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.

GTP binding controls complex formation by the human ROCO protein MASL1

The human ROCO proteins are a family of multi-domain proteins sharing a conserved ROC-COR supra-domain. The family has four members: leu- cine-rich repeat kinase 1 (LRRK1), leucine-rich repeat kinase 2 (LRRK2), death-associated protein kinase 1 (DAPK1) and malignant fibrous histiocy- toma amplified sequences with leucine-rich tandem repeats 1 (MASL1). Previous studies of LRRK1/2 and DAPK1 have shown that the ROC (Ras of complex proteins) domain can bind and hydrolyse GTP, but the cellular consequences of this activity are still unclear. Here, the first biochemical characterization of MASL1 and the impact of GTP binding on MASL1 complex formation are reported. The results demonstrate that MASL1, similar to other ROCO proteins, can bind guanosine nucleotides via its ROC domain. Furthermore, MASL1 exists in two distinct cellular com- plexes associated with heat shock protein 60, and the formation of a low molecular weight pool of MASL1 is modulated by GTP binding. Finally, loss of GTP enhances MASL1 toxicity in cells. Taken together, these data point to a central role for the ROC/GTPase domain of MASL1 in the reg- ulation of its cellular function.

GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1

The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.

Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

Inhibition of LRRK2 kinase activity stimulates macroautophagy

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific target for disease modifying therapies in patients.

The pgip family in soybean and three other legume species: evidence for a birth-and-death model of evolution

Background Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) plant cell wall glycoproteins involved in plant immunity. They are typically encoded by gene families with a small number of gene copies whose evolutionary origin has been poorly investigated. Here we report the complete characterization of the full complement of the pgip family in soybean (Glycine max [L.] Merr.) and the characterization of the genomic region surrounding the pgip family in four legume species. Results BAC clone and genome sequence analyses showed that the soybean genome contains two pgip loci. Each locus is composed of three clustered genes that are induced following infection with the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary, and remnant sequences of pgip genes. The analyzed homeologous soybean genomic regions (about 126 Kb) that include the pgip loci are strongly conserved and this conservation extends also to the genomes of the legume species Phaseolus vulgaris L., Medicago truncatula Gaertn. and Cicer arietinum L., each containing a single pgip locus. Maximum likelihood-based gene trees suggest that the genes within the pgip clusters have independently undergone tandem duplication in each species. Conclusions The paleopolyploid soybean genome contains two pgip loci comprised in large and highly conserved duplicated regions, which are also conserved in bean, M. truncatula and C. arietinum. The genomic features of these legume pgip families suggest that the forces driving the evolution of pgip genes follow the birth-and-death model, similar to that proposed for the evolution of resistance (R) genes of NBS-LRR-type.

Genetic, structural, and molecular insights into the function of ras of complex proteins domains

Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

The influence of viscous and latent heating on crystal-rich magma flow in a conduit

The flow dynamics of crystal-rich high-viscosity magma is likely to be strongly influenced by viscous and latent heat release. Viscous heating is observed to play an important role in the dynamics of fluids with temperature-dependent viscosities. The growth of microlite crystals and the accompanying release of latent heat should play a similar role in raising fluid temperatures. Earlier models of viscous heating in magmas have shown the potential for unstable (thermal runaway) flow as described by a Gruntfest number, using an Arrhenius temperature dependence for the viscosity, but have not considered crystal growth or latent heating. We present a theoretical model for magma flow in an axisymmetric conduit and consider both heating effects using Finite Element Method techniques. We consider a constant mass flux in a 1-D infinitesimal conduit segment with isothermal and adiabatic boundary conditions and Newtonian and non-Newtonian magma flow properties. We find that the growth of crystals acts to stabilize the flow field and make the magma less likely to experience a thermal runaway. The additional heating influences crystal growth and can counteract supercooling from degassing-induced crystallization and drive the residual melt composition back towards the liquidus temperature. We illustrate the models with results generated using parameters appropriate for the andesite lava dome-forming eruption at Soufriere Hills Volcano, Montserrat. These results emphasize the radial variability of the magma. Both viscous and latent heating effects are shown to be capable of playing a significant role in the eruption dynamics of Soufriere Hills Volcano. Latent heating is a factor in the top two kilometres of the conduit and may be responsible for relatively short-term (days) transients. Viscous heating is less restricted spatially, but because thermal runaway requires periods of hundreds of days to be achieved, the process is likely to be interrupted. Our models show that thermal evolution of the conduit walls could lead to an increase in the effective diameter of flow and an increase in flux at constant magma pressure.

Characteristics of silica-rich pedofeatures in a buried paleosol

Whitish and whitish-light brown milky-like textural pedofeatures and impregnations were found in the voids and the matrix of buried paleosols older than 2.7 million years in a site in Sardinia, Italy. The pedofeatures were described and analysed using micromorphology, X-ray diffraction and microprobe techniques, and their spatial distribution correlated with field evidence. The suite of analyses showed that the main components of the pedofeatures were more or less ordered silica phases. As well as forming a matrix cement, these pedofeatures also occurred as coatings and infillings in pores. Significant amounts of alumina and, less, Mg, Ca and Fe were also present in the pedofeatures, possibly in the form of silicate coatings and inclusions/impurities, or alumino-silicates of the adjacent soil matrix. A number of hypotheses are drawn on the possible mechanisms of formation of these silica-rich pedofeatures, including the possibility of prolonged weathering of volcanic materials and the resulting formation of colloids and more or less ordered silica phases, with successive dehydration and progressive ordering of phases during the at least 2.5 million years. (C) 2003 Elsevier B.V. All rights reserved.

Fe-sulphate-rich evaporative mineral precipitates from the Rio Tinto, southwest Spain

The soluble metal sulphate salts melanterite, rozenite, rhomboclase, szornolnokite, copiapite, coquimbite, hexahydrite and halotrichite, together with gypsum, have been identified, some for the first time oil the banks of the Rio Tinto, SW Spain. Secondary Fe-sulphate minerals call form directly from evaporating acid, sulphate-rich Solutions as a result of pyrite oxidation. Chemical analyses of mixtures of these salt minerals indicate concentrations of Fe (up to 31 wt.%), Mg (up to 4 wt.%), Cu (up to 2 wt.%) and Zn (up to wt.%). These minerals are shown to act as transient storage Cor metals and can store on average up to 10% (9.5 - 11%) and 22% (20-23%) Zn and Cu respectively, of the total discharge of the Rio Tinto during the summer period. Melanterite and rozenite precipitates at Rio Tinto are only found in association with very acidic drainage waters (pH <1.0) draining directly from pyritic waste piles. Copiapite precipitates abundantly oil the banks of the Rio Tinto by (1) direct evaporation of the river water; or (2) as part of a paragenetic sequence with the inclusion of minor halotrichite, indicating natural dehydration and decomposition. The natural occurrences are comparable with the process of paragenesis from the evaporation of Rio Tinto river water under laboratory experiments resulting in the formation of aluminocopiapite, halotrichite, coquimbite, voltaite and gypsum.

The influence of Fe-rich coatings on the dissolution of anorthite at pH 2.6

Surface coatings are very common on mineral grains in soils but most laboratory dissolution experiments are carried out on pristine, uncoated mineral grains. An experiment designed to unambiguously isolate the effect of surface coatings on mineral dissolution from any influence of solution saturation state is reported. Two aliquots of 53 to 63 mum anorthite feldspar powder were used. One was dissolved in pH 2.6 HCl, the other in pH 2.6 FeCl3 solution, both for similar to6000 h in flow-through reactors. An amorphous Fe-rich, Al-, Ca- and Si-free orange precipitate coated the anorthite dissolved in the FeCl3 solution. BET surface area of the anorthite increased from 0.16 to 1.65 m(2) g(-1) in the HCl experiment and to 3.89 m(2) g(-1) in the FeCl3 experiment. The increase in surface area in the HCl experiment was due to the formation of etch pits on the anorthite grain surface whilst the additional increase in the FeCl3 experiment was due to the micro- and meso-porous nature of the orange precipitate. This precipitate did not inhibit or slow the dissolution of the anorthite. Steady state dissolution rates for the anorthite dissolved in the HCl and FeCl3 were similar to2.5 and 3.2 X 10(-10) mol(feldspar) m(-2) s(-1) respectively. These rates are not significantly different after the cumulative uncertainty of 17% in their value due to uncertainty in the inputs parameters used in their calculation is taken into account. Results from this experiment support previous theoretical and inference-based conclusions that porous coatings should not inhibit mineral dissolution. Copyright (C) 2003 Elsevier Ltd.

Characteristics of silica-rich pedofeatures in a buried paleosol

Whitish and whitish-light brown milky-like textural pedofeatures and impregnations were found in the voids and the matrix of buried paleosols older than 2.7 million years in a site in Sardinia, Italy. The pedofeatures were described and analysed using micromorphology, X-ray diffraction and microprobe techniques, and their spatial distribution correlated with field evidence. The suite of analyses showed that the main components of the pedofeatures were more or less ordered silica phases. As well as forming a matrix cement, these pedofeatures also occurred as coatings and infillings in pores. Significant amounts of alumina and, less, Mg, Ca and Fe were also present in the pedofeatures, possibly in the form of silicate coatings and inclusions/impurities, or alumino-silicates of the adjacent soil matrix. A number of hypotheses are drawn on the possible mechanisms of formation of these silica-rich pedofeatures, including the possibility of prolonged weathering of volcanic materials and the resulting formation of colloids and more or less ordered silica phases, with successive dehydration and progressive ordering of phases during the at least 2.5 million years. (C) 2003 Elsevier B.V. All rights reserved.

Triacylglycerol-rich lipoprotein-gene interactions in endothelial cells

Lipoproteins such as LDL (low-density lipoprotein) and oxidized LDL have potentially adverse effects on endothelial cells due to their ability to activate pro-inflammatory pathways regulated via the transcription factor NF-kappaB (nuclear factor kappaB). Triacylglycerol-rich lipoproteins (the chylomicrons, very-low-density lipoprotein and their respective remnant particles) have also been implicated in the induction of a pro-inflammatory phenotype and up-regulation of adhesion molecule expression. Although early studies supported the proposal that LPL (lipoprotein lipase)-mediated hydrolysis of TRLs (triglyceride-rich lipoproteins) at the endothelium could activate the NFkappaB pathway, more recent studies provide evidence of pro-and anti-inflammatory responses when cells are exposed to fatty acids of TRL particles. A large number of genes are up- and down-regulated when cells are exposed to TRL, with the net effect reflecting receptor- and nonreceptor-mediated pathways that are activated or inhibited depending on fatty acid type, the lipid and apolipoprotein composition of the TRL and the presence or absence of LPL. Early concepts of TRL particles as essentially pro-inflammatory stimuli to the endothelium provide an overly simplistic view of their impact on the vascular compartment.

Characterisation of an s-type low molecular weight glutenin subunit of wheat and its proline and glutamine-rich repetitive domain

The low molecular weight glutenin subunits (LMW-GS) are major components of the glutenin polymers which determine the elastomeric properties of wheat (Triticum aestivum L.) gluten and dough. They comprise a complex mixture of components and have proved to be difficult to purify for detailed characterisation. The mature LMW subunit proteins comprise two structural domains, with one domain consisting of repeated sequences based on short peptide motifs. DNA sequences encoding this domain and a whole subunit were expressed in Escherichia coli and the recombinant proteins purified. Detailed comparisons by spectroscopy (CD, FT-IR) and dynamic light scattering indicated that the repetitive and non-repetitive domains of the proteins formed different structures with the former having an extended conformation with an equilibrium between poly-L-proline II-like structure and type II’ b-turns, and the latter a more compact globular structure rich in a-helix. Although the structures of these two domains appear to form independently, dynamic light scattering of the whole subunit dissolved in trifluoroethanol(TFE) suggested that they interact, leading to a more compact conformation. These observations may have relevance to the role of the LMW-GS in gluten structure and functionality.