Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms
Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltra...
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| Veröffentlicht in: | PloS one 2013-04, Vol.8 (4), p.e61442-e61442 |
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| description | Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein. |
| doi_str_mv | 10.1371/journal.pone.0061442 |
| format | Article |
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Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0061442</identifier><identifier>PMID: 23630590</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>alpha-Synuclein - chemistry ; alpha-Synuclein - genetics ; alpha-Synuclein - metabolism ; Alzheimer's disease ; Alzheimers disease ; Amino Acid Sequence ; Amino acids ; Animals ; Autoradiography ; Basal ganglia ; beta-Synuclein - chemistry ; beta-Synuclein - genetics ; beta-Synuclein - metabolism ; Biology ; Brain - metabolism ; Carbon ; Central nervous system diseases ; Chromatography ; Chromatography, Gel ; Cytoplasm - metabolism ; Damage ; Dementia ; Enzymes ; Esters ; Gel electrophoresis ; Humans ; Immunoprecipitation ; Incubation ; Isoaspartic Acid - chemistry ; Isoelectric Point ; Medicine ; Metabolism ; Metabolites ; Methionine ; Methylation ; Methyltransferase ; Mice ; Mice, Knockout ; Molecular Sequence Data ; Movement disorders ; Mutants ; Mutation, Missense ; Nervous system ; Nervous system diseases ; Neurodegenerative diseases ; Neurological diseases ; Oligomers ; Parkinson Disease - genetics ; Parkinson's disease ; Parkinsons disease ; Pathology ; Peptides ; pH effects ; Phosphorylation ; Physiological aspects ; Physiology ; Post-translation ; Post-translational modifications ; Protein D-Aspartate-L-Isoaspartate Methyltransferase - genetics ; Protein D-Aspartate-L-Isoaspartate Methyltransferase - metabolism ; Protein L ; Protein Processing, Post-Translational ; Protein-L-isoaspartate(D-aspartate) O-methyltransferase ; Proteins ; Repair ; Scintillation counters ; Sequence Homology, Amino Acid ; Size exclusion chromatography ; Synuclein ; Transferases</subject><ispartof>PloS one, 2013-04, Vol.8 (4), p.e61442-e61442</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Vigneswara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Vigneswara et al 2013 Vigneswara et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-d6e320eb14bc11a94d44c1713c9e86fff1d88ce6bbb1b4a2aacaca6f7ea8edd23</citedby><cites>FETCH-LOGICAL-c692t-d6e320eb14bc11a94d44c1713c9e86fff1d88ce6bbb1b4a2aacaca6f7ea8edd23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632608/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632608/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23630590$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vigneswara, Vasanthy</creatorcontrib><creatorcontrib>Cass, Simon</creatorcontrib><creatorcontrib>Wayne, Declan</creatorcontrib><creatorcontrib>Bolt, Edward L</creatorcontrib><creatorcontrib>Ray, David E</creatorcontrib><creatorcontrib>Carter, Wayne G</creatorcontrib><title>Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.</description><subject>alpha-Synuclein - chemistry</subject><subject>alpha-Synuclein - genetics</subject><subject>alpha-Synuclein - metabolism</subject><subject>Alzheimer's disease</subject><subject>Alzheimers disease</subject><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Animals</subject><subject>Autoradiography</subject><subject>Basal ganglia</subject><subject>beta-Synuclein - chemistry</subject><subject>beta-Synuclein - genetics</subject><subject>beta-Synuclein - metabolism</subject><subject>Biology</subject><subject>Brain - metabolism</subject><subject>Carbon</subject><subject>Central nervous system diseases</subject><subject>Chromatography</subject><subject>Chromatography, Gel</subject><subject>Cytoplasm - metabolism</subject><subject>Damage</subject><subject>Dementia</subject><subject>Enzymes</subject><subject>Esters</subject><subject>Gel electrophoresis</subject><subject>Humans</subject><subject>Immunoprecipitation</subject><subject>Incubation</subject><subject>Isoaspartic Acid - chemistry</subject><subject>Isoelectric Point</subject><subject>Medicine</subject><subject>Metabolism</subject><subject>Metabolites</subject><subject>Methionine</subject><subject>Methylation</subject><subject>Methyltransferase</subject><subject>Mice</subject><subject>Mice, Knockout</subject><subject>Molecular Sequence Data</subject><subject>Movement disorders</subject><subject>Mutants</subject><subject>Mutation, Missense</subject><subject>Nervous system</subject><subject>Nervous system diseases</subject><subject>Neurodegenerative diseases</subject><subject>Neurological diseases</subject><subject>Oligomers</subject><subject>Parkinson Disease - genetics</subject><subject>Parkinson's disease</subject><subject>Parkinsons disease</subject><subject>Pathology</subject><subject>Peptides</subject><subject>pH effects</subject><subject>Phosphorylation</subject><subject>Physiological aspects</subject><subject>Physiology</subject><subject>Post-translation</subject><subject>Post-translational modifications</subject><subject>Protein D-Aspartate-L-Isoaspartate Methyltransferase - genetics</subject><subject>Protein D-Aspartate-L-Isoaspartate Methyltransferase - metabolism</subject><subject>Protein L</subject><subject>Protein Processing, Post-Translational</subject><subject>Protein-L-isoaspartate(D-aspartate) O-methyltransferase</subject><subject>Proteins</subject><subject>Repair</subject><subject>Scintillation counters</subject><subject>Sequence Homology, Amino Acid</subject><subject>Size exclusion chromatography</subject><subject>Synuclein</subject><subject>Transferases</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNkktv1DAUhSMEoqXwDxBEQkKwyOBXnIQFUql4jFRUCQpb68Z2Zjxy4tROEP33OJ20mqAukBex7O-c63tzkuQ5RitMC_xu50bfgV31rtMrhDhmjDxIjnFFScYJog8P9kfJkxB2COW05PxxckQopyiv0HFy-c1ZLUcLPoWNNt0mdU0Ktt9ClkKn0o96gCxcd6O08Ta8T3vvhrhLFbRRcMN43YPxaavlFjoT2vA0edSADfrZ_D1Jfn7-dHn2NTu_-LI-Oz3PJK_IkCmuKUG6xqyWGEPFFGMSF5jKSpe8aRqsylJqXtc1rhkQABkXbwoNpVaK0JPk5d63ty6IeSBBYEpzlDOMUSTWe0I52Inemxb8tXBgxM2B8xsBfjCxOVErViEMUGCkGKEMsJQclznUeSErWkWvD3O1sW61krobPNiF6fKmM1uxcb9FHDbhqIwGb2YD765GHQbRmiC1tdBpN07vZgXjtCynzl79g97f3UxtIDZgusbFunIyFaesKHFBSDWVXd1DxaV0a2RMT2Pi-ULwdiGIzKD_DBsYQxDrH9__n734tWRfH7BbDXbYBmfHwbguLEG2B6V3IXjd3A0ZIzGF_3YaYgq_mMMfZS8Of9Cd6Dbt9C_g0f9i</recordid><startdate>20130422</startdate><enddate>20130422</enddate><creator>Vigneswara, Vasanthy</creator><creator>Cass, Simon</creator><creator>Wayne, Declan</creator><creator>Bolt, Edward L</creator><creator>Ray, David E</creator><creator>Carter, Wayne G</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130422</creationdate><title>Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms</title><author>Vigneswara, Vasanthy ; Cass, Simon ; Wayne, Declan ; Bolt, Edward L ; Ray, David E ; Carter, Wayne G</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-d6e320eb14bc11a94d44c1713c9e86fff1d88ce6bbb1b4a2aacaca6f7ea8edd23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>alpha-Synuclein - 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Academic</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vigneswara, Vasanthy</au><au>Cass, Simon</au><au>Wayne, Declan</au><au>Bolt, Edward L</au><au>Ray, David E</au><au>Carter, Wayne G</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2013-04-22</date><risdate>2013</risdate><volume>8</volume><issue>4</issue><spage>e61442</spage><epage>e61442</epage><pages>e61442-e61442</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23630590</pmid><doi>10.1371/journal.pone.0061442</doi><tpages>e61442</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2013-04, Vol.8 (4), p.e61442-e61442 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1335054110 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | alpha-Synuclein - chemistry alpha-Synuclein - genetics alpha-Synuclein - metabolism Alzheimer's disease Alzheimers disease Amino Acid Sequence Amino acids Animals Autoradiography Basal ganglia beta-Synuclein - chemistry beta-Synuclein - genetics beta-Synuclein - metabolism Biology Brain - metabolism Carbon Central nervous system diseases Chromatography Chromatography, Gel Cytoplasm - metabolism Damage Dementia Enzymes Esters Gel electrophoresis Humans Immunoprecipitation Incubation Isoaspartic Acid - chemistry Isoelectric Point Medicine Metabolism Metabolites Methionine Methylation Methyltransferase Mice Mice, Knockout Molecular Sequence Data Movement disorders Mutants Mutation, Missense Nervous system Nervous system diseases Neurodegenerative diseases Neurological diseases Oligomers Parkinson Disease - genetics Parkinson's disease Parkinsons disease Pathology Peptides pH effects Phosphorylation Physiological aspects Physiology Post-translation Post-translational modifications Protein D-Aspartate-L-Isoaspartate Methyltransferase - genetics Protein D-Aspartate-L-Isoaspartate Methyltransferase - metabolism Protein L Protein Processing, Post-Translational Protein-L-isoaspartate(D-aspartate) O-methyltransferase Proteins Repair Scintillation counters Sequence Homology, Amino Acid Size exclusion chromatography Synuclein Transferases |
| title | Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms |
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