Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization*
Identifieur interne : 000124 ( Pmc/Corpus ); précédent : 000123; suivant : 000125Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization*
Auteurs : Filsy Samuel ; William P. Flavin ; Sobia Iqbal ; Consiglia Pacelli ; Sri Dushyaanthan Sri Renganathan ; Louis-Eric Trudeau ; Edward M. Campbell ; Paul E. Fraser ; Anurag TandonSource :
- The Journal of Biological Chemistry [ 0021-9258 ] ; 2015.
Abstract
Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.
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DOI: 10.1074/jbc.M115.705095
PubMed: 26719332
PubMed Central: 4813466
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<idno type="ISSN">0021-9258</idno>
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<front><div type="abstract" xml:lang="en"><p>Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.</p>
</div>
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<pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id>
<journal-id journal-id-type="iso-abbrev">J. Biol. Chem</journal-id>
<journal-id journal-id-type="hwp">jbc</journal-id>
<journal-id journal-id-type="pmc">jbc</journal-id>
<journal-id journal-id-type="publisher-id">JBC</journal-id>
<journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title>
</journal-title-group>
<issn pub-type="ppub">0021-9258</issn>
<issn pub-type="epub">1083-351X</issn>
<publisher><publisher-name>American Society for Biochemistry and Molecular Biology</publisher-name>
<publisher-loc>11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A.</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">26719332</article-id>
<article-id pub-id-type="pmc">4813466</article-id>
<article-id pub-id-type="publisher-id">M115.705095</article-id>
<article-id pub-id-type="doi">10.1074/jbc.M115.705095</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Molecular Bases of Disease</subject>
</subj-group>
</article-categories>
<title-group><article-title>Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization<xref ref-type="fn" rid="FN1">*</xref>
</article-title>
<alt-title alt-title-type="short">Effects of α-Syn Phosphorylation</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Samuel</surname>
<given-names>Filsy</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
<xref ref-type="author-notes" rid="FN2"><sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="FN3"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Flavin</surname>
<given-names>William P.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
<xref ref-type="author-notes" rid="FN3"><sup>2</sup>
</xref>
<xref ref-type="author-notes" rid="FN4"><sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Iqbal</surname>
<given-names>Sobia</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
<xref ref-type="author-notes" rid="FN2"><sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="FN3"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Pacelli</surname>
<given-names>Consiglia</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>¶</sup>
</xref>
<xref ref-type="author-notes" rid="FN5"><sup>4</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Sri Renganathan</surname>
<given-names>Sri Dushyaanthan</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Trudeau</surname>
<given-names>Louis-Eric</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>¶</sup>
</xref>
<xref ref-type="author-notes" rid="FN6"><sup>5</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Campbell</surname>
<given-names>Edward M.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
<xref ref-type="aff" rid="aff3"><sup>‖</sup>
</xref>
<xref ref-type="author-notes" rid="FN7"><sup>6</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Fraser</surname>
<given-names>Paul E.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
<xref ref-type="aff" rid="aff5">**</xref>
<xref ref-type="author-notes" rid="FN8"><sup>7</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Tandon</surname>
<given-names>Anurag</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
<xref ref-type="aff" rid="aff6"><sup>‡‡</sup>
</xref>
<xref ref-type="corresp" rid="cor1"><sup>8</sup>
</xref>
</contrib>
<aff id="aff1">From the<label>‡</label>
Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5T 2S8, Canada,</aff>
<aff id="aff2">the<label>§</label>
Integrative Cell Biology Program,</aff>
<aff id="aff3"><label>‖</label>
Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153,</aff>
<aff id="aff4">the<label>¶</label>
Departments of Pharmacology and Neurosciences, Central Nervous System Research Group (GRSNC), Faculty of Medicine, Université de Montréal, Québec H4T 1J4, Canada, and</aff>
<aff id="aff5">the Departments of<label>**</label>
Medical Biophysics and</aff>
<aff id="aff6"><label>‡‡</label>
Medicine, University of Toronto, Toronto, Ontario M5T 2S8, Canada</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>8</label>
To whom correspondence should be addressed: <addr-line>Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, 60 Leonard Ave., Toronto, Ontario M5T 2S8, Canada.</addr-line>
Tel.: <phone>416-507-6889</phone>
; Fax: <fax>416-603-6435</fax>
; E-mail: <email>a.tandon@utoronto.ca</email>
.</corresp>
<fn fn-type="supported-by" id="FN2"><label>1</label>
<p>Supported in part by Edmond J. Safra Foundation fellowships.</p>
</fn>
<fn fn-type="equal" id="FN3"><label>2</label>
<p>These authors contributed equally to this work.</p>
</fn>
<fn fn-type="supported-by" id="FN4"><label>3</label>
<p>Supported by the Illinois Chapter of the ARCS® Foundation.</p>
</fn>
<fn fn-type="supported-by" id="FN5"><label>4</label>
<p>Supported by a fellowship from Parkinson Society of Canada.</p>
</fn>
<fn fn-type="supported-by" id="FN6"><label>5</label>
<p>Supported by the Krembil Foundation and Brain Canada.</p>
</fn>
<fn fn-type="supported-by" id="FN7"><label>6</label>
<p>Supported by a Rapid Response Innovation Award from the Michael J. Fox Foundation.</p>
</fn>
<fn fn-type="supported-by" id="FN8"><label>7</label>
<p>Supported by the Alzheimer Society of Ontario.</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><day>26</day>
<month>2</month>
<year>2016</year>
</pub-date>
<pub-date pub-type="epub"><day>30</day>
<month>12</month>
<year>2015</year>
</pub-date>
<volume>291</volume>
<issue>9</issue>
<fpage>4374</fpage>
<lpage>4385</lpage>
<history><date date-type="received"><day>17</day>
<month>11</month>
<year>2015</year>
</date>
<date date-type="rev-recd"><day>29</day>
<month>12</month>
<year>2015</year>
</date>
</history>
<permissions><copyright-statement>© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.</copyright-statement>
<copyright-year>2016</copyright-year>
<copyright-holder>The American Society for Biochemistry and Molecular Biology, Inc.</copyright-holder>
</permissions>
<self-uri content-type="pdf" xlink:href="zbc00916004374.pdf"></self-uri>
<abstract><p>Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.</p>
</abstract>
<kwd-group><kwd>endocytosis</kwd>
<kwd>fibril</kwd>
<kwd>Parkinson disease</kwd>
<kwd>post-translational modification (PTM)</kwd>
<kwd>protein kinase</kwd>
<kwd>protein misfolding</kwd>
<kwd>protein self-assembly</kwd>
<kwd>vesicles</kwd>
</kwd-group>
<funding-group><award-group id="award1"><funding-source>Canadian Institutes of Health Research
<named-content content-type="funder-id">http://dx.doi.org/10.13039/501100000024</named-content>
</funding-source>
<award-id>MOP-84501</award-id>
<award-id>MOP-115056</award-id>
</award-group>
<award-group id="award2"><funding-source>Alzheimer Society of Ontario</funding-source>
</award-group>
<award-group id="award3"><funding-source>Foundation Brain Canada
<named-content content-type="funder-id">http://dx.doi.org/10.13039/100009408</named-content>
</funding-source>
</award-group>
<award-group id="award4"><funding-source>Michael J. Fox Foundation for Parkinson's Research
<named-content content-type="funder-id">http://dx.doi.org/10.13039/100000864</named-content>
</funding-source>
</award-group>
</funding-group>
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</front>
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