Correction: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis
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PROVIDER: S-EPMC10795974 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.
| S-EPMC4310589 | biostudies-literature
Project description:OBJECTIVE:SCA12 is a progressive autosomal-dominant disorder, caused by a CAG/CTG repeat expansion in PPP2R2B on chromosome 5q32, and characterized by tremor, gait ataxia, hyperreflexia, dysmetria, abnormal eye movements, anxiety, depression, and sometimes cognitive impairment. Neuroimaging has demonstrated cerebellar and cortical atrophy. We now present the neuropathology of the first autopsied SCA12 brain and utilize cell models to characterize potential mechanisms of SCA12 neurodegeneration. METHODS:A fixed SCA12 brain was examined using gross, microscopic, and immunohistochemical methods. The effect of the repeat expansion on PPP2R2B Bβ1 expression was examined in multiple cell types by transient transfection of constructs containing the PPP2R2B Bβ1 promoter region attached to a luciferase reporter. The neurotoxic effect of PPP2R2B overexpression was examined in transfected rat primary neurons. RESULTS:Neuropathological investigation revealed enlarged ventricles, marked cerebral cortical atrophy and Purkinje cell loss, less-prominent cerebellar and pontine atrophy, and neuronal intranuclear ubiquitin-positive inclusions, consistent with Marinesco bodies, which did not stain for long polyglutamine tracts, alpha-synuclein, tau, or transactive response DNA-binding protein 43. Reporter assays demonstrated that the region of PPP2R2B containing the repeat functions as a promoter, and that promoter activity increases with longer repeat length and is dependent on cell type, repeat sequence, and sequence flanking the repeat. Overexpression of PPP2R2B in primary cortical neurons disrupted normal morphology. CONCLUSIONS:SCA12 involves extensive, but selective, neurodegeneration distinct from Alzheimer's disease, synucleinopathies, tauopathies, and glutamine expansion diseases. SCA12 neuropathology may arise from the neurotoxic effect of repeat-expansion-induced overexpression of PPP2R2B.
| S-EPMC5127409 | biostudies-literature
Project description:BackgroundWe investigated whether balance impairments caused by cerebellar disease are associated with specific sensorimotor processing deficits that generalize across all sensory modalities. Experiments focused on the putative cerebellar functions of scaling and coordinate transformation of balance responses evoked by stimulation of single sensory channels.MethodsVestibular, visual, and proprioceptive sensory channels were stimulated in isolation using galvanic vestibular stimulation, moving visual scenery, and muscle vibration, respectively, in 16 subjects with spinocerebellar ataxia type 6 (SCA6) and 16 matched healthy controls. Two polarities of each stimulus type evoked postural responses of similar form in the forward and backward directions. Disease severity was assessed using the Scale for Assessment and Rating of Ataxia.ResultsImpaired balance of SCA6 subjects during unperturbed stance was reflected in faster than normal body sway (P = 0.009), which correlated with disease severity (r = 0.705, P < 0.001). Sensory perturbations revealed a sensorimotor processing abnormality that was specific to response scaling for the visual channel. This manifested as visually evoked postural responses that were approximately three times larger than normal (backward, P < 0.001; forward P = 0.005) and correlated with disease severity (r = 0.543, P = 0.03). Response direction and habituation properties were no different from controls for all three sensory modalities.ConclusionCerebellar degeneration disturbs the scaling of postural responses evoked by visual motion, possibly through disinhibition of extracerebellar visuomotor centers. The excessively high gain of the visuomotor channel without compensatory decreases in gains of other sensorimotor channels provides a potential mechanism for instability of the balance control system in cerebellar disease.
| S-EPMC4949507 | biostudies-literature
Project description:Machado-Joseph disease (MJD) or spinocerebellar ataxia 3 (SCA3) is a rare, inherited, monogenic, neurodegenerative disease, and the most common SCA worldwide. MJD/SCA3 causative mutation is an abnormal expansion of the triplet CAG at exon 10 within the ATXN3 gene. The gene encodes for ataxin-3, which is a deubiquitinating protein that is also involved in transcriptional regulation. In normal conditions, the ataxin-3 protein polyglutamine stretch has between 13 and 49 glutamines. However, in MJD/SCA3 patients, the size of the stretch increases from 55 to 87, contributing to abnormal protein conformation, insolubility, and aggregation. The formation of aggregates, which is a hallmark of MJD/SCA3, compromises different cell pathways, leading to an impairment of cell clearance mechanisms, such as autophagy. MJD/SCA3 patients display several signals and symptoms in which the most prominent is ataxia. Neuropathologically, the regions most affected are the cerebellum and the pons. Currently, there are no disease-modifying therapies, and patients rely only on supportive and symptomatic treatments. Due to these facts, there is a huge research effort to develop therapeutic strategies for this incurable disease. This review aims to bring together current state-of-the-art strategies regarding the autophagy pathway in MJD/SCA3, focusing on evidence for its impairment in the disease context and, importantly, its targeting for the development of pharmacological and gene-based therapies.
| S-EPMC10138583 | biostudies-literature
Project description:Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.
| S-EPMC4820033 | biostudies-literature
Project description:Polynucleotide kinase/phosphatase (PNKP) is a DNA strand break repair enzyme that uses separate 5' kinase and 3' phosphatase active sites to convert damaged 5'-hydroxyl/3'-phosphate strand termini to ligatable 5'-phosphate/3'-hydroxyl ends. The phosphatase active site has received particular attention as a target of inhibition in cancer therapy development. The phosphatase domain dephosphorylates a range of single- and double-stranded substrates; however, structural studies have shown that the phosphatase catalytic cleft can bind only single-stranded substrates. Here we use a catalytically inactive but structurally intact phosphatase mutant to probe interactions between PNKP and a variety of single- and double-stranded DNA substrates using an electrophoretic mobility shift assay. This work indicates that the phosphatase domain binds 3'-phosphorylated single-stranded DNAs in a manner that is highly dependent on the presence of the 3'-phosphate. Double-stranded substrate binding, in contrast, is not as dependent on the 3'-phosphate. Experiments comparing blunt-end, 3'-overhanging, and frayed-end substrates indicate that the predicted loss of energy due to base pair disruption upon binding of the phosphatase active site is likely balanced by favorable interactions between the liberated complementary strand and PNKP. Comparison of the effects on substrate binding of mutations within the phosphatase active site cleft with mutations in surrounding positively charged surfaces suggests that the surrounding surfaces are important for binding to double-stranded substrates. We further show that while fluorescence polarization methods can detect specific binding of single-stranded DNAs with the phosphatase domain, this method does not detect specific interactions between the PNKP phosphatase and double-stranded substrates.
| S-EPMC5469602 | biostudies-literature
Project description:BackgroundSpinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis.ObjectiveHere, we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis.MethodsThe toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if ribosomal RNA (rRNA) processing is disrupted in SCA2 and Huntington's disease (HD) human brain tissue.ResultsexpATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin β-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue.ConclusionThese findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role of expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
| S-EPMC8884117 | biostudies-literature
Project description:Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disease caused by a polyglutamine-encoding trinucleotide repeat expansion in the gene of transcription factor TATA box-binding protein (TBP). While its underlying pathomechanism is elusive, polyglutamine-expanded TBP fragments of unknown origin mediate the mutant protein's toxicity. Calcium-dependent calpain proteases are protagonists in neurodegenerative disorders. Here, we demonstrate that calpains cleave TBP, and emerging C-terminal fragments mislocalize to the cytoplasm. SCA17 cell and rat models exhibited calpain overactivation, leading to excessive fragmentation and depletion of neuronal proteins in vivo. Transcriptome analysis of SCA17 cells revealed synaptogenesis and calcium signaling perturbations, indicating the potential cause of elevated calpain activity. Pharmacological or genetic calpain inhibition reduced TBP cleavage and aggregation, consequently improving cell viability. Our work underlines the general significance of calpains and their activating pathways in neurodegenerative disorders and presents these proteases as novel players in the molecular pathogenesis of SCA17.
| S-EPMC9050766 | biostudies-literature
Project description:T4 polynucleotide kinase-phosphatase (Pnkp) exemplifies a family of enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The polynucleotide 3'-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3'-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid-base catalyst Asp167. We report that Pnkp also has RNA 2'-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2',3'-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNA(OH), mutant D167N converts RNA > p to RNA 3'-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNA(OH). Our results suggest that healing of 2',3'-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3'-phosphoaspartyl)-Pnkp → RNA(3')p + Pnkp → RNA(OH) + phosphoaspartyl-Pnkp → P(i) + Pnkp.
| S-EPMC3592404 | biostudies-literature
Project description:Mammalian polynucleotide kinase (mPNK) is a critical DNA repair enzyme whose 5'-kinase and 3'-phoshatase activities function with poorly understood but striking specificity to restore 5'-phosphate/3'-hydroxyl termini at sites of DNA damage. Here we integrated site-directed mutagenesis and small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of mPNK. The flexible attachment of the FHA domain to the catalytic segment, elucidated by SAXS, enables the interactions of mPNK with diverse DNA substrates and protein partners required for effective orchestration of DNA end repair. Point mutations surrounding the kinase active site identified two substrate recognition surfaces positioned to contact distinct regions on either side of the phosphorylated 5'-hydroxyl. DNA substrates bind across the kinase active site cleft to position the double-stranded portion upstream of the 5'-hydroxyl on one side, and the 3'-overhang on the opposite side. The bipartite DNA-binding surface of the mPNK kinase domain explains its preference for recessed 5'-termini, structures that would be encountered in the course of DNA strand break repair.
| S-EPMC2764422 | biostudies-literature