layer present in the root of all vascular plants, except Lycophytes. This tissue layer establishes a protective diffusion barrier surrounding the vasculature and is expected to prevent passive, uncontrolled flow of nutrients through the root. This barrier property is achieved by the production of Casparian strips (CS), a localized cell wall impregnation of lignin in the anticlinal walls of each endodermal cell, forming a belt-like structure sealing the extracellular space. The CS act as a selective barrier between the external cell layers and the vascular cylinder and are thought to be important in many aspects of root function. For instance, selective nutrient uptake and sequestration from the soil, resistance to different abiotic and biotic stresses are expected to involve functional CS. Although discovered 150 years ago, nothing was known about the genes involved in CS establishment until recently. The use of the model plant Arabidopsis thaliana together with both reverse and forward genetic approaches led to the discovery of an increasing number of genes involved in different steps of CS formation during the last few years. One of these genes encodes SCHENGEN3 (SGN3), a leucine-rich repeat receptor-like kinase (LRR-RLK). SGN3 was discovered first by reverse genetic due to its endodermis-enriched expression, and the corresponding mutant displays strong endodermal permeability of the apoplastic tracer Propidium Iodide (PI) indicative of defective CS. One aim of this thesis is to study the role of SGN3 at the molecular level in order to understand its involvement in establishing an impermeable CS. The endodermal permeability of sgn3 is shown to be the result of incorrect localization of key proteins involved in CS establishment (the "Casparian strip domain proteins", CASPs), leading to non-functional CS interrupted by discontinuities. CASPs localize in the plasma membrane domain subjacent to the CS, named the Casparian Strip membrane Domain (CSD). The CSD discontinuities in sgn3 together with SGN3 localization in close proximity to the CASPs lead to the assumption that SGN3 is involved in the formation of a continuous CSD. In addition, SGN3 might have a second role, acting as a kinase reporting CSD integrity leading to lignin and suberin production in CSD/CS defective plants. Up to now, sgn3 is the strongest and most specific CS mutant available, displaying tracer penetration along the whole length of the seedling root. For this reason, this mutant is well suited in order to characterize the physiological behaviour of CS affected plants. Due to the lack of such mutants in the past, it was not possible to test the presumed functions of CS by using plants lacking this structure. We decided to use sgn3 for this purpose. Surprisingly, sgn3 overall growth is only slightly affected. Nevertheless, processes expected to rely on functional CS, such as water transport through the root, nutrient homeostasis, salt tolerance and resistance to an excess of some nutrients are altered in this mutant. On the other hand, homeostasis for most elements and drought tolerance are not affected in sgn3. It is surprising to observe that homeostatic defects are specific, with a decrease in potassium and an increase in magnesium levels. It indicates a backup system, set up by the plant in order to counteract free diffusion of nutrients into the stele. For instance, potassium shortage in sgn3 upregulates the transcription of potassium influx transport proteins and genes known to be induced by potassium starvation. Moreover, sgn3 mutant is hypersensitive to low potassium conditions. Hopefully, these results about SGN3 will help our understanding of CS establishment at the molecular level. In addition, physiological experiments using sgn3 should give us a framework for future experiments and help us to understand the different roles of CS and their involvement during nutrient radial transport through the root. -- L'endoderme est un tissu présent dans les racines de toutes les plantes vasculaires à l'exception des Lycophytes. Ce tissu établit une barrière protectrice entourant les tissus vasculaires dans le but d'éviter la diffusion passive et incontrôlée des nutriments au travers de la racine. Cette propriété de barrière provient de la production des cadres de Caspary, une imprégnation localisée de lignine des parties anticlinales de la paroi de chaque cellule d'endoderme. Cela donne naissance à un anneau/cadre qui rend étanche l'espace extracellulaire. Les cadres de Caspary agissent comme une barrière sélective entre les couches externes de la racine et le cylindre central et sont supposés être importants dans beaucoup d'aspects du fonctionnement de la racine. Par exemple, l'absorption sélective de nutriments et leur séquestration à partir du sol ainsi que la résistance contre différents stress abiotiques et biotiques sont supposés impliquer des cadres de Caspary fonctionnels. Bien que découverts il y a 150 ans, rien n'était connu concernant les gènes impliqués dans Ja formation des cadres de Caspary jusqu'à récemment. Durant ces dernière années, l'utilisation de la plante modèle Arabidopsis thaliana ainsi que des approches de génétique inverse et classique ont permis la découverte d'un nombre croissant de gènes impliqués à différentes étapes de la formation de cette structure. Un des ces gènes code pour SCHENGEN3 (SGN3), un récepteur kinase "leucine-rich repeat receptor-like kinase" (LRR-RLK). SGN3 a été découvert en premier par génétique inverse grâce à son expression enrichie dans l'endoderme. Les cadres de Caspary ne sont pas fonctionnels dans le mutant correspondant, ce qui est visible à cause de la perméabilité de l'endoderme au traceur apoplastique Propidium Iodide (PI). Un des objectifs de cette thèse est d'étudier la fonction de SGN3 au niveau moléculaire dans le but de comprendre son rôle dans la formation des cadres de Caspary. J'ai pu démontrer que la perméabilité de l'endoderme du mutant sgn3 est le résultat de la localisation incorrecte de protéines impliquées dans la formation des cadres de Caspary, les "Casparian strip domain proteins" (CASPs). Cela induit des cadres de Caspary non fonctionnels, contenant de nombreuses interruptions. Les CASPs sont localisés à la membrane plasmique dans un domaine sous-jacent les cadres de Caspary appelé Casparian Strip membrane Domain (CSD). Les interruptions du CSD dans le mutant sgn3, ainsi que la localisation de SGN3 à proximité des CASPs nous font penser à un rôle de SGN3 dans l'élaboration d'un CSD ininterrompu. De plus, SGN3 pourrait avoir un second rôle, agissant en tant que kinase reportant l'intégrité du CSD et induisant la production de lignine et de subérine dans des plantes contenant des cadres de Caspary non fonctionnels. Jusqu'à ce jour, sgn3 est le mutant en notre possession le plus fort et le plus spécifique, ayant un endoderme perméable tout le long de la racine. Pour cette raison, ce mutant est adéquat dans le but de caractériser la physiologie de plantes ayant des cadres de Caspary affectés. De manière surprenante, la croissance de sgn3 est seulement peu affectée. Néanmoins, des processus censés nécessiter des cadres de Caspary fonctionnels, comme le transport de l'eau au travers de la racine, l'homéostasie des nutriments, la tolérance au sel et la résistance à l'excès de certains nutriments sont altérés dans ce mutant. Malgré tout, l'homéostasie de la plupart des nutriments ainsi que la résistance au stress hydrique ne sont pas affectés dans sgn3. De manière surprenante, les altérations de l'ionome de sgn3 sont spécifiques, avec une diminution de potassium et un excès de magnésium. Cela implique un système de compensation établi par la plante dans le but d'éviter la diffusion passive des nutriments en direction du cylindre central. Par exemple, le manque de potassium dans sgn3 augmente la transcription de transporteurs permettant l'absorption de cet élément. De plus, des gènes connus pour être induits en cas de carence en potassium sont surexprimés dans sgn3 et la croissance de ce mutant est sévèrement affectée dans un substrat pauvre en potassium. Ces résultats concernant SGN3 vont, espérons-le, aider à la compréhension du processus de formation des cadres de Caspary au niveau moléculaire. De plus, les expériences de physiologie utilisant sgn3 présentées dans cette thèse devraient nous donner une base pour des expériences futures et nous permettre de comprendre mieux le rôle des cadres de Caspary, et plus particulièrement leur implication dans le transport radial des nutriments au travers de la racine. -- Les plantes terrestres sont des organismes puisant l'eau et les nutriments dont elles ont besoin pour leur croissance dans le sol grâce à leurs racines. De par leur immobilité, elles doivent s'adapter à des sols contenant des quantités variables de nutriments et il leur est crucial de sélectionner ce dont elles ont besoin afin de ne pas s'intoxiquer. Cette sélection est faite grâce à un filtre formé d'un tissu racinaire interne appelé endoderme. L'endoderme fabrique une barrière imperméable entourant chaque cellule appelée "cadre de Caspary". Ces cadres de Caspary empêchent le libre passage des nutriments, permettant un contrôle précis de leur passage. De plus, ils sont censés permettre de résister contre différents stress environnementaux comme la sécheresse, la salinité du sol ou l'excès de nutriments. Bien que découverts il y a 150 ans, rien n'était connu concernant les gènes impliqués dans la formation des cadres de Caspary jusqu'à récemment. Durant ces dernière années, l'utilisation de la plante modèle Arabidopsis thaliana a permis la découverte d'un nombre croissant de gènes impliqués à différentes étapes de la formation de cette structure. Un de ces gènes code pour SCHENGEN3 (SGN3), un récepteur kinase "leucine-rich repeat receptor-like kinase" (LRR- RLK). Nous montrons dans cette étude que le gène SGN3 est impliqué dans la formation des cadres de Caspary, et que le mutant correspondant sgn3 a des cadres de Caspary interrompus. Ces interruptions rendent l'endoderme perméable, l'empêchant de bloquer le passage des molécules depuis le sol vers le centre de la racine. En utilisant ce mutant, nous avons pu caractériser la physiologie de plantes ayant des cadres de Caspary affectés. Cela a permis de découvrir que le transport de l'eau au travers de la racine était affecté dans le mutant sgn3. De plus, l'accumulation de certains éléments dans les feuilles de ce mutant est altérée. Nous avons également pu montrer une sensibilité de ce mutant à un excès de sel ou de certains nutriments comme le fer et le manganèse.

The receptor-like kinase SlSOBIR1 is differentially modulated by virus infection but its overexpression in tobacco has no significant impact on virus accumulation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information

The three-dimensional structures of leucine-rich repeat (LRR) -containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor. using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general. cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, interior/exterior orientations of side chains. and backbone conformation of the LRR structures can be predicted correctly. On the other hand. the analysis revealed that, in some cases. it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Receptor tipo quinase de tomate: caracterização molecular e implicações nas infecções virais

Pós-graduação em Ciências Biológicas (Genética) - IBB