The R1441C mutation of LRRK2 disrupts GTP hydrolysis

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant form of LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.     

The parkinsonian LRRK2 R1441G mutation shows macroautophagy-mitophagy dysregulation concomitant with endoplasmic reticulum stress

Cell Biology and Toxicology - Autophagy is a mechanism responsible for the degradation of cellular components to maintain their homeostasis. However, autophagy is commonly altered and compromised...     

Efficient allele-specific targeting of LRRK2 R1441 mutations mediated by RNAi

Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75-80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease.     

Computational analysis of folding and mutation properties of C5 domain of myosin binding protein C

Thermal folding molecular dynamics simulations of the domain C5 of Myosin binding protein C were performed using a native-centric model to study the role of three mutations related to Familial Hypertrophic Cardiomyopathy. Mutation of Asn755 causes the largest shift of the folding temperature, and the residue is located in the CFGA' beta-sheet featuring the highest phi-values. The mutation thus appears to reduce the thermodynamic stability in agreement with experimental data. The mutations on Arg654 and Arg668, conversely, cause little change in the folding temperature and they reside in the low phi-value BDE beta-sheet, so that their pathological role cannot be related to impairment of the folding process but possibly to the binding with target molecules. As the typical signature of Domain C5 is the presence of a longer and destibilizing CD-loop with respect to the other Ig-like domains, we completed the work with a bioinformatic analysis of this loop showing a high density of negative charge and low hydrophobicity. This indicates the CD-loop as a natively unfolded sequence with a likely coupling between folding and ligand binding.     

Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson's disease R1441C/G mutants

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are linked to the most common familial forms and some sporadic forms of Parkinson's disease (PD). The LRRK2 protein contains two well-known functional domains, MAPKKK-like kinase and Rab-like GTPase domains. Emerging evidence shows that LRRK2 contains kinase activity which is enhanced in several PD-associated mutants of LRRK2. However, the GTPase activity of LRRK2 has yet to be formally demonstrated. Here, we produced and purified the epitope-tagged LRRK2 protein from transgenic mouse brain, and showed that purified brain LRRK2 possesses both kinase and GTPase activity as assayed by GTP binding and hydrolysis. The brain LRRK2 is associated with elevated kinase activity in comparison to that from transgenic lung or transfected cultured cells. In transfected cell cultures, we detected GTP hydrolysis activity in full-length as well as in GTPase domain of LRRK2. This result indicates that LRRK2 GTPase can be active independent of LRRK2 kinase activity (while LRRK2 kinase activity requires the presence of LRRK2 GTPase as previously shown). We further found that PD mutation R1441C/G in the GTPase domain causes reduced GTP hydrolysis activity, consistent with the altered enzymatic activity in the mutant LRRK2 carrying PD familial mutations. Therefore, our study shows the biochemical characteristics of brain-specific LRRK2 which is associated with robust kinase and GTPase activity. The distinctive levels of kinase/GTPase activity in brain LRRK2 may help explain LRRK2-associated neuronal functions or dysfunctions in the pathogenesis of PD.     

The kinase domain alters the kinetic properties of the myosin IIIA motor

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.     

A core mutation affecting the folding properties of a soluble domain of the ATPase protein CopA from Bacillus subtilis

The two N-terminal domains of the P-type copper ATPase, CopAa and CopAb, from Bacillus subtilis differ in their folding capabilities in vitro. Whereas CopAb has the typical betaalphabetabetaalphabeta structure and is a rigid protein, CopAa is found to be largely unfolded. A sequence analysis of the two and of orthologue homologous proteins indicates that Ser46 in CopAa may destabilise the hydrophobic core, as also confirmed through a bioinformatic energy study. CopAb has a Val in the corresponding position. The S46V and S46A mutants are found to be folded, although the latter displays multiple conformations. S46VCopAa, in both apo and copper(I) loaded forms, has very similar structural and dynamic properties with respect to CopAb, besides a different length of strand beta2 and beta4. It is intriguing that the oxygen of Thr16 is found close, though at longer than bonding distance, to copper in both domains, as it also occurs in a human orthologue domain. This study contributes to understanding the behaviour of proteins that do not properly fold in vitro. A possible biological significance of the peculiar folding behaviour of this domain is discussed.     

Unaltered Striatal Dopamine Release Levels in Young Parkin Knockout,Pink1 Knockout,DJ-1 Knockout and LRRK2 R1441G Transgenic Mice

Parkinson''s disease (PD) is one of the most prevalent neurodegenerative brain diseases; it is accompanied by extensive loss of dopamine (DA) neurons of the substantia nigra that project to the putamen, leading to impaired motor functions. Several genes have been associated with hereditary forms of the disease and transgenic mice have been developed by a number of groups to produce animal models of PD and to explore the basic functions of these genes. Surprisingly, most of the various mouse lines generated such as Parkin KO, Pink1 KO, DJ-1 KO and LRRK2 transgenic have been reported to lack degeneration of nigral DA neuron, one of the hallmarks of PD. However, modest impairments of motor behavior have been reported, suggesting the possibility that the models recapitulate at least some of the early stages of PD, including early dysfunction of DA axon terminals. To further evaluate this possibility, here we provide for the first time a systematic comparison of DA release in four different mouse lines, examined at a young age range, prior to potential age-dependent compensations. Using fast scan cyclic voltammetry in striatal sections prepared from young, 6–8 weeks old mice, we examined sub-second DA overflow evoked by single pulses and action potential trains. Unexpectedly, none of the models displayed any dysfunction of DA overflow or reuptake. These results, compatible with the lack of DA neuron loss in these models, suggest that molecular dysfunctions caused by the absence or mutation of these individual genes are not sufficient to perturb the function and survival of mouse DA neurons.     

The novel C-terminal KCNQ1 mutation M520R alters protein trafficking

The long QT-syndrome is characterized by a prolongation of the QT-interval and tachyarrhythmias causing syncopes and sudden death. We identified the missense mutation M520R in the calmodulin binding domain of the Kv7.1 channel from a German family with long QT-syndrome. Heterologous expression of the mutant did not reveal any whole-cell currents independent of the auxiliary subunit KCNE1. Co-expression of the wild-type Kv7.1 channels and the mutant showed that the mutant did not have a dominant negative effect. In immunocytochemical assays of transfected COS-1 cells wild-type Kv7.1 showed an immunopositive labeling of the plasma membrane. For M520R no plasma membrane staining was visible, instead a strong signal in the ER was observed. These results indicate that the LQT1 mutation M520R leads to ER-retention and dysfunctional trafficking of the mutant channel resulting in haploinsufficiency.     

Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

Background

Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.

Principle Findings

Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.

Conclusion

Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.     

Structure of the Roc-COR domain tandem of C. tepidum, a prokaryotic homologue of the human LRRK2 Parkinson kinase

Correction to: The EMBO Journal (2008) 27, 2239-2249. doi:10.1038/emboj.2008.150.     

A 'hot-spot' mutation alters the mechanical properties of keratin filament networks

Keratins 5 and 14 polymerize to form the intermediate filament network in the progenitor basal cells of many stratified epithelia including epidermis, where it provides crucial mechanical support. Inherited mutations in K5 or K14 result in epidermolysis bullosa simplex (EBS), a skin-fragility disorder. The impact that such mutations exert on the intrinsic mechanical properties of K5/K14 filaments is unknown. Here we show, by using differential interference contrast microscopy, that a 'hot-spot' mutation in K14 greatly reduces the ability of reconstituted mutant filaments to bundle under crosslinking conditions. Rheological assays measure similar small-deformation mechanical responses for crosslinked solutions of wild-type and mutant keratins. The mutation, however, markedly reduces the resilience of crosslinked networks against large deformations. Single-particle tracking, which probes the local organization of filament networks, shows that the mutant polymer exhibits highly heterogeneous structures compared to those of wild-type filaments. Our results indicate that the fragility of epithelial cells expressing mutant keratin may result from an impaired ability of keratin polymers to be crosslinked into a functional network.     

Increased LRRK2 kinase activity alters neuronal autophagy by disrupting the axonal transport of autophagosomes

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R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca²⁺ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (P_o) are associated with Ca²⁺ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca²⁺]_o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca²⁺]_o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca²⁺ transient was normal, despite the reduced SR Ca²⁺ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca²⁺ transients because they are incapable of properly increasing the amount of Ca²⁺ stored in the SR because of a larger SR Ca²⁺ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca²⁺ channel current (I_Ca-L) and SR Ca²⁺ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 P_o and fractional Ca²⁺ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca²⁺ content, but cannot compensate for the inability to increase SR Ca²⁺ content during inotropic challenge.     

Structure of the Roc-COR domain tandem of C. tepidum, a prokaryotic homologue of the human LRRK2 Parkinson kinase

Ras of complex proteins (Roc) belongs to the superfamily of Ras-related small G-proteins that always occurs in tandem with the C-terminal of Roc (COR) domain. This Roc-COR tandem is found in the bacterial and eukaryotic world. Its most prominent member is the leucine-rich repeat kinase LRRK2, which is mutated and activated in Parkinson patients. Here, we investigated biochemically and structurally the Roco protein from Chlorobium tepidum. We show that Roc is highly homologous to Ras, whereas the COR domain is a dimerisation device. The juxtaposition of the G-domains and mutational analysis suggest that the Roc GTPase reaction is stimulated and/or regulated by dimerisation in a nucleotide-dependent manner. The region most conserved between bacteria and man is the interface between Roc and COR, where single-point Parkinson mutations of the Roc and COR domains are in close proximity. The analogous mutations in C. tepidum Roc-COR decrease the GTPase reaction rate, most likely due to a modification of the interaction between the Roc and COR domains.     

Expansion of the octarepeat domain alters the misfolding pathway but not the folding pathway of the prion protein

A misfolded conformation of the prion protein (PrP), PrP (Sc), is the essential component of prions, the infectious agents that cause transmissible neurodegenerative diseases. Insertional mutations that lead to an increase in the number of octarepeats (ORs) in PrP are linked to familial human prion disease. In this study, we investigated how expansion of the OR domain causes PrP to favor a prion-like conformation. Therefore, we compared the conformational and aggregation modulating properties of wild-type versus expanded OR domains, either as a fusion construct with the protein G B1 domain (GB1-OR) or as an integral part of full-length mouse PrP (MoPrP). Using circular dichroism spectroscopy, we first demonstrated that ORs are not unfolded but exist as an ensemble of three distinct conformers: polyproline helix-like, beta-turn, and "Trp-related". Domain expansion had little effect on the conformation of GB1-OR fusion proteins. When part of MoPrP however, OR domain expansion changed PrP's folding landscape, not by hampering the production of native alpha-helical monomers but by greatly reducing the propensity to form amyloid and by altering the assembly of misfolded, beta-rich aggregates. These features may relate to subtle pH-dependent conformational differences between wild-type and mutant monomers. In conclusion, we propose that PrP insertional mutations are pathogenic because they enhance specific misfolding pathways of PrP rather than by undermining native folding. This idea was supported by a trial bioassay in transgenic mice overexpressing wild-type MoPrP, where intracerebral injection of recombinant MoPrP with an expanded OR domain but not wild-type MoPrP caused prion disease.     

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.