Alzheimer Disease-specific Conformation of Hyperphosphorylated Paired Helical Filament-Tau Is Polyubiquitinated through Lys-48, Lys-11, and Lys-6 Ubiquitin Conjugation
One of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of paired helical filaments (PHFs) of hyperphosphorylated microtubule-associated protein Tau. Tandem mass spectrometry was employed to examine PHF-Tau post-translational modifications, in particular protein phosphorylation and ubiquitination, to shed light on their role in the early stages of Alzheimer disease. PHF-Tau from Alzheimer disease brain was affinity-purified by MC1 monoclonal antibody to isolate a soluble fraction of PHF-Tau in a conformation unique to human AD brain. A large number of phosphorylation sites were identified by employing a data-dependent neutral loss algorithm to trigger MS3 scans of phosphopeptides. It was found that soluble PHF-Tau is ubiquitinated at its microtubule-binding domain at residues Lys-254, Lys-311, and Lys-353, suggesting that ubiquitination of PHF-Tau may be an earlier pathological event than previously thought and that ubiquitination could play a regulatory role in modulating the integrity of microtubules during the course of AD. Tandem mass spectrometry data for ubiquitin itself indicate that PHF-Tau is modified by three polyubiquitin linkages, at Lys-6, Lys-11, and Lys-48. Relative quantitative analysis indicates that Lys-48-linked polyubiquitination is the primary form of polyubiquitination with a minor portion of ubiquitin linked at Lys-6 and Lys-11. Because modification by Lys-48-linked polyubiquitin chains is known to serve as the essential means of targeting proteins for degradation by the ubiquitin-proteasome system, and it has been reported that modification at Lys-6 inhibits ubiquitin-dependent protein degradation, a failure of the ubiquitin-proteasome system could play a role in initiating the formation of degradation-resistant PHF tangles. One of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of paired helical filaments (PHFs) of hyperphosphorylated microtubule-associated protein Tau. Tandem mass spectrometry was employed to examine PHF-Tau post-translational modifications, in particular protein phosphorylation and ubiquitination, to shed light on their role in the early stages of Alzheimer disease. PHF-Tau from Alzheimer disease brain was affinity-purified by MC1 monoclonal antibody to isolate a soluble fraction of PHF-Tau in a conformation unique to human AD brain. A large number of phosphorylation sites were identified by employing a data-dependent neutral loss algorithm to trigger MS3 scans of phosphopeptides. It was found that soluble PHF-Tau is ubiquitinated at its microtubule-binding domain at residues Lys-254, Lys-311, and Lys-353, suggesting that ubiquitination of PHF-Tau may be an earlier pathological event than previously thought and that ubiquitination could play a regulatory role in modulating the integrity of microtubules during the course of AD. Tandem mass spectrometry data for ubiquitin itself indicate that PHF-Tau is modified by three polyubiquitin linkages, at Lys-6, Lys-11, and Lys-48. Relative quantitative analysis indicates that Lys-48-linked polyubiquitination is the primary form of polyubiquitination with a minor portion of ubiquitin linked at Lys-6 and Lys-11. Because modification by Lys-48-linked polyubiquitin chains is known to serve as the essential means of targeting proteins for degradation by the ubiquitin-proteasome system, and it has been reported that modification at Lys-6 inhibits ubiquitin-dependent protein degradation, a failure of the ubiquitin-proteasome system could play a role in initiating the formation of degradation-resistant PHF tangles. The major pathological hallmarks of Alzheimer disease (AD) 3The abbreviations used are: AD, Alzheimer disease; LC-MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry; NFT, neurofibrillary tangle; NL, neutral loss; PHF, paired helical filament; PTM, post-translational modification; SRM, selected reaction monitoring; UPS, ubiquitin-proteasome system; Ub, ubiquitin. are the extracellular formation of senile plaques composed of the amyloid β peptide and the intraneuronal formation of neurofibrillary tangles (NFTs), which have a degradation-resistant core made up of paired helical filaments (PHFs) of the microtubule-associated protein Tau (PHF-Tau). In the AD brain, the degree of NFT formation has been shown to correlate more closely to the loss of neuronal function than the degree of senile plaque accumulation (1Arriagada P.V. Growdon J.H. Hedley-Whyte E.T. Hyman B.T. Neurology. 1992; 42: 631-639Crossref PubMed Google Scholar). A recent study by SantaCruz et al. (2SantaCruz K. Lewis J. Spires T. Paulson J. Kotilinek L. Ingelsson M. Guimaraes A. DeTure M. Ramsden M. McGowan E. Forster C. Yue M. Orne J. Janus C. Mariash A. Kuskowski M. Hyman B. Hutton M. Ashe K.H. Science. 2005; 309: 476-481Crossref PubMed Scopus (1563) Google Scholar) has shown, however, that the existence of NFTs alone does not cause neuronal death, implying that a pre-tangle form of Tau may be responsible for the neuronal loss and other pathological symptoms characteristic of disorders involving the Tau protein (tauopathies), including AD. Tau was initially discovered as a phosphoprotein that promotes assembly of microtubules (3Lindwall G. Cole R.D. J. Biol. Chem. 1984; 259: 5301-5305Abstract Full Text PDF PubMed Google Scholar); it was later found that hyperphosphorylated Tau is the major protein comprising the PHFs in AD (4Grundke-Iqbal I. Iqbal K. Tung Y.C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2877) Google Scholar, 5Grundke-Iqbal I. Iqbal K. Quinlan M. Tung Y.C. Zaidi M.S. Wisniewski H.M. J. Biol. Chem. 1986; 261: 6084-6089Abstract Full Text PDF PubMed Google Scholar). Because hyperphosphorylated Tau is found in all other tauopathies in addition to AD (6Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2145) Google Scholar), interest then developed in the role that phosphorylation may play in the formation of PHFs and the development of NFTs. The stabilization of microtubules is the major known function of Tau. Tau has six isoforms in the adult human brain, which vary in length from 352 to 441 amino acid residues and result from alternative splicing of the tau gene, which is located on human chromosome 17 at 17q21 (7Buée L. Bussiere T. Buee-Scherrer V. Delacourte A. Hof P.R. Brain Res. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1559) Google Scholar). Tau has an N-terminal projection domain (so named because it projects from the microtubule surface) composed of an acidic region at the N terminus and a proline-rich region in the have of the projection including microtubule J. 1992; PubMed Scopus Google Scholar) and with S. J. Biol. PubMed Scopus Google Scholar, 1984; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, M. E. J. Biol. Chem. 1986; 261: Full Text PDF PubMed Google Scholar), A. V. J. PubMed Scopus Google Scholar), and the J. G. J. Biol. PubMed Scopus Google Scholar, M. M. J. Res. 2000; PubMed Scopus Google Scholar). its Tau has a microtubule-binding domain with three microtubule-binding on the and an acidic region at the It has been that phosphorylation may cause Tau to from microtubules and form PHFs M. Neurosci. Full Text PDF PubMed Scopus Google Scholar). because Tau is J. Sci. PubMed Scopus Google Scholar) and in that Tau not be to PHFs S. E. PubMed Scopus Google Scholar), the and PHF formation is not the Tau and neuronal (7Buée L. Bussiere T. Buee-Scherrer V. Delacourte A. Hof P.R. Brain Res. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1559) Google Scholar, J. Sci. PubMed Scopus Google Scholar, J. M. Rev. PubMed Scopus Google Scholar, I. Iqbal K. J. 2005; PubMed Scopus Google Scholar). Tau in PHFs has been reported to be ubiquitinated M. M. K. M. K. Full Text PDF PubMed Scopus Google Scholar). Because proteins are for degradation by the ubiquitin-proteasome system PubMed Scopus Google Scholar) and indicate that PHFs the S. T. J. PubMed Scopus Google Scholar), the PHFs and the the role of Tau in is a to the Tau Tau ubiquitination, Tau and PHF formation and to of during are responsible for disease In soluble PHF-Tau was the antibody which PHF-Tau from AD brain not Tau from human brain J. Neurosci. Res. PubMed Scopus Google Scholar). The affinity-purified PHF-Tau was by liquid mass spectrometry a mass to post-translational phosphorylation and ubiquitination on PHF-Tau and proteins with The of a data-dependent algorithm to trigger MS3 scans in the event of a neutral loss to the of a the of mass used to and a for ubiquitinated ubiquitination sites were found on PHF-Tau Lys-311, and as as polyubiquitination at Lys-6, Lys-11, and of ubiquitin a of Relative by was used to the of polyubiquitination at of The indicate that the is by the form of Tau that Tau may be for degradation by the UPS, with for the role of Tau ubiquitination in the accumulation of PHFs in AD. analysis identified PHF-Tau as with proteins not with suggesting that PHF Tau may an early of PHF-Tau in A of mass and was employed to and on soluble PHF-Tau of indicate that PHF-Tau be modified at its microtubule-binding domain by at three of polyubiquitin the of study to the of Tau and the of AD and a for the development of AD and of PHF-Tau and was affinity-purified from AD brain by MC1 monoclonal antibody as previously J. Neurosci. Res. PubMed Scopus Google Scholar). of affinity-purified PHF-Tau was as of PHF-Tau of protein were at in by three of was for of and The reaction was by the addition of The was a employing in to isolate phosphopeptides. of affinity-purified PHF-Tau was as protein of interest were from and were with with and and in a was with in for at by with in for at in the were with with and in a with of of of and of was on for was and of was for at were at for with with and with acid of protein from an affinity-purified PHF-Tau was used for were and were in in were with a monoclonal antibody at were then in a antibody by with and MS3 analysis of and were a mass with by a system with a from were a peptide the with A and and were then with a of for and for The mass was with a an The of the mass was and the was and MS3 were the in were selected for an to were for in MS3 was then the three in the a neutral loss of was to loss of acid on and mass data were as mass and analysis of proteins that with PHF-Tau was a mass with with an system the of as were a peptide at A and and then with a of for and for Tandem were with A was by three scans of the three from the was that three of an the was on the for mass data were as mass The mass was with a an The of the mass was and the was Relative of by a quantitative of polyubiquitin an was developed in which was on with the of and modified to the polyubiquitination sites that been identified previously in study by The six and were the as in as to to to to to and to were as to the a of in of the that the peptide to be from was then used to scans for in to the The in the were the of with used in of polyubiquitination used to in a of of on a from the algorithm was used to and a human protein in the of Tau a of the six Tau isoforms as phosphorylation were used of for and and for and to and ubiquitinated a modification of was to the of ubiquitin that to the of the peptide J. G. J. PubMed Scopus Google Scholar). and by peptide and a of for for and for were then to that all major were identified in the of that residues identified with a modification of were from MS3 of and of peptide and protein made by was by the and in A. E. Chem. PubMed Scopus Google and A. E. Chem. PubMed Scopus Google Scholar). to and peptide from the data by an The then for and peptide a of from of peptide for tandem mass are then on the of of peptide and their from are then used to a of proteins that the data and then to a that protein is in the peptide is the which a algorithm to The of protein is then from to that are by are then of PHF-Tau by Tandem from human AD brain the MC1 antibody J. Neurosci. Res. PubMed Scopus Google Scholar) was the was a employing in to isolate phosphopeptides. PHF-Tau was by the was mass of and The to region was then to as and The from the were by liquid chromatography and by a tandem mass The from the and are The of and phosphorylation sites by tandem mass spectrometry is because the are by an to the acid may be the neutral loss M. J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the of of the data-dependent MS3 of the to trigger an MS3 an to loss of and to and was during the A. S. M. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of Tau phosphorylation at and that the of amino acid residues is to the Tau the number The in the and the the MS3 from data-dependent of MS3 scans a to loss of The and in the MS3 are by the of amino acid and In the is by a large neutral loss of The peptide has an mass of mass of the peptide for the The of the is then acid is from the of the is The amino acid at the is of data-dependent MS3 of phosphorylation Tau identified by of the Tau PHF-Tau with phosphorylation sites identified in study by and MS3 are shown in phosphorylation sites were identified at and and are in the proline-rich region of and the microtubule-binding of and phosphorylation sites identified in PHF-Tau by and indicate on Tau with number more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as peptide is found in and Tau The of peptide is to residues of number by residues of that indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as peptide is found in and Tau The of peptide is to residues of number by residues of that indicates number of in phosphorylation sites could not be from more of the phosphorylation sites on as indicate on Tau with number more of the phosphorylation sites on as indicates number of in phosphorylation sites could not be from peptide is found in and Tau The of peptide is to residues of number by residues of that in a were identified residues and In the indicate more of the phosphorylation sites in peptide of residues may be at and at and In indicates the modification The MS3 of have a to the neutral loss as in the of not indicate phosphorylation at and in the proline-rich region and at and in the microtubule-binding A peptide located in the N-terminal acidic region of PHF-Tau was with of the phosphorylation that phosphorylation may at A number of other were as by the of with neutral to the loss of the were of to the of the peptide not of Tau at Lys-254, Lys-311, and by a protein is the terminus of ubiquitin to a on the protein by an Because the three residues on the terminus of ubiquitin are a ubiquitinated protein is by the a with a mass of to the sites may then be identified by for a modification at in a at the modified on the ubiquitinated protein J. G. J. PubMed Scopus Google Scholar). found three ubiquitination sites on PHF-Tau at Lys-254, Lys-311, and the domain of and a of Alzheimer sites identified in in a for modified and at ubiquitination sites on PHF-Tau and The for ubiquitination at to at a ubiquitinated ubiquitination at ubiquitination, does not at and the ubiquitinated peptide is three amino acid residues In the of ubiquitination at Lys-311, the peptide is not by at Lys-311, because is by a a the peptide the amino acid as the ubiquitinated peptide In to the for the the in the for the ubiquitinated peptide the of of the of ubiquitin for the and of PHF-Tau and ubiquitination by at peptide at ubiquitinated peptide not at Lys-254, and a is to at peptide does not at because it is by a ubiquitinated peptide the modified are by in to of the of at Lys-6, Lys-11, and identified of amino acid residues in ubiquitin with and found three polyubiquitination sites Lys-11, and suggesting PHF-Tau be modified by three of for the polyubiquitin at Lys-6 and of ubiquitin. The for polyubiquitination at is by a of the amino acid of the peptide and the and that were which are composed of a of and The found polyubiquitin are in ubiquitination sites were found by in of the other proteins as that with PHF-Tau in MC1 that the degree of ubiquitination on proteins the of of Lys-6, Lys-11, and polyubiquitin by polyubiquitination at Lys-48. polyubiquitination at polyubiquitination at identified in analysis of in a of PHF-Tau protein acidic in a of in PHF-Tau by mass data of the ubiquitination of the affinity-purified PHF-Tau was to analysis by with a monoclonal antibody was in a mass of is with data the of the of a ubiquitin proteins of which the and from is protein of that is by the antibody and the antibody and are a number of protein of PHF-Tau proteins by and of protein from an affinity-purified PHF-Tau were and by to and to of are shown at Relative of by the of polyubiquitination at Lys-6, Lys-11, and a mass employing was to an event for of the of the of the to the polyubiquitination sites as as the of the and and for the of the six to polyubiquitination at Lys-6, Lys-11, and for a indicates the of as shown by their for the the for the modified on the for of their that the of polyubiquitination at the three sites are Lys-6 Lys-11. were for with mass of and the of the have not been an quantitative of Tau polyubiquitination of modified and from PHF-Tau polyubiquitination indicates of ubiquitinated indicates of indicates of ubiquitinated indicates of in a The of the and are with the of the as for in the for polyubiquitination at Lys-6 the peptide has a of and the peptide which is as has a of It be that the peptide for polyubiquitination at a and is a fraction of the ubiquitin at that and were not of with the of proteins that with Tau in MC1 an affinity-purified PHF-Tau from human AD brain was by and the proteins were by The was then that were by with and The were a human data and the of the peptide and protein was the of and of the proteins that were identified and by are in the of protein suggesting the of proteins that with PHF-Tau The isoforms of PHF-Tau in from to and PHF-Tau is known to have and has been reported to in three at and T. Hof P.R. C. L. Delacourte A. PubMed Scopus Google Scholar), to and The of ubiquitin in of the data the ubiquitination and polyubiquitination of PHF-Tau and The of with Tau that Tau with microtubules the used to PHF-Tau in The of Tau could indicate the existence of a form of as the mass of the proteins in is which is than the mass of the of Tau. In a of and mass to the of phosphorylation and ubiquitination in soluble PHF-Tau from AD brain by the MC1 which a of Tau unique to human AD brain J. Neurosci. Res. PubMed Scopus Google Scholar) that be to an early in the of PHFs in AD M. 2000; PubMed Scopus Google Scholar, I. J.H. PubMed Scopus Google Scholar). The that of Tau has been to at a the microtubule-binding domain is by implying that the conformation of Tau for study could be as reported in of Tau phosphorylation L. Hutton G. Brain Res. 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The three of projection from alternative splicing of the tau gene, as a isoforms and isoforms and isoforms It may be that six isoforms are on isoforms of Tau are in particular of particular on isoforms of Tau are in to particular as in stages of neuronal development in reaction to In the the may to Tau with in to it is that phosphorylation of Tau is a event in to of the Tau to microtubules in to it with then the Tau to be Tau degradation-resistant the degradation Tau it is to the in degradation of and accumulation of Tau is because of a failure in Tau is a is by the of by et al. S. T. J. PubMed Scopus Google Scholar), a in function in AD and that PHF-Tau from AD brain inhibits in found that soluble PHF-Tau by the MC1 antibody from AD brain is ubiquitinated at Lys-254, Lys-311, and Lys-353, all of which the microtubule-binding that ubiquitination as a of and polyubiquitination and that polyubiquitination at at and ubiquitination and polyubiquitination the of Tau that may be a of degradation Because polyubiquitination of proteins by Lys-48-linked ubiquitin chains proteins for degradation by the UPS, and because it has been shown that Tau is a of the J. 2005; PubMed Scopus Google Scholar), Tau are not is an The of study a number of other as the polyubiquitin chains in as Lys-11, and Lys-6 as chains of and ubiquitin as chains with ubiquitin linked to more than of the ubiquitin and isoforms of Tau are of the ubiquitin which is found in AD and from a in of have been shown to the and to of has an terminus that it from to proteins does not it from which to the J. PubMed Scopus Google Scholar). a the to to the It has been shown that ubiquitin in NFTs is to and by B. J. L. J. PubMed Scopus Google Scholar). not that particular of is a later data a of could be an early event in the formation of a of Tau that inhibits the because it has been shown that ubiquitin as by is an of ubiquitin-dependent G. B. A. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that ubiquitination at Lys-6 could ubiquitin-dependent an event that which be of by the UPS, from study that and quantitative of the Tau be by tandem mass spectrometry in the of sites are and the of data-dependent neutral loss MS3 the of phosphorylation data indicate that with the of it be to the of post-translational as a mass it to the of Tau in pathological of AD and to a for are to in may of Tau to and may a for development of for early AD. of for on the and of mass spectrometry