A validated antibody panel for the characterization of tau post-translational modifications
Tóm tắt
Tau is a microtubule-binding protein, which is subject to various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Aberrant PTMs such as hyperphosphorylation result in tau aggregation and the formation of neurofibrillary tangles, which are a hallmark of Alzheimer’s disease (AD). In order to study the importance of PTMs on tau function, antibodies raised against specific modification sites are widely used. However, quality control of these antibodies is lacking and their specificity for particular modifications is often unclear. In this study, we first designed an online tool called ‘TauPTM’, which enables the visualization of PTMs and their interactions on human tau. Using TauPTM, we next searched for commercially available antibodies against tau PTMs and characterized their specificity by peptide array, immunoblotting, electrochemiluminescence ELISA and immunofluorescence technologies. We demonstrate that commercially available antibodies can show a significant lack of specificity, and PTM-specific antibodies in particular often recognize non-modified versions of the protein. In addition, detection may be hindered by other PTMs in close vicinity, complicating the interpretation of results. Finally, we compiled a panel of specific antibodies and show that they are useful to detect PTM-modified endogenous tau in hiPSC-derived neurons and mouse brains. This study has created a platform to reliably and robustly detect changes in localization and abundance of post-translationally modified tau in health and disease. A web-based version of TauPTM is fully available at
http://www.tauptm.org
.
Tài liệu tham khảo
Lee VM, Goedert M, Trojanowski JQ. Neurodegenerative tauopathies. Annu Rev Neurosci. 2001;24:1121–59.
Mukrasch MD, Bibow S, Korukottu J, Jeganathan S, Biernat J, Griesinger C, Mandelkow E, Zweckstetter M. Structural polymorphism of 441-residue tau at single residue resolution. PLoS Biol. 2009;7:e34.
Kadavath H, Hofele RV, Biernat J, Kumar S, Tepper K, Urlaub H, Mandelkow E, Zweckstetter M. Tau stabilizes microtubules by binding at the interface between tubulin heterodimers. Proc Natl Acad Sci U S A. 2015;112:7501–6.
Lee G, Cowan N, Kirschner M. The primary structure and heterogeneity of tau protein from mouse brain. Science. 1988;239:285–8.
Goedert M, Spillantini MG, Jakes R, Rutherford D, Crowther RA. Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron. 1989;3:519–26.
Wang Y, Mandelkow E. Tau in physiology and pathology. Nat Rev Neurosci. 2016;17:5–21.
Baker M. Reproducibility crisis: blame it on the antibodies. Nature. 2015;521:274–6.
Andorfer C, Kress Y, Espinoza M, de Silva R, Tucker KL, Barde YA, Duff K, Davies P. Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms. J Neurochem. 2003;86:582–90.
Goedert M, Jakes R. Expression of separate isoforms of human tau protein: correlation with the tau pattern in brain and effects on tubulin polymerization. EMBO J. 1990;9:4225–30.
Li W, Sun W, Zhang Y, Wei W, Ambasudhan R, Xia P, Talantova M, Lin T, Kim J, Wang X, et al. Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc Natl Acad Sci U S A. 2011;108:8299–304.
Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell. 2007;28:730–8.
Tucker KL, Meyer M, Barde YA. Neurotrophins are required for nerve growth during development. Nat Neurosci. 2001;4:29–37.
Kuster DW, Barefield D, Govindan S, Sadayappan S. A sensitive and specific quantitation method for determination of serum cardiac myosin binding protein-C by electrochemiluminescence immunoassay. J Visualized Experiments : JoVE. 2013;
ES O, Mielke MM, Rosenberg PB, Jain A, Fedarko NS, Lyketsos CG, Mehta PD. Comparison of conventional ELISA with electrochemiluminescence technology for detection of amyloid-beta in plasma. J Alzheimer’s disease : JAD. 2010;21:769–73.
Begley CG, Ellis LM. Drug development: raise standards for preclinical cancer research. Nature. 2012;483:531–3.
Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J, Anagnostou V, Rimm D. Antibody validation. BioTechniques. 2010;48:197–209.
Egelhofer TA, Minoda A, Klugman S, Lee K, Kolasinska-Zwierz P, Alekseyenko AA, Cheung MS, Day DS, Gadel S, Gorchakov AA, et al. An assessment of histone-modification antibody quality. Nat Struct Mol Biol. 2011;18:91–3.
Harmsen MM, De Haard HJ. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol. 2007;77:13–22.
Lista S, Faltraco F, Prvulovic D, Hampel H. Blood and plasma-based proteomic biomarker research in Alzheimer's disease. Prog Neurobiol. 2013;101-102:1–17.
Acker CM, Forest SK, Zinkowski R, Davies P, d'Abramo C. Sensitive quantitative assays for tau and phospho-tau in transgenic mouse models. Neurobiol Aging. 2013;34:338–50.
Song L, SX L, Ouyang X, Melchor J, Lee J, Terracina G, Wang X, Hyde L, Hess JF, Parker EM, Zhang L. Analysis of tau post-translational modifications in rTg4510 mice, a model of tau pathology. Mol Neurodegener. 2015;10:14.
Morris M, Knudsen GM, Maeda S, Trinidad JC, Ioanoviciu A, Burlingame AL, Mucke L. Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat Neurosci. 2015;18:1183–9.
Cook C, Carlomagno Y, Gendron TF, Dunmore J, Scheffel K, Stetler C, Davis M, Dickson D, Jarpe M, DeTure M, Petrucelli L. Acetylation of the KXGS motifs in tau is a critical determinant in modulation of tau aggregation and clearance. Hum Mol Genet. 2014;23:104–16.
Cohen TJ, Guo JL, Hurtado DE, Kwong LK, Mills IP, Trojanowski JQ, Lee VM. The acetylation of tau inhibits its function and promotes pathological tau aggregation. Nat Commun. 2011;2:252.
Reynolds MR, Reyes JF, Fu Y, Bigio EH, Guillozet-Bongaarts AL, Berry RW, Binder LI. Tau nitration occurs at tyrosine 29 in the fibrillar lesions of Alzheimer's disease and other tauopathies. J Neurosci. 2006;26:10636–45.
Cripps D, Thomas SN, Jeng Y, Yang F, Davies P, Yang AJ. Alzheimer disease-specific conformation of hyperphosphorylated paired helical filament-tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation. J Biol Chem. 2006;281:10825–38.
Cavallini A, Brewerton S, Bell A, Sargent S, Glover S, Hardy C, Moore R, Calley J, Ramachandran D, Poidinger M, et al. An unbiased approach to identifying tau kinases that phosphorylate tau at sites associated with Alzheimer disease. J Biol Chem. 2013;288:23331–47.
Kelleher I, Garwood C, Hanger DP, Anderton BH, Noble W. Kinase activities increase during the development of tauopathy in htau mice. J Neurochem. 2007;103:2256–67.
Gunawardana CG, Mehrabian M, Wang X, Mueller I, Lubambo IB, Jonkman JE, Wang H, Schmitt-Ulms G. The human tau Interactome: binding to the Ribonucleoproteome, and impaired binding of the proline-to-leucine mutant at position 301 (P301L) to chaperones and the proteasome. Molecular & cellular proteomics : MCP. 2015;14:3000–14.
Ittner LM, Ke YD, Delerue F, Bi M, Gladbach A, van Eersel J, Wolfing H, Chieng BC, Christie MJ, Napier IA, et al. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer’s disease mouse models. Cell. 2010;142:387–97.