scholarly journals Parkinson’s Disease-linked LRRK2 structure and model for microtubule interaction

2020 ◽  
Author(s):  
Colin K. Deniston ◽  
John Salogiannis ◽  
Sebastian Mathea ◽  
David M. Snead ◽  
Indarjit Lahiri ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Membrane Trafficking ◽  
Kinase Inhibitors ◽  
Electron Tomography ◽  
Kinase Domain ◽  
Closed Conformation ◽  
Lrrk2 Kinase

AbstractLeucine Rich Repeat Kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson’s disease. LRRK2 is proposed to function in membrane trafficking and co-localizes with microtubules. We report the 3.5Å structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14Å cryo-electron tomography in situ structure. We propose that the conformation of LRRK2’s kinase domain regulates its microtubule interaction, with a closed conformation favoring binding. We show that the catalytic half of LRRK2 is sufficient for microtubule binding and blocks the motility of the microtubule-based motors kinesin and dynein in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 is a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

scholarly journals From structure to ætiology: a new window on the biology of leucine-rich repeat kinase 2 and Parkinson's disease

2021 ◽  
Vol 478 (14) ◽  
pp. 2945-2951
Author(s):  
Susanne Herbst ◽  
Patrick A. Lewis
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Inhibitors ◽  
Leucine Rich Repeat ◽  
Large Size ◽  
Domain Organisation ◽  
Lrrk2 Kinase ◽  
Resolution Structure

Since the discovery of mutations in leucine-rich repeat kinase 2 (LRRK2) as an underlying genetic cause for the development of Parkinson's disease (PD) in 2004 (Neuron 44, 601–607; Neuron 44, 595–600), and subsequent efforts to develop LRRK2 kinase inhibitors as a therapy for Parkinson's (Expert Opin. Ther. Targets 21, 751–753), elucidating the atomic resolution structure of LRRK2 has been a major goal of research into this protein. At over 250 kDa, the large size and complicated domain organisation of LRRK2 has made this a highly challenging target for structural biologists, however, a number of recent studies using both in vitro and in situ approaches (Nature 588, 344–349; Cell 182, 1508–1518.e1516; Cell 184, 3519–3527.e3510) have provided important new insights into LRRK2 structure and the complexes formed by this protein.

scholarly journals A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson’s Disease Using RapidFire Mass Spectrometry

2015 ◽  
Vol 21 (2) ◽  
pp. 145-155 ◽  
Author(s):  
Melanie Leveridge ◽  
Lee Collier ◽  
Colin Edge ◽  
Phil Hardwicke ◽  
Bill Leavens ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
High Throughput ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Label Free ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson’s disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.

scholarly journals Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Martin Steger ◽  
Francesca Tonelli ◽  
Genta Ito ◽  
Paul Davies ◽  
Matthias Trost ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Inhibitors ◽  
Rab Gtpases ◽  
Common Amino Acid ◽  
Bona Fide ◽  
Lrrk2 Kinase ◽  
Conserved Residue

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.

Role of LRRK2 kinase activity in the pathogenesis of Parkinson's disease

2012 ◽  
Vol 40 (5) ◽  
pp. 1058-1062 ◽  
Author(s):  
Elisa Greggio
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Complex Molecule ◽  
Disease Onset ◽  
Missense Mutations ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

Interest in studying the biology of LRRK2 (leucine-rich repeat kinase 2) started in 2004 when missense mutations in the LRRK2 gene were linked to an inherited form of Parkinson's disease with clinical and pathological presentation resembling the sporadic syndrome. LRRK2 is a complex molecule containing domains implicated in protein interactions, as well as kinase and GTPase activities. The observation that the common G2019S mutation increases kinase activity in vitro suggests that altered phosphorylation of LRRK2 targets may have pathological outcomes. Given that protein kinases are ideal targets for drug therapies, much effort has been directed at understanding the role of LRRK2 kinase activity on disease onset. However, no clear physiological substrates have been identified to date, indicating that much research is still needed to fully understand the signalling pathways orchestrated by LRRK2 and deregulated under pathological conditions.

scholarly journals Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110012 ◽  
Author(s):  
Helen I. Woodroof ◽  
Joe H. Pogson ◽  
Mike Begley ◽  
Lewis C. Cantley ◽  
Maria Deak ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Substrate Specificity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Missense Mutations ◽  
Pediculus Humanus ◽  
Tensin Homolog

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro . We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated—namely Drosophila melanogaster (dPINK1) , Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)—are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.

scholarly journals The in Situ structure of a Parkinson's Disease Mutant LRRK2 Bound to Cellular Microtubules Revealed by Cryo-electron Tomography

2020 ◽  
Vol 26 (S2) ◽  
pp. 800-802
Author(s):  
Reika Watanabe ◽  
Robert Buschauer ◽  
Jan Böhning ◽  
Martina Audagnotto ◽  
Keren Lasker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Mutant Lrrk2

scholarly journals Pharmacological rescue of impaired mitophagy in Parkinson’s disease-related LRRK2 G2019S knock-in mice

2020 ◽  
Author(s):  
Francois Singh ◽  
Alan R. Prescott ◽  
Graeme Ball ◽  
Alastair D. Reith ◽  
Ian G. Ganley
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Neurodegenerative Disorder ◽  
Reporter Mice ◽  
Lrrk2 G2019s ◽  
Lrrk2 Kinase

AbstractParkinson’s disease (PD) is a major and progressive neurodegenerative disorder, yet the biological mechanisms involved in its aetiology are poorly understood. Evidence links this disorder with mitochondrial dysfunction and/or impaired lysosomal degradation – key features of the autophagy of mitochondria, known as mitophagy. Here we investigated the role of LRRK2, a protein kinase frequently mutated in PD, on this process in vivo. Using mitophagy and autophagy reporter mice, bearing either knockout of LRRK2 or expressing the pathogenic kinase-activating G2019S LRRK2 mutation, we found that basal mitophagy was specifically altered in clinically relevant cells and tissues. Our data show that basal mitophagy inversely correlates with LRRK2 kinase activity in vivo. In support of this, use of distinct LRRK2 kinase inhibitors in cells increased basal mitophagy, and a CNS penetrant LRRK2 kinase inhibitor, GSK3357679A, rescued the mitophagy defects observed in LRRK2 G2019S mice. This study provides the first in vivo evidence that pathogenic LRRK2 directly impairs basal mitophagy, a process with strong links to idiopathic Parkinson’s disease, and demonstrates that pharmacological inhibition of LRRK2 is a rational mitophagy-rescue approach and potential PD therapy.

scholarly journals Nanobodies as allosteric modulators of Parkinson′s disease-associated LRRK2

2021 ◽  
Author(s):  
Ranjan K. Singh ◽  
Ahmed Soliman ◽  
Giambattista Guaitoli ◽  
Eliza Störmer ◽  
Felix von Zweydorf ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Common Disease ◽  
Type I ◽  
Rab Protein ◽  
Lrrk2 Kinase ◽  
Inhibitory Drug ◽  
Lrrk2 Kinase Activity

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson′s disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

Sign in / Sign up

Export Citation Format

Share Document