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1.
Cell Rep ; 42(12): 113529, 2023 12 26.
Article in English | MEDLINE | ID: mdl-38060380

ABSTRACT

Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.


Subject(s)
Chaperone-Mediated Autophagy , Microautophagy , Autophagy , Endosomes/metabolism , Lysosomes/metabolism , Molecular Chaperones/metabolism
2.
Circ Res ; 133(8): 674-686, 2023 09 29.
Article in English | MEDLINE | ID: mdl-37675562

ABSTRACT

BACKGROUND: The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis. METHODS: Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. RESULTS: In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site. CONCLUSIONS: ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events.


Subject(s)
ADAMTS7 Protein , Atherosclerosis , Coronary Artery Disease , Plaque, Atherosclerotic , Tissue Inhibitor of Metalloproteinase-1 , Animals , Humans , Mice , ADAMTS7 Protein/genetics , Atherosclerosis/genetics , Collagen/metabolism , Coronary Artery Disease/genetics , Matrix Metalloproteinase 9 , Plaque, Atherosclerotic/metabolism , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Mice, Knockout, ApoE
3.
Cardiovasc Res ; 118(4): 1088-1102, 2022 03 16.
Article in English | MEDLINE | ID: mdl-33878186

ABSTRACT

AIMS: Coronary artery disease (CAD) has a strong genetic predisposition. However, despite substantial discoveries made by genome-wide association studies (GWAS), a large proportion of heritability awaits identification. Non-additive genetic effects might be responsible for part of the unaccounted genetic variance. Here, we attempted a proof-of-concept study to identify non-additive genetic effects, namely epistatic interactions, associated with CAD. METHODS AND RESULTS: We tested for epistatic interactions in 10 CAD case-control studies and UK Biobank with focus on 8068 SNPs at 56 loci with known associations with CAD risk. We identified a SNP pair located in cis at the LPA locus, rs1800769 and rs9458001, to be jointly associated with risk for CAD [odds ratio (OR) = 1.37, P = 1.07 × 10-11], peripheral arterial disease (OR = 1.22, P = 2.32 × 10-4), aortic stenosis (OR = 1.47, P = 6.95 × 10-7), hepatic lipoprotein(a) (Lp(a)) transcript levels (beta = 0.39, P = 1.41 × 10-8), and Lp(a) serum levels (beta = 0.58, P = 8.7 × 10-32), while individual SNPs displayed no association. Further exploration of the LPA locus revealed a strong dependency of these associations on a rare variant, rs140570886, that was previously associated with Lp(a) levels. We confirmed increased CAD risk for heterozygous (relative OR = 1.46, P = 9.97 × 10-32) and individuals homozygous for the minor allele (relative OR = 1.77, P = 0.09) of rs140570886. Using forward model selection, we also show that epistatic interactions between rs140570886, rs9458001, and rs1800769 modulate the effects of the rs140570886 risk allele. CONCLUSIONS: These results demonstrate the feasibility of a large-scale knowledge-based epistasis scan and provide rare evidence of an epistatic interaction in a complex human disease. We were directed to a variant (rs140570886) influencing risk through additive genetic as well as epistatic effects. In summary, this study provides deeper insights into the genetic architecture of a locus important for cardiovascular diseases.


Subject(s)
Cardiovascular Diseases , Coronary Artery Disease , Cardiovascular Diseases/diagnosis , Cardiovascular Diseases/genetics , Coronary Artery Disease/diagnostic imaging , Coronary Artery Disease/genetics , Epistasis, Genetic , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Lipoprotein(a)/genetics , Polymorphism, Single Nucleotide
4.
Basic Res Cardiol ; 115(6): 67, 2020 11 13.
Article in English | MEDLINE | ID: mdl-33185739

ABSTRACT

A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (ApoE-/-Svep1+/-) compared to Svep1 wild-type mice (ApoE-/-Svep1+/+) and ApoE-/-Svep1+/- mice displayed elevated plaque neutrophil, Ly6Chigh monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE-/-Svep1+/- mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE-/-Svep1+/- mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency.


Subject(s)
Calcium-Binding Proteins/metabolism , Cell Adhesion Molecules/metabolism , Coronary Artery Disease/metabolism , Coronary Vessels/metabolism , Proteins/metabolism , Animals , Antigens, Ly/metabolism , Calcium-Binding Proteins/deficiency , Calcium-Binding Proteins/genetics , Cell Adhesion Molecules/deficiency , Cell Adhesion Molecules/genetics , Cells, Cultured , Chemokine CXCL1/genetics , Chemokine CXCL1/metabolism , Chemotaxis, Leukocyte , Coronary Artery Disease/genetics , Coronary Artery Disease/pathology , Coronary Vessels/pathology , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/pathology , Genetic Association Studies , Genetic Predisposition to Disease , Haploinsufficiency , Humans , Macrophages/metabolism , Mice, Inbred C57BL , Mice, Knockout, ApoE , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Neutrophil Infiltration , Neutrophils/pathology , Plaque, Atherosclerotic , Polymorphism, Single Nucleotide , Proteins/genetics
5.
Clin Res Cardiol ; 107(Suppl 2): 2-9, 2018 Aug.
Article in English | MEDLINE | ID: mdl-30022276

ABSTRACT

As clinicians, we understand the development of atherosclerosis as a consequence of cholesterol deposition and inflammation in the arterial wall, both being triggered by traditional risk factors such as hypertension, hyperlipidaemia or diabetes mellitus. Another risk factor is genetic predisposition, as indicated by the predictive value of a positive family history. However, we had to wait until recently to appreciate the abundant contribution of genetic variation to the manifestation of atherosclerosis. Indeed, by now 164 chromosomal loci have been identified by genome-wide association studies (GWAS) to affect the risk of coronary artery disease. By design, practically all risk variants discovered by GWAS are frequently found in our population, resulting in the fact that principally every Western European individual carries between 130 and 190 risk alleles at the known, genome-wide significant loci (there are 0, 1, or 2 risk alleles per locus). One can assume that it is this widespread disposition that makes mankind susceptible to the detrimental effects of lifestyle factors, which likewise increase the risk of atherosclerosis. In this review, we summarize the recent genetic discoveries and attempt to group the multiple genetic risk variants in functional groups that may become actionable from a preventive or therapeutic perspective.


Subject(s)
Coronary Artery Disease/genetics , Genetic Predisposition to Disease , Genome-Wide Association Study/methods , Genetic Variation , Genotype , Humans
7.
Aging Cell ; 17(1)2018 02.
Article in English | MEDLINE | ID: mdl-29024336

ABSTRACT

Loss of neuronal proteostasis, a common feature of the aging brain, is accelerated in neurodegenerative disorders, including different types of tauopathies. Aberrant turnover of tau, a microtubule-stabilizing protein, contributes to its accumulation and subsequent toxicity in tauopathy patients' brains. A direct toxic effect of pathogenic forms of tau on the proteolytic systems that normally contribute to their turnover has been proposed. In this study, we analyzed the contribution of three different types of autophagy, macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy to the degradation of tau protein variants and tau mutations associated with this age-related disease. We have found that the pathogenic P301L mutation inhibits degradation of tau by any of the three autophagic pathways, whereas the risk-associated tau mutation A152T reroutes tau for degradation through a different autophagy pathway. We also found defective autophagic degradation of tau when using mutations that mimic common posttranslational modifications in tau or known to promote its aggregation. Interestingly, although most mutations markedly reduced degradation of tau through autophagy, the step of this process preferentially affected varies depending on the type of tau mutation. Overall, our studies unveil a complex interplay between the multiple modifications of tau and selective forms of autophagy that may determine its physiological degradation and its faulty clearance in the disease context.


Subject(s)
Autophagy/drug effects , Neurons/drug effects , Tauopathies/pathology , tau Proteins/pharmacology , Animals , Autophagy/genetics , Brain/drug effects , Brain/metabolism , Humans , Male , Mutation/genetics , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Phosphorylation/drug effects , Proteolysis/drug effects , Rats, Wistar , tau Proteins/metabolism
8.
Hum Mol Genet ; 26(20): 4055-4066, 2017 10 15.
Article in English | MEDLINE | ID: mdl-29016863

ABSTRACT

Congenital cranial dysinnervation disorders (CCDDs) comprise a heterogeneous spectrum of diseases characterized by congenital, non-progressive impairment of eye, eyelid and/or facial movements including Möbius syndrome, Duane retraction syndrome, congenital ptosis, and congenital fibrosis of the extraocular muscles. Over the last 20 years, several CCDDs have been identified as neurodevelopmental disorders that are caused by mutations of genes involved in brain and cranial nerve development, e.g. KIF21A and TUBB3 that each plays a pivotal role for microtubule function. In a five-generation pedigree, we identified a heterozygous mutation of TUBB6, a gene encoding a class V tubulin which has not been linked to a human hereditary disease so far. The missense mutation (p.Phe394Ser) affects an amino acid residue highly conserved in evolution, and co-segregates with a phenotype characterized by congenital non-progressive bilateral facial palsy and congenital velopharyngeal dysfunction presenting with varying degrees of hypomimia, rhinophonia, impaired gag reflex and bilateral ptosis. Expression of the mutated protein in yeast led to an impaired viability compared to wildtype cells when exposed to the microtubule-poison benomyl. Our findings enlarge the spectrum of tubulinopathies and emphasize that mutations of TUBB6 should be considered in patients with congenital non-progressive facial palsy. Further studies are needed to verify whether this phenotype is indeed part of the CCDD spectrum.


Subject(s)
Blepharoptosis/complications , Blepharoptosis/genetics , Facial Paralysis/congenital , Facial Paralysis/genetics , Tubulin/genetics , Velopharyngeal Insufficiency/congenital , Velopharyngeal Insufficiency/genetics , Blepharoptosis/pathology , Child, Preschool , Facial Paralysis/pathology , Female , Genes, Dominant , Humans , Male , Middle Aged , Mutation , Oculomotor Muscles/pathology , Pedigree , Velopharyngeal Insufficiency/pathology
9.
J Biol Chem ; 291(35): 18096-106, 2016 08 26.
Article in English | MEDLINE | ID: mdl-27405763

ABSTRACT

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 µm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.


Subject(s)
Autophagy/physiology , Endosomes/metabolism , HSC70 Heat-Shock Proteins/metabolism , Intracellular Membranes/metabolism , Phosphatidylserines/metabolism , Animals , Cell Line , Endosomes/chemistry , Endosomes/genetics , HSC70 Heat-Shock Proteins/chemistry , HSC70 Heat-Shock Proteins/genetics , Intracellular Membranes/chemistry , Mice , Phosphatidylserines/chemistry , Phosphatidylserines/genetics
10.
Biochem Biophys Res Commun ; 476(4): 528-533, 2016 08 05.
Article in English | MEDLINE | ID: mdl-27262440

ABSTRACT

The Saccharomyces cerevisiae gene VPS35 encodes a component of the retromer complex which is involved in vesicle transport from endosomes to the trans-Golgi network. Yeast and human VPS35 orthologs are highly conserved and mutations in human VPS35 cause an autosomal dominant form of late-onset Parkinson disease (PD). We now show that deletion of VPS35 in yeast (vps35Δ) leads to a dose-dependent growth defect towards copper. This increased sensitivity could be rescued by transformation with yeast wild-type VPS35 but not by the expression of a construct harboring the yeast equivalent (i.e. D686N) of the most commonly identified VPS35-associated PD mutation, p.D620N. In addition, we show that expression of one copy of α-synuclein, which is known to directly interact with copper, leads to a pronounced aggravation of copper toxicity in vps35Δ cells, thereby linking the regulation of copper homeostasis by Vps35p in yeast to one of the key molecules in PD pathophysiology.


Subject(s)
Copper/toxicity , Parkinson Disease/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , Gene Knockout Techniques , Genes, Fungal , Genetic Complementation Test , Humans , Mutation , Parkinson Disease Associated Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Vesicular Transport Proteins/deficiency , alpha-Synuclein/genetics , alpha-Synuclein/metabolism
11.
J Neurosci ; 35(14): 5724-42, 2015 Apr 08.
Article in English | MEDLINE | ID: mdl-25855184

ABSTRACT

Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.


Subject(s)
Adenosine Triphosphatases/deficiency , Brain/metabolism , Lysosomes/metabolism , Membrane Proteins/deficiency , Parkinsonian Disorders/genetics , Parkinsonian Disorders/pathology , alpha-Synuclein/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/ultrastructure , Animals , Brain/pathology , Brain/ultrastructure , Cytosol/metabolism , Cytosol/ultrastructure , Disease Models, Animal , Dopaminergic Neurons/pathology , Endosomes/metabolism , Endosomes/ultrastructure , Exploratory Behavior/physiology , Hindlimb Suspension/psychology , Hydrogen-Ion Concentration , Lipids/analysis , Lysosomes/ultrastructure , Male , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/genetics , Nerve Tissue Proteins/metabolism , Parkinsonian Disorders/physiopathology , Postural Balance/genetics , Proton-Translocating ATPases
12.
Mol Cell ; 48(4): 641-6, 2012 Nov 30.
Article in English | MEDLINE | ID: mdl-23063529

ABSTRACT

Together with GTP and initiator methionyl-tRNA, translation initiation factor eIF2 forms a ternary complex that binds the 40S ribosome and then scans an mRNA to select the AUG start codon for protein synthesis. Here, we show that a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly is caused by a missense mutation in eIF2γ (encoded by EIF2S3), the core subunit of the heterotrimeric eIF2 complex. Biochemical studies of human cells overexpressing the eIF2γ mutant and of yeast eIF2γ with the analogous mutation revealed a defect in binding the eIF2ß subunit to eIF2γ. Consistent with this loss of eIF2 integrity, the yeast eIF2γ mutation impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpressing eIF2ß. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function.


Subject(s)
Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Intellectual Disability/genetics , Peptide Chain Initiation, Translational/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Eukaryotic Initiation Factor-2/chemistry , Humans , Models, Molecular , Mutation, Missense , Saccharomyces cerevisiae Proteins/chemistry
13.
Am J Hum Genet ; 91(5): 919-27, 2012 Nov 02.
Article in English | MEDLINE | ID: mdl-23084290

ABSTRACT

A subset of nuclear-encoded RNAs has to be imported into mitochondria for the proper replication and transcription of the mitochondrial genome and, hence, for proper mitochondrial function. Polynucleotide phosphorylase (PNPase or PNPT1) is one of the very few components known to be involved in this poorly characterized process in mammals. At the organismal level, however, the effect of PNPase dysfunction and impaired mitochondrial RNA import are unknown. By positional cloning, we identified a homozygous PNPT1 missense mutation (c.1424A>G predicting the protein substitution p.Glu475Gly) of a highly conserved PNPase residue within the second RNase-PH domain in a family affected by autosomal-recessive nonsyndromic hearing impairment. In vitro analyses in bacteria, yeast, and mammalian cells showed that the identified mutation results in a hypofunctional protein leading to disturbed PNPase trimerization and impaired mitochondrial RNA import. Immunohistochemistry revealed strong PNPase staining in the murine cochlea, including the sensory hair cells and the auditory ganglion neurons. In summary, we show that a component of the mitochondrial RNA-import machinery is specifically required for auditory function.


Subject(s)
Exoribonucleases/genetics , Hearing Loss, Sensorineural/genetics , Mutation , RNA Transport/genetics , RNA/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chromosome Mapping , Cochlea/metabolism , Cochlea/pathology , Consanguinity , Exons , Exoribonucleases/chemistry , Exoribonucleases/metabolism , Female , Gene Expression , Hearing Loss, Sensorineural/metabolism , Humans , Male , Mice , Models, Molecular , Molecular Sequence Data , Pedigree , Protein Conformation , RNA, Mitochondrial , Zebrafish/genetics , Zebrafish/metabolism
14.
Hum Mutat ; 32(8): 956-64, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21542062

ABSTRACT

Kufor-Rakeb syndrome (KRS) is a rare form of autosomal recessive juvenile or early-onset, levodopa responsive parkinsonism and has been associated with mutations in ATP13A2(also known as PARK9), a lysosomal type 5 P-type ATPase. Recently, we identified novel compound heterozygous mutations, c.3176T>G (p.L1059R) and c.3253delC (p.L1085WfsX1088) in ATP13A2 of two siblings affected with KRS. When overexpressed, wild-type ATP13A2 localized to Lysotracker-positive and LAMP2-positive lysosomes while both truncating and missense mutated ATP13A2 were retained in the endoplasmic reticulum (ER). Both mutant proteins were degraded by the proteasomal but not the lysosomal pathways. In addition, ATP13A2 mRNA with c.3253delC was degraded by nonsense-mediated mRNA decay (NMD), which was protected by cycloheximide treatment. To validate our findings in a biologically relevant setting, we used patient-derived human olfactory neurosphere cultures and fibroblasts and demonstrated persistent ER stress by detecting upregulation of unfolded protein response-related genes in the patient-derived cells. We also confirmed NMD degraded ATP13A2 c.3253delC mRNA in the cells. These findings indicate that these novel ATP13A2 mutations are indeed pathogenic and support the notion that mislocalization of the mutant ATP13A2, resultant ER stress, alterations in the proteasomal pathways and premature degradation of mutant ATP13A2 mRNA contribute to the aetiology of KRS.


Subject(s)
Mutation/genetics , Parkinsonian Disorders/genetics , Parkinsonian Disorders/metabolism , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/metabolism , Adult , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Chlorocebus aethiops , Endoplasmic Reticulum/metabolism , Female , Humans , Lysosomes/metabolism , Male , Molecular Sequence Data , Pedigree , Protein Transport/genetics , RNA Stability , Sequence Alignment , Stress, Physiological/genetics , Young Adult
15.
Biochem Biophys Res Commun ; 383(2): 198-202, 2009 May 29.
Article in English | MEDLINE | ID: mdl-19345671

ABSTRACT

The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P(5B) ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor-Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor-Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.


Subject(s)
Cadmium/toxicity , Manganese/toxicity , Nickel/toxicity , Proton-Translocating ATPases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Selenium/toxicity , Cadmium/metabolism , Cations, Divalent/metabolism , Cations, Divalent/toxicity , Histidine/metabolism , Humans , Manganese/metabolism , Nickel/metabolism , Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Selenium/metabolism , Vacuoles/enzymology
16.
Mov Disord ; 24(3): 429-33, 2009 Feb 15.
Article in English | MEDLINE | ID: mdl-19097176

ABSTRACT

A role of ATP13A2 in early-onset Parkinsonism (EOP) has been proposed. Conversely, the contribution of this ATPase to late-onset Parkinson's disease (PD) remains unexplored. We therefore conducted a case-control association study in this age-of-onset group with PD. The initial sample was of German origin and consisted of 220 patients with late-onset PD (mean age of onset 60.1 years) and 232 age-matched unrelated controls. Five single nucleotide polymorphisms (SNPs) covering ATP13A2 and its common haplotypes were genotyped. The overall association results in this sample were negative. Interestingly, gender stratification gave a positive result for SNP rs11203280 (P(UNC) = 0.016) in men. This result could not be reproduced in a replication sample of German and Serbian origin composed of 161 patients with late-onset PD (mean age of onset 51.7 years) and 150 age- and ethnic-matched controls. In conclusion, we found no consistent evidence for an association between ATP13A2 and late-onset PD.


Subject(s)
Parkinson Disease/genetics , Proton-Translocating ATPases/genetics , Adult , Age of Onset , Aged , Female , Genotype , Haplotypes , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics
17.
Nat Genet ; 38(10): 1184-91, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16964263

ABSTRACT

Neurodegenerative disorders such as Parkinson and Alzheimer disease cause motor and cognitive dysfunction and belong to a heterogeneous group of common and disabling disorders. Although the complex molecular pathophysiology of neurodegeneration is largely unknown, major advances have been achieved by elucidating the genetic defects underlying mendelian forms of these diseases. This has led to the discovery of common pathophysiological pathways such as enhanced oxidative stress, protein misfolding and aggregation and dysfunction of the ubiquitin-proteasome system. Here, we describe loss-of-function mutations in a previously uncharacterized, predominantly neuronal P-type ATPase gene, ATP13A2, underlying an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia (PARK9, Kufor-Rakeb syndrome). Whereas the wild-type protein was located in the lysosome of transiently transfected cells, the unstable truncated mutants were retained in the endoplasmic reticulum and degraded by the proteasome. Our findings link a class of proteins with unknown function and substrate specificity to the protein networks implicated in neurodegeneration and parkinsonism.


Subject(s)
Adenosine Triphosphatases/genetics , Dementia/etiology , Lysosomes/enzymology , Mutation , Parkinsonian Disorders/genetics , Proton-Translocating ATPases/genetics , Adenosine Triphosphatases/metabolism , Dementia/genetics , Endoplasmic Reticulum/enzymology , Female , Humans , Male , Mesencephalon/enzymology , Mesencephalon/pathology , Neurons/enzymology , Neurons/pathology , Parkinsonian Disorders/complications
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