Browse
Submit Data
Databases
API
Help

Dataset Information

13 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2.

ABSTRACT: Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson's disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals.

SUBMITTER: Langston RG

PROVIDER: S-EPMC6450775 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity.

Project description:Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease (PD) with a clinical appearance indistinguishable from idiopathic PD. Initial studies suggest that LRRK2 mutations are the most common yet identified determinant of PD susceptibility, transmitted in an autosomal-dominant mode of inheritance. Herein, we characterize the LRRK2 gene and transcript in human brain and subclone the predominant ORF. Exogenously expressed LRRK2 protein migrates at approximately 280 kDa and is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Familial-linked mutations G2019S or R1441C do not have an obvious effect on protein steady-state levels, turnover, or localization. However, in vitro kinase assays using full-length recombinant LRRK2 reveal an increase in activity caused by familial-linked mutations in both autophosphorylation and the phosphorylation of a generic substrate. These results suggest a gain-of-function mechanism for LRRK2-linked disease with a central role for kinase activity in the development of PD.

| S-EPMC1283829 | biostudies-literature

The G2019S pathogenic mutation disrupts sensitivity of leucine-rich repeat kinase 2 to manganese kinase inhibition.

Project description:Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common cause of late-onset Parkinson disease. Previously, we showed that the G2019S pathogenic mutation can cause a dramatic increase (approximately 10-fold) in kinase activity, far above other published studies. A notable experimental difference was the use of Mn-ATP as a substrate. Therefore, the effects of metal cation-ATP cofactors on LRRK2 kinase activity were investigated. It is shown, using several divalent metal cations, that only Mg(2+) or Mn(2+) can support LRRK2 kinase activity. However, for wild-type, I2020T, and R1441C LRRK2, Mn(2+) was significantly less effective at supporting kinase activity. In sharp contrast, both Mn(2+) and Mg(2+) were effective at supporting the activity of G2019S LRRK2. These divergent effects associated with divalent cation usage and the G2019S mutation were predominantly because of differences in catalytic rates. However, LRRK2 was shown to have much lower (approximately 40-fold) ATP K(m) for Mn-ATP compared with Mg-ATP. Consequently, sub-stoichiometric concentrations of Mn(2+) can act to inhibit the kinase activity of wild-type, but not G2019S LRRK2 in the presence of Mg(2+) . From these findings, a new model is proposed for a possible function of LRRK2 and the consequence of the G2019S LRRK2 pathogenic mutation.

| S-EPMC2963190 | biostudies-literature

Leucine Rich Repeat Kinase 2 and Innate Immunity.

Project description:For more than a decade, researchers have sought to uncover the biological function of the enigmatic leucine rich repeat kinase 2 (LRRK2) enzyme, a large multi-domain protein with dual GTPase and kinase activities. Originally identified as a familial Parkinson's disease (PD) risk gene, variations in LRRK2 are also associated with risk of idiopathic PD, inflammatory bowel disease and susceptibility to bacterial infections. LRRK2 is highly expressed in peripheral immune cells and the potential of LRRK2 to regulate immune and inflammatory pathways has emerged as common link across LRRK2-implicated diseases. This review outlines the current genetic and biochemical evidence linking LRRK2 to the regulation of innate immune inflammatory pathways, including the toll-like receptor and inflammasome pathways. Evidence suggests a complex interplay between genetic risk and protective alleles acts to modulate immune outcomes in a manner dependent on the particular pathogen and cell type invaded.

| S-EPMC7077357 | biostudies-literature

The chaperone activity of heat shock protein 90 is critical for maintaining the stability of leucine-rich repeat kinase 2.

Project description:Parkinson's disease (PD), a progressive neurodegenerative disease characterized by bradykinesia, rigidity, and resting tremor, is the most common neurodegenerative movement disorder. Although the majority of PD cases are sporadic, some are inherited, including those caused by leucine-rich repeat kinase 2 (LRRK2) mutations. The substitution of serine for glycine at position 2019 (G2019S) in the kinase domain of LRRK2 represents the most prevalent genetic mutation in both familial and apparently sporadic cases of PD. Because mutations in LRRK2 are likely associated with a toxic gain of function, destabilization of LRRK2 may be a novel way to limit its detrimental effects. Here we show that LRRK2 forms a complex with heat shock protein 90 (Hsp90) in vivo and that inhibition of Hsp90 disrupts the association of Hsp90 with LRRK2 and leads to proteasomal degradation of LRRK2. Hsp90 inhibitors may therefore limit the mutant LRRK2-elicited toxicity to neurons. As a proof of principle, we show that Hsp90 inhibitors rescue the axon growth retardation caused by overexpression of the LRRK2 G2019S mutation in neurons. Therefore, inhibition of LRRK2 kinase activity can be achieved by blocking Hsp90-mediated chaperone activity and Hsp90 inhibitors may serve as potential anti-PD drugs.

| S-EPMC2564280 | biostudies-literature

Substrate phosphorylation of protein microarray by leucine-rich repeat kinase 2

Project description:Numerous leucine-rich repeat kinase 2 mutations identified throughout the protein are associated with Parkinson disease, however the activating G2019S kinase domain mutation is currently regarded as the most common cause of familial and sporadic forms of this disorder. Despite studies demonstrating the prominent role that its kinase activity plays in the pathobiology of leucine-rich repeat kinase 2, few substrates have been identified and only a subset of these have been linked to disease. Therefore, we utilized protein microarrays to screen over 9,000 human proteins in an unbiased radiometric assay for potential targets of the kinase. ProtoArrayM-bM-^DM-" Human Protein Microarrays v5.0 (Invitrogen, Carlsbad, CA, USA) were used following the manufactureM-bM-^@M-^Ys protocol (ProtoArray Kinase Substrate Identification Kit). Briefly, slides were equilibrated at 4C for 15 min before blocking in 1% BSA in PBS for 1 h at 4oC with gentle shaking. Recombinant G2019S or D1994A glutathione-S-transferase (GST)-LRRK2 (970-2527) (Invitrogen) was diluted to 50nM in 20mM Tris (pH 7.5), 10mM MgCl2, 1mM EGTA, 1mM Na3VO4, 5mM beta-glycerophosphate, 2mM DTT, 0.02% polysorbate 20, and 10 mCi /mL of [gamma- 33P]ATP (33 nM final concentration) in a total volume of 120uL. Slides were overlayed with buffer alone, or buffer containing G2019S or D1994A LRRK2, then covered with a coverslip and placed in a 50 mL conical tube for 1 h at 30oC. Afterwards, slides were washed with 0.5% SDS buffer and water followed by centrifugation. Dried slides were exposed to a PhosphorImager plate (Amersham Biosciences, Piscataway, NJ, USA), and scanned on a Storm 840 (Molecular Dynamics, Inc., Sunnyvale, CA, USA) at 50 microns.

2012-07-13 | E-GEOD-30496 | biostudies-arrayexpress

Substrate phosphorylation of protein microarray by leucine-rich repeat kinase 2

2012-07-14 | GSE30496 | GEO

CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation, and toxicity.

Project description:Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.

| S-EPMC2650345 | biostudies-literature

The Parkinson's disease-associated protein, leucine-rich repeat kinase 2 (LRRK2), is an authentic GTPase that stimulates kinase activity.

Project description:Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of autosomal dominant Parkinson's disease (PD). LRRK2, a member of the ROCO protein family, contains both Ras GTPase-like (Roc) and kinase (MAPKKK) domains, as well as other functional motifs. Here, we have identified LRRK2 as the first mammalian ROCO protein that is an authentic and functional GTPase, defined by the ability to bind GTP and undergo intrinsic GTP hydrolysis. Furthermore, the Roc domain is sufficient for this native GTPase activity and binds and hydrolyzes GTP indistinguishably from the Ras-related small GTPase, Rac1. The PD-associated mutation, R1441C, located within the Roc domain, leads to an increase in LRRK2 kinase activity and a decrease in the rate of GTP hydrolysis, compared to the wild-type protein, in an in vitro assay. This finding suggests that the R1441C mutation may help stabilize an activated state of LRRK2. Additionally, LRRK2-mediated phosphorylation is stimulated upon binding of non-hydrolyzable GTP analogs, suggesting that LRRK2 is an MAPKKK-activated intramolecularly by its own GTPase. Since GTPases and MAPKKKs are upstream regulators of multiple signal transduction cascades, LRRK2 may play a central role in integrating pathways involved in neuronal cell signaling and the pathogenesis of PD.

| S-EPMC2083285 | biostudies-literature

The I2020T Leucine-rich repeat kinase 2 transgenic mouse exhibits impaired locomotive ability accompanied by dopaminergic neuron abnormalities.

Project description:BackgroundLeucine-rich repeat kinase 2 (LRRK2) is the gene responsible for autosomal-dominant Parkinson's disease (PD), PARK8, but the mechanism by which LRRK2 mutations cause neuronal dysfunction remains unknown. In the present study, we investigated for the first time a transgenic (TG) mouse strain expressing human LRRK2 with an I2020T mutation in the kinase domain, which had been detected in the patients of the original PARK8 family.ResultsThe TG mouse expressed I2020T LRRK2 in dopaminergic (DA) neurons of the substantia nigra, ventral tegmental area, and olfactory bulb. In both the beam test and rotarod test, the TG mice exhibited impaired locomotive ability in comparison with their non-transgenic (NTG) littermates. Although there was no obvious loss of DA neurons in either the substantia nigra or striatum, the TG brain showed several neurological abnormalities such as a reduced striatal dopamine content, fragmentation of the Golgi apparatus in DA neurons, and an increased degree of microtubule polymerization. Furthermore, the tyrosine hydroxylase-positive primary neurons derived from the TG mouse showed an increased frequency of apoptosis and had neurites with fewer branches and decreased outgrowth in comparison with those derived from the NTG controls.ConclusionsThe I2020T LRRK2 TG mouse exhibited impaired locomotive ability accompanied by several dopaminergic neuron abnormalities. The TG mouse should provide valuable clues to the etiology of PD caused by the LRRK2 mutation.

| S-EPMC3467184 | biostudies-literature

HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission.

Project description:Abcission, the natural shedding of leaves, flowers and fruits, is a fundamental component of plant development. Abscission is a highly regulated process that occurs at distinct zones of cells that undergo enlargement and subsequent separation. Although some components of abscission, including accumulation of the hormone ethylene and cell wall-degrading enzymes, have been described, the regulatory pathways remain largely unknown. In this paper we describe a critical component required for floral organ abscission in Arabidopsis thaliana, the receptor-like protein kinase HAESA. Histochemical analysis of transgenic plants harboring a HAESA promoter:: beta-glucuronidase reporter gene and in situ RNA hybridization experiments show HAESA expression in the abscission zones where the sepals, petals, and stamens attach to the receptacle, at the base of pedicels, and at the base of petioles where leaves attach to the stem. Immunodetection, immunoprecipitation, and protein kinase activity assays reveal HAESA is a plasma membrane serine/threonine protein kinase. The reduction of function of HAESA in transgenic plants harboring an antisense construct results in delayed abscission of floral organs, and the severity of the phenotype is directly correlated with the level of HAESA protein. These results demonstrate that HAESA functions in developmentally regulated floral organ abscission.

| S-EPMC316334 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data