DJ-1 AND ATP13A2: TWO PROTEINS INVOLVED IN PARKINSON’S DISEASE

Parkinson\u27s disease (PD) is the second most common neurodegenerative disorder after Alzheimer\u27s disease, affecting approximately 0.3% of the total U.S. population, and its prevalence increases with age. Two neuropathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta, a region in the midbrain involved in initiating and sustaining movement, and the presence of cytosolic inclusions called Lewy bodies (LBs) in various brain regions. LBs are enriched with fibrillar forms of the presynaptic protein &agr;-synuclein (aSyn). Two autosomal recessive genes implicated in familial PD are PARK9, encoding the P-type ATPase ATP13A2, a lysosomal ATPase; and PARK7, encoding DJ-1, a protein with proposed antioxidant and chaperone activities. Understanding the biochemical mechanisms underlying the neuroprotective functions of DJ-1 and mechanistic details accounting for functional overlap between ATP13A2 and DJ-1 can provide insight into the cellular pathways relevant to PD pathogenesis. ^ DJ-1 belongs to the DJ-1/ThiJ/PfpI superfamily, consisting of proteins that typically function as proteases and chaperones. Most members of this superfamily have shared characteristics including a conserved cysteine residue, a catalytic triad, and the ability to form oligomers. Whereas PH1704, a bacterial protease and a member of the DJ-1/ThiJ/PfpI superfamily, adopts a hexameric structure that is necessary for the formation of a catalytic triad, DJ-1 exists as a homodimer and apparently has a catalytic diad rather than a catalytic triad. A recent study has shown that DJ-1Δ15, a truncated form of DJ-1 (15 amino acids cleaved at the C terminus, resulting in the removal of helix H8, has a much greater protease activity compared to full length DJ-1. The structure of DJ-1Δ15 is similar to that of PH1704, which lacks a C-terminal helix corresponding to helix H8 of full-length DJ-1. We chose to focus on DJ-1Δ15 because it was not known whether this variant has the ability to form a hexamer, or whether such a hexameric structure is responsible for the observed protease activity (similar to PH1704). We analyzed DJ-1Δ15 via computer modelling using PyMOL to determine whether it can form a hexamer with favorable intersubunit interactions. In addition, we prepared recombinant DJ-1Δ15 and analyzed the protein via analytical ultra-centrifugation (AUC), native PAGE electrophoresis, and size exclusion chromatography coupled with multi-angle light scattering. Our PyMOL results showed that (i) DJ-1Δ15 can from a hexamer which may be stabilized by two inter-subunit interfaces, referred to as patch 1\u27 and patch 2 and, (ii) hexamer formation may lead to the formation of a catalytic triad involving residues C106 and H126 from one subunit and E84 from another subunit. The full length DJ-1 was unable to form the hexamer because helix H8 caused steric hindrances. Data from AUC and size exclusion analysis of DJ-1Δ15 showed peaks representing species with different assembly states. There was evidence of a species with a molecular weight greater than that of the DJ-1Δ15 dimer. In addition the native gel data of DJ-1Δ15 showed a higher molecular weight species. Taken together, our results suggest that the DJ-1Δ15 protease activity may be due to the formation of an oligomer that is larger than the homodimer—potentially a hexamer. ^ Analysis of the DJ-1 crystal structure revealed a previously undescribed potential ATP binding site that included two arginine residues, Arg 28 and Arg 48, near the oxidizable Cys106 residue. We therefore focused on elucidating whether DJ-1 binds and hydrolyzes ATP. We found that our preparations of recombinant, human WT DJ-1 had ATPase activity that was lost upon filtration or ultracentrifugation. Analysis of our protein samples via sucrose gradient sedimentation coupled with immunoblotting revealed the presence of high molecular weight (high-MW) species immunoreactive with a DJ-1 antibody in our DJ-1 preparations. Additional studies revealed that the high-MW protein fraction of our DJ-1 preparations had ATPase activity and consisted of ring-like structures that could be visualized by electron microscopy. Furthermore, a DJ-1-positive high-MW complex isolated from these preparations by sucrose gradient sedimentation was shown via mass spectrometry analysis to contain F1 ATPase subunits, which are also known to assemble into ring-like structures, suggesting that the ATPase activity in our high molecular weight fraction might be associated with assembled F1 ATPase. Consistent with this idea, the ATPase activity in our high molecular weight protein fraction was abolished in the presence of sodium azide or piceatannol, classical F1 ATPase inhibitors. Furthermore, we obtained evidence that dimeric DJ-1 may interact with purified E-coli F1 ATP synthase, and this interaction was apparently dependent on the presence of a reduced cysteine residue at position 106. These results imply that (i) F1 ATP synthase (in mitochondria) may be a target of the DJ-1 chaperone activity, and (ii) this interaction may be modulated by DJ-1 oxidation. ^ Loss-of-function mutations in the ATP13A2 or DJ-1 gene have been shown to disrupt lysosomal autophagy and interfere with mitochondrial function and quality control. We hypothesized that dysfunction of either protein elicits neurotoxicity by triggering defects in autophagy coupled with an accumulation of dysfunctional mitochondria. To address this hypothesis, we investigated the functional interplay between ATP13A2 and DJ-1 in terms of their ability to protect neuronal cells against a PD related stress, methamphetamine (METH), an abused drug that disrupts autophagy in N27 dopaminergic neuronal cells. Our results showed that knocking down ATP13A2 or DJ-1 results in a buildup of LC3 II, an autophagic marker, whereas ATP13A2 over-expression reduces the accumulation of autophagic vesicles, termed autophagosomes. In addition, ATP13A2 or DJ-1 KD N27 cells showed a decreased rate of O2 consumption. Strikingly, the level of ATP13A2 mRNA was increased in DJ-1 knockdown in primary midbrain cultures; in contrast the level of DJ-1 mRNA was decreased in ATP13A2 knockdown in the same cultures. These results suggest DJ-1 and ATP13A2 interact functionally in regulating lysosomal degradation and mitochondrial function. ^ Overall, these studies have yielded insights into biochemical mechanisms of DJ-1-mediated neuroprotection and of the functional interplay between DJ-1 and ATP13A2. Not only do these findings advance our understanding of neuroprotective mechanisms relevant to PD, but they also suggest new strategies to treat this devastating syndrome

Effect of spermidine on misfolding and interactions of alpha-synuclein.

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation--misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxicshift reprogramming and has glyoxalase activity. We show thatsubstoichiometric concentrations of Hsp31 can abrogate aggrega-tion of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of -synuclein ( Syn), a target of the chaperoneactivity of human DJ-1, an Hsp31 homolog. We demonstrate thatHsp31 is able to suppress the in vitro fibrillization or aggregation of Syn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of Syn cytoplasmic foci, and yeast cells were rescued from Syn-generated proteotoxicity upon Hsp31overexpression. Moreover, we showed that Hsp31 protein levels are increased byH2O2, in the diauxic phase of normal growth con-ditions, and in cells under Syn-mediated proteotoxic stress. Weshow that Hsp31 chaperone activity and not the methylglyoxalaseactivity or the autophagy pathway drives the protective effects.Wealso demonstrate reduced aggregation of the Sup35 prion domain,PrD-Sup35, as visualized by fluorescent protein fusions. In addi-tion, Hsp31 acts on its substrates prior to the formation of largeaggregates because Hsp31 does not mutually localize with prionaggregates, and it prevents the formation of detectable in vitro Syn fibrils. These studies establish that the protective role ofHsp31 against cellular stress is achieved by chaperone activity thatintervenes early in the protein misfolding process and is effectiveona wide spectrum of substrate proteins, including Synandprion proteins

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

DJ-1 and ATP13A2: Two proteins involved in Parkinson\u27s disease

Parkinson\u27s disease (PD) is the second most common neurodegenerative disorder after Alzheimer\u27s disease, affecting approximately 0.3% of the total U.S. population, and its prevalence increases with age. Two neuropathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta, a region in the midbrain involved in initiating and sustaining movement, and the presence of cytosolic inclusions called Lewy bodies (LBs) in various brain regions. LBs are enriched with fibrillar forms of the presynaptic protein &agr;-synuclein (aSyn). Two autosomal recessive genes implicated in familial PD are PARK9, encoding the P-type ATPase ATP13A2, a lysosomal ATPase; and PARK7, encoding DJ-1, a protein with proposed antioxidant and chaperone activities. Understanding the biochemical mechanisms underlying the neuroprotective functions of DJ-1 and mechanistic details accounting for functional overlap between ATP13A2 and DJ-1 can provide insight into the cellular pathways relevant to PD pathogenesis. DJ-1 belongs to the DJ-1/ThiJ/PfpI superfamily, consisting of proteins that typically function as proteases and chaperones. Most members of this superfamily have shared characteristics including a conserved cysteine residue, a catalytic triad, and the ability to form oligomers. Whereas PH1704, a bacterial protease and a member of the DJ-1/ThiJ/PfpI superfamily, adopts a hexameric structure that is necessary for the formation of a catalytic triad, DJ-1 exists as a homodimer and apparently has a catalytic diad rather than a catalytic triad. A recent study has shown that DJ-1Δ15, a truncated form of DJ-1 (15 amino acids cleaved at the C terminus, resulting in the removal of helix H8, has a much greater protease activity compared to full length DJ-1. The structure of DJ-1Δ15 is similar to that of PH1704, which lacks a C-terminal helix corresponding to helix H8 of full-length DJ-1. We chose to focus on DJ-1Δ15 because it was not known whether this variant has the ability to form a hexamer, or whether such a hexameric structure is responsible for the observed protease activity (similar to PH1704). We analyzed DJ-1Δ15 via computer modelling using PyMOL to determine whether it can form a hexamer with favorable intersubunit interactions. In addition, we prepared recombinant DJ-1Δ15 and analyzed the protein via analytical ultra-centrifugation (AUC), native PAGE electrophoresis, and size exclusion chromatography coupled with multi-angle light scattering. Our PyMOL results showed that (i) DJ-1Δ15 can from a hexamer which may be stabilized by two inter-subunit interfaces, referred to as patch 1\u27 and patch 2 and, (ii) hexamer formation may lead to the formation of a catalytic triad involving residues C106 and H126 from one subunit and E84 from another subunit. The full length DJ-1 was unable to form the hexamer because helix H8 caused steric hindrances. Data from AUC and size exclusion analysis of DJ-1Δ15 showed peaks representing species with different assembly states. There was evidence of a species with a molecular weight greater than that of the DJ-1Δ15 dimer. In addition the native gel data of DJ-1Δ15 showed a higher molecular weight species. Taken together, our results suggest that the DJ-1Δ15 protease activity may be due to the formation of an oligomer that is larger than the homodimer—potentially a hexamer. Analysis of the DJ-1 crystal structure revealed a previously undescribed potential ATP binding site that included two arginine residues, Arg 28 and Arg 48, near the oxidizable Cys106 residue. We therefore focused on elucidating whether DJ-1 binds and hydrolyzes ATP. We found that our preparations of recombinant, human WT DJ-1 had ATPase activity that was lost upon filtration or ultracentrifugation. Analysis of our protein samples via sucrose gradient sedimentation coupled with immunoblotting revealed the presence of high molecular weight (high-MW) species immunoreactive with a DJ-1 antibody in our DJ-1 preparations. Additional studies revealed that the high-MW protein fraction of our DJ-1 preparations had ATPase activity and consisted of ring-like structures that could be visualized by electron microscopy. Furthermore, a DJ-1-positive high-MW complex isolated from these preparations by sucrose gradient sedimentation was shown via mass spectrometry analysis to contain F1 ATPase subunits, which are also known to assemble into ring-like structures, suggesting that the ATPase activity in our high molecular weight fraction might be associated with assembled F1 ATPase. Consistent with this idea, the ATPase activity in our high molecular weight protein fraction was abolished in the presence of sodium azide or piceatannol, classical F1 ATPase inhibitors. Furthermore, we obtained evidence that dimeric DJ-1 may interact with purified E-coli F1 ATP synthase, and this interaction was apparently dependent on the presence of a reduced cysteine residue at position 106. These results imply that (i) F1 ATP synthase (in mitochondria) may be a target of the DJ-1 chaperone activity, and (ii) this interaction may be modulated by DJ-1 oxidation. Loss-of-function mutations in the ATP13A2 or DJ-1 gene have been shown to disrupt lysosomal autophagy and interfere with mitochondrial function and quality control. We hypothesized that dysfunction of either protein elicits neurotoxicity by triggering defects in autophagy coupled with an accumulation of dysfunctional mitochondria. To address this hypothesis, we investigated the functional interplay between ATP13A2 and DJ-1 in terms of their ability to protect neuronal cells against a PD related stress, methamphetamine (METH), an abused drug that disrupts autophagy in N27 dopaminergic neuronal cells. Our results showed that knocking down ATP13A2 or DJ-1 results in a buildup of LC3 II, an autophagic marker, whereas ATP13A2 over-expression reduces the accumulation of autophagic vesicles, termed autophagosomes. In addition, ATP13A2 or DJ-1 KD N27 cells showed a decreased rate of O2 consumption. Strikingly, the level of ATP13A2 mRNA was increased in DJ-1 knockdown in primary midbrain cultures; in contrast the level of DJ-1 mRNA was decreased in ATP13A2 knockdown in the same cultures. These results suggest DJ-1 and ATP13A2 interact functionally in regulating lysosomal degradation and mitochondrial function. Overall, these studies have yielded insights into biochemical mechanisms of DJ-1-mediated neuroprotection and of the functional interplay between DJ-1 and ATP13A2. Not only do these findings advance our understanding of neuroprotective mechanisms relevant to PD, but they also suggest new strategies to treat this devastating syndrome

Interaction model of alpha-synuclein molecules.

<p>The model describes three major peaks in the contour length histograms as identical sites in each monomer interacting with each other with the formation of a dimer. A) Position of the interacting site further from the C-terminus (point of attachment) results in longer contour length value. B) Positions at the beginning of the detected interaction sites. Colored arrows correspond to three detected interaction sites schematically shown in A), and the black arrow shows the position of the A30P mutation in alpha-synuclein; C), D) and E) superposition of representative force-distance curves for the detected rupture events corresponding to L_C1, L_C2 and L_C3, red, green and blue lines on the graphs are WLC curves calculated with L_C values from Fig. 4.</p

Schematic representation of the experimental approach.

<p>A) The unstructured form of alpha-synuclein immobilized on the tip and a substrate reveals no interaction. B) Misfolding of alpha-synuclein results in the formation of aggregation-prone conformation with elevated propensity of intermolecular interactions. C) A representative force-distance curve measured in the absence of spermidine. D) A representative force –distance curve with a rupture event in the presence of spermidine: (1) an adhesion peak due to short-range non-specific interactions between the tip and the surface, (2) gradual increase in force characteristic of polymer stretching, (3) complete rupture at 110 pN and (4) region where tip comes free from the surface. The inset shows worm-like chain fitting yielding contour length of 31 nm.</p

Complex force-distance curves with multiple rupture peaks detected with WT alpha-synuclein (A, B) and A30P mutant (C, D).

<p>Red, green and blue lines on the graphs are calculated WLC curves using maxima of Gaussians determined from contour length distributions corresponding to 26, 36 and 47 nm (WT) and 22, 34 and 49 nm (A30P). E) Pie charts showing relative contribution of multiple rupture events to total rupture events: 31% (WT) and 15% (A30P).</p

Effect of spermidine on the appearance of force-distance curves with A30P alpha-synuclein.

<p>Superposition of representative force-distance curves measured upon probing of interactions between A30P alpha-synuclein molecules: A) in the absence of spermidine (40 curves), B) with addition of 5 mM spermidine (58 curves).</p

α‑Synuclein Misfolding Assessed with Single Molecule AFM Force Spectroscopy: Effect of Pathogenic Mutations

Misfolding and subsequent aggregation of alpha-synuclein (α-Syn) protein are critically involved in the development of several neurodegenerative diseases, including Parkinson’s disease (PD). Three familial single point mutations, A30P, E46K, and A53T, correlate with early onset PD; however, the molecular mechanism of the effects of these mutations on the structural properties of α-Syn and its propensity to misfold remains unclear. Here, we address this issue utilizing a single molecule AFM force spectroscopy approach in which structural details of dimers formed by all four variants of α-Syn are characterized. Analysis of the force spectroscopy data reflecting contour length distribution for α-Syn dimer dissociation suggests that multiple segments are involved in the assembly of the dimer. The interactions are not limited to the central nonamyloid-beta component (NAC) of the protein but rather expand beyond this segment. All three mutations alter the protein’s folding and interaction patterns affecting interactions far beyond their immediate locations. Implementation of these findings to our understanding of α-Syn aggregation pathways is discussed