Recombinant Tau Research Articles

Novel mutation in MAPT exon 13 (p.N410H) causes corticobasal degeneration

In order to determine the frequency of microtubule-associated protein tau gene (MAPT) mutations and rare variants in CBD, we performed a systematic sequence analysis of MAPT coding and 3′ untranslated region (3′UTR) in a large cohort of autopsy-confirmed CBD patients (N = 109). This identified a novel MAPT mutation in exon 13, p.N410H, in a case that is neuropathologically indistinguishable from sporadic CBD. On immunoblot, the p.N410H mutation carrier had the same insoluble tau profile as seen in CBD. Additionally, tau expression analysis in brain tissue found a significant increase in the 4R/3R tau mRNA ratio (P = 0.04), indicating that p.N410H disrupts tau isoform homeostasis. Biochemically, recombinant tau protein with p.N410H showed a marked increase in tau filament formation compared to wild-type tau (P < 0.001), had a 19.2% decrease in rate of microtubule assembly (P < 0.05), and a 10.3% reduction in the extent of total microtubule polymerization (P < 0.01). Sequence analysis of the complete MAPT 3′UTR in autopsy-confirmed CBD cases further identified two rare variants with nominally significant association with CBD. An ATC nucleotide insertion (“MAPTv8”) was found in 4.6% of CBD patients compared to 1.2% of controls (P = 0.031, OR = 3.71), and rs186977284 in 4.6% CBD patients, but only 0.9% of controls (P = 0.04, OR = 3.58). Rs186977284 was also present in 2.7% of a large cohort of autopsy-confirmed PSP patients (N = 566) and only 0.9% of an additional control series (P = 0.034, OR = 3.08), extending the association to PSP. Our findings show that mutations in MAPT can cause CBD and MAPT non-coding variants may increase the risk of complex 4R tauopathies.

LRRK2 phosphorylates novel tau epitopes and promotes tauopathy

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson’s disease (PD). The neuropathology of LRRK2-related PD is heterogeneous and can include aberrant tau phosphorylation or neurofibrillary tau pathology. Recently, LRRK2 has been shown to phosphorylate tau in vitro; however, the major epitopes phosphorylated by LRRK2 and the physiological or pathogenic consequences of these modifications in vivo are unknown. Using mass spectrometry, we identified multiple sites on recombinant tau that are phosphorylated by LRRK2 in vitro, including pT149 and pT153, which are phospho-epitopes that to date have been largely unexplored. Importantly, we demonstrate that expression of transgenic LRRK2 in a mouse model of tauopathy increased the aggregation of insoluble tau and its phosphorylation at T149, T153, T205, and S199/S202/T205 epitopes. These findings indicate that tau can be a LRRK2 substrate and that this interaction can enhance salient features of human disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1188-4) contains supplementary material, which is available to authorized users.

TOC1: Characterization of a Selective Oligomeric Tau Antibody

The work presented herein addresses a specific portion of the tau pathology, pre-fibrillar oligomers, now thought to be important pathological components in Alzheimer's disease and other neurodegenerative tauopathies. In previous work, we generated an antibody against purified recombinant cross-linked tau dimers, called Tau Oligomeric Complex 1 (TOC1). TOC1 recognizes tau oligomers and its immunoreactivity is elevated in Alzheimer's disease brains. In this report, we expand upon the previous study to show that TOC1 selectively labels tau oligomers over monomers or polymers, and that TOC1 is also reactive in other neurodegenerative tauopathies. Using a series of deletion mutants spanning the tau molecule, we further demonstrate that TOC1 has one continuous epitope located within amino acids 209-224, in the so-called proline rich region. Together with the previous study, our data indicates that TOC1 is a conformation-dependent antibody whose epitope is revealed upon dimerization and oligomerization, but concealed again as polymers form. This characterization of the TOC1 antibody further supports its potential as a powerful biochemical tool that can be used to better investigate the involvement of tau in neurodegenerative diseases.

Evaluation of pre-analytical and analytical factors that contribute to variability in CSF biomarker levels

development for tau that shows a marked improvement in precision, linearity and reproducibility when compared to currently available tests. Methods: An in vitro diagnostic test for tau is being developed for the VITROS Immunodiagnostic Systems, and analytical performance was evaluated following CLSI guidelines using two VITROS Tau assay reagent lots, 3 CSF pools, and 3 controls. Linearity was tested with 11 admixtures of endogenous tau in CSF and recombinant tau 441 in buffer. Interference testing included commonly prescribed drugs, recombinant tau, Ab peptides, and endogenous substances. Limit of detection (LoD) and lower limit of quantitation were confirmed with 20 replicates per day on 3 days. Results: The median within-laboratory coefficient of variation (CV) for the 3 CSF pools was 3.3% with a range of 2.4% 4.2%. The controls’ within-laboratory CV was 1.2%. Repeatability CVs were 1.9% for CSF pools and 1.2% for controls. The assay showed good linearity across themeasuring range (08,000pg/mL) with the admixture of endogenous and synthetic peptide. Bias introduced by commonly prescribed drugs at high levels was <610%. The interference from Ab peptides at greater than known physiological concentrations as well as from endogenous substances was <5%. The limit of detection was 9.5pg/mL; the lower limit of quantitation was 40pg/mL. Conclusions: VITROS Immunodiagnostic Products Tau Assay 1 demonstrates excellent analytical performance: it is precise, linear and the tested interferences cause no significant bias. The fully automated format and ability to perform these assays in a clinical laboratory should make testing for these important markers more reliable and routine.

Characterization of a novel monoclonal antibody specific for a tau autoproteolytic cut site that differentiates between Alzheimer's disease and non-Alzheimer's specimens

Tau is a microtubule binding protein that normally functions in axons to maintain microtubule structure but is hyperphosphorylated in AD facilitating its dissociation from microtubules and accumulation in the somatodendritic compartment where it can aggregate (Iqbal et al. 2010). Oligomeric forms of tau appear to be most closely associated with neuronal loss and memory impairment in mouse models of tauopathy (Berger et al. 2007; Yoshiyama et al. 2007). Tau oligomers were also found to accumulate in human AD specimens (Maeda et al. 2006; Patterson et al. 2011; Lasagna-Reeves et al. 2012). Importantly, extracellular tau oligomers have been shown to cause memory impairment and toxicity in mice inhibiting formation of long-term potentiation in hippocampal slices and formation of associative fear memory (Fá et al. 2010) and induce neurodegeneration by affecting mitochondrial and synaptic function (Lasagna-Reeves et al. 2012). However, the particular structure and mechanism by which tau oligomers exert their deleterious effects has not been determined. We have found that upon the formation of oligomeric structures tau acquires the additional function of a protease leading to self-cleavage and the ability to cut other proteins. Here, we present initial characterization of a novel monoclonal antibody specific for an end of a tau fragment generated by the autoproteolytic activity of tau oligomers. N-terminal sequencing of autoproteolytic fragments of tau oligomers was performed using mass spectrometry. A cut site was used to generate peptides to produce polyclonal antibodies specific to the cut ends. A rabbit was also chosen for the production of hybridomas to select a clone producing monoclonal antibodies specific for the cut end of a fragment that has tau oligomer protease activity. ELISAs and immunoblots were performed using peptides, recombinant tau and brain specimens for selection and characterization. A hybridoma line showing specificity was selected and monoclonal antibody was purified. Immunoblot analysis of AD and non-AD brain specimens using this antibody showed oligomeric and fragment bands that were specific to the AD specimens. This approach was successful in producing a monoclonal antibody that demonstrates the disease-relevance of a tau oligomer autoproteolytic cut site. This antibody may also have biomarker and immunotherapeutic potential.

Intravenous immunoglobulin products contain specific antibodies to recombinant human tau protein

Intravenous immunoglobulin (IVIG) products are prepared from plasma immunoglobulins from healthy donors. Pilot studies suggest that IVIG may stabilize cognitive functioning in patients with mild-to-moderate Alzheimer's disease. This study measured antibodies to recombinant human tau protein in the IVIG products Gammagard (Baxter), Gamunex (Talecris), and Flebogamma (Grifols). Anti-tau antibodies were measured by ELISA, subtracting IVIG's polyvalent binding from its binding to tau-coated wells to calculate specific anti-tau antibody levels. Because polyvalent binding of IVIG products may interfere with ELISA measurement of their specific antibody levels, the percentage of binding of each IVIG product to tau-coated wells that was specific for tau was also determined. Specific anti-tau antibodies were detected in all three IVIG products, with significant differences between these products (p<0.001) even when Flebogamma's anti-tau antibodies were doubled to account for its preparation as a 5% solution vs. 10% solutions for Gammagard and Gamunex (means: Gammagard, 3.1 μg/ml; Gamunex, 2.5 μg/ml; Flebogamma, 1.2 μg/ml). The percentages of each IVIG product's specific binding to tau-coated wells also varied between the various products (p<0.001) and between all pairs of IVIG products (means: Gammagard, 73.1%; Flebogamma, 54.5%; Gamunex, 37.4%; p<0.01 for all pairwise comparisons). These findings indicate that IVIG products contain specific anti-tau antibodies. The concentrations of these antibodies and the percentages of specific binding of IVIG to tau-coated wells vary between IVIG products. Further studies are indicated to determine if IVIG also contains antibodies to pathologic forms of tau.

Global Analysis of Phosphorylation of Tau by the Checkpoint Kinases Chk1 and Chk2 in vitro

Hyperphosphorylation of microtubule-associated protein tau is thought to contribute to Alzheimer's disease (AD) pathogenesis. We previously showed that DNA damage-activated cell cycle checkpoint kinases Chk1 and Chk2 phosphorylate tau at an AD-related site and enhance tau toxicity, suggesting potential roles of these kinases in AD. The purpose of this study is to systematically identify which sites in tau are directly phosphorylated by Chk1 and Chk2. Using recombinant human tau phosphorylated by Chk1 and Chk2 in vitro, we first analyzed tau phosphorylation at the AD-related sites by Western blot with phospho-tau-specific antibodies. Second, to globally identify phosphorylated sites in tau, liquid chromatography-tandem mass spectrometry (LC-MS(3)) was employed. These systematic analyses identified a total of 27 Ser/Thr residues as Chk1- or Chk2- target sites. None of them were proline-directed kinase targets. Many of these sites are located within the microtubule-binding domain and C-terminal domain, whose phosphorylation has been shown to reduce tau binding to microtubules and/or has been implicated in tau toxicity. Among these 27 sites, 13 sites have been identified to be phosphorylated in AD brains. Since DNA damage is accumulated in diseased brains, Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in AD pathogenesis.

Synthesis and biological evaluation of novel styryl benzimidazole derivatives as probes for imaging of neurofibrillary tangles in Alzheimer’s disease

This paper describes the synthesis and biological evaluation of styrylbenzimidazole (SBIM) derivatives as agents for imaging neurofibrillary tangles (NFT) in patients with Alzheimer’s disease (AD). SBIM derivatives were prepared with 4-iodobenzene-1,2-diamine and substituted cinnamaldehydes. In binding experiments using recombinant tau and Aβ1–42 aggregates, SBIM-3 showed higher affinity for the tau aggregates than Aβ1–42 aggregates (ratio of Kd values was 2.73). In in vitro autoradiography and fluorescent staining, [125I]SBIM-3 (or SBIM-3) bound NFT in sections of AD brain tissue. In biodistribution experiments using normal mice, all [125I]SBIM derivatives showed high initial uptake into (3.20–4.11%ID/g at 2min after the injection) and rapid clearance from (0.12–0.33%ID/g at 60min after the injection) the brain. In conclusion, appropriate structural modifications of SBIM derivatives could lead to more useful agents for the in vivo imaging of NFT in AD brains.

The effect of a single intrauterine injection of recombinant bovine interferon tau on blood cell count, CL lifespan and inter-estrus interval in cattle

The effect of a single intrauterine injection of recombinant bovine interferon tau on blood cell count, CL lifespan and inter-estrus interval in cattle

Rapamycin decreases tau phosphorylation at Ser214 through regulation of cAMP-dependent kinase

Preventing or reducing tau hyperphosphorylation is considered to be a therapeutic strategy in the treatment of Alzheimer’s disease (AD). Rapamycin may be a potential therapeutic agent for AD, because the rapamycin-induced autophagy may enhance the clearance of the hyperphosphorylated tau. However, recent rodent studies show that the protective effect of rapamycin may not be limited in the autophagic clearance of the hyperphosphorylated tau. Because some tau-related kinases are targets of the mammalian target of rapamycin (mTOR), we assume that rapamycin may regulate tau phosphorylation by regulating these kinases. Our results showed that in human neuroblastoma SH-SY5Y cells, treatment with rapamycin induced phosphorylation of the type IIα regulatory (RIIα) subunit of cAMP-dependent kinase (PKA). Rapamycin also induced nuclear translocation of the catalytic subunits (Cat) of PKA and decreases in tau phosphorylation at Ser214 (pS214). The above effects of rapamycin were prevented by pretreatment with the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126. In addition, these effects of rapamycin might not depend on the level of tau expression, because similar results were obtained in both the non-tau-expressing wild type human embryonic kidney 293 (HEK293) cells and HEK293 cells stably transfected with the longest isoform of recombinant human tau (tau441; HEK293/tau441). These findings suggest that rapamycin decreases pS214 via regulation of PKA. Because tau phosphorylation at Ser214 may prime tau for further phosphorylation by other kinases, our findings provide a novel possible mechanism by which rapamycin reduces or prevents tau hyperphosphorylation.

Synthetic Tau Fibrils Mediate Transmission of Neurofibrillary Tangles in a Transgenic Mouse Model of Alzheimer's-Like Tauopathy

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau pathologies, are neurodegenerative diseases characterized by neurofibrillary tangles (NFTs) comprising filamentous tau protein. Although emerging evidence suggests that tau pathology may be transmitted, we demonstrate here that synthetic tau fibrils are sufficient to transmit tau inclusions in a mouse model. Specifically, intracerebral inoculation of young PS19 mice overexpressing mutant human tau (P301S) with synthetic preformed fibrils (pffs) assembled from recombinant full-length tau or truncated tau containing four microtubule binding repeats resulted in rapid induction of NFT-like inclusions that propagated from injected sites to connected brain regions in a time-dependent manner. Interestingly, injection of tau pffs into either hippocampus or striatum together with overlaying cortex gave rise to distinct pattern of spreading. Moreover, unlike tau pathology that spontaneously develops in old PS19 mice, the pff-induced tau inclusions more closely resembled AD NFTs because they were Thioflavin S positive, acetylated, and more resistant to proteinase K digestion. Together, our study demonstrates that synthetic tau pffs alone are capable of inducing authentic NFT-like tau aggregates and initiating spreading of tau pathology in a tauopathy mouse model.

Trimeric Tau Is Toxic to Human Neuronal Cells at Low Nanomolar Concentrations

In Alzheimer's disease (AD), tau aggregates into fibrils and higher order neurofibrillary tangles, a key histopathological feature of AD. However, soluble oligomeric tau species may play a more critical role in AD progression since these tau species correlate better with neuronal loss and cognitive dysfunction. Recent studies show that extracellular oligomeric tau can inhibit memory formation and synaptic function and also transmit pathology to neighboring neurons. However, the specific forms of oligomeric tau involved in toxicity are still unknown. Here, we used two splice variants of recombinant human tau and generated monomeric, dimeric, and trimeric fractions of each isoform. The composition of each fraction was verified chromatographically and also by atomic force microscopy. The toxicity of each fraction toward both human neuroblastoma cells and cholinergic-like neurons was assessed. Trimeric, but not monomeric or dimeric, tau oligomers of both splice variants were neurotoxic at low nanomolar concentrations. Further characterization of tau oligomer species with disease-specific modifications and morphologies is necessary to identify the best targets for the development of biomarker and therapeutic development for AD and related tauopathies.

Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons

The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845-12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909-913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

Non-Adhesive Behavior of New Nanostructured PNIPAM Surfaces Towards Specific Neurodegenerative Proteins: Application to Storage and Titration of Tau Proteins

New nonfouling tubes are developed and their influence on the adhesion of neuroproteins is studied. The biomarkers are considered as single components (recombinant prion and Tau proteins) or in a solution of native and pathological forms. The samples are stored for 24 h at 4 °C in virgin and treated tubes layered with two different nanostructured coatings based on poly(N-isopropylacrylamide) with either a positive or a neutral charge, and the protein adhesion is monitored. The recombinant protein with a high pI is repelled from the nanostructured surface that has a negative ζ potential, whereas the recombinant protein with the lower pI is attracted. Furthermore, in the case of complex solutions, neutral nanostructured surfaces are able to retain all amyloid biomarkers.

Fibril-Forming Motifs Are Essential and Sufficient for the Fibrillization of Human Tau

BackgroundThe misfolding of amyloidogenic proteins including human Tau protein, human prion protein, and human α-synuclein is involved in neurodegenerative diseases such as Alzheimer disease, prion disease, and Parkinson disease. Although a lot of research on such amyloidogenic proteins has been done, we do not know the determinants that drive these proteins to form fibrils and thereby induce neurodegenerative diseases. In this study, we want to know the role of fibril-forming motifs from such amyloidogenic proteins in the fibrillization of human Tau protein.Methodology/Principal FindingsAs evidenced by thioflavin T binding and turbidity assays, transmission electron microscopy, and circular dichroism, fibril-forming motifs are essential and sufficient for the fibrillization of microtubule-associated protein Tau: only when both of its fibril-forming motifs, PHF6 and PHF6*, are deleted can recombinant human Tau fragment Tau244–372 lose its ability to form fibrils, and the insertion of unrelated fibril-forming motifs from other amyloidogenic proteins, such as human prion protein, yeast prion protein, human α-synuclein, and human amyloid β, into the disabled Tau protein can retrieve its ability to form fibrils. Furthermore, this retrieval is independent of the insertion location on Tau244–372.Conclusions/SignificanceWe demonstrate for the first time that insertion of fibril-forming motifs can replace PHF6/PHF6* motifs, driving human Tau protein to form fibrils with different morphologies and different kinetic parameters. Our results suggest that fibril-forming motifs play a key role in the fibrillization of human Tau protein and could be the determinants of amyloidogenic proteins tending to misfold, thereby causing the initiation and development of neurodegenerative diseases. Our study also touches on the importance of amyloid “strains”: changes to the amyloidgenic driver region results in altered structural morphologies at the macromolecular level.

Tau Isoform Composition Influences Rate and Extent of Filament Formation

The risk of developing tauopathic neurodegenerative disease depends in part on the levels and composition of six naturally occurring Tau isoforms in human brain. These proteins, which form filamentous aggregates in disease, vary only by the presence or absence of three inserts encoded by alternatively spliced exons 2, 3, and 10 of the Tau gene (MAPT). To determine the contribution of alternatively spliced segments to Tau aggregation propensity, the aggregation kinetics of six unmodified, recombinant human Tau isoforms were examined in vitro using electron microscopy assay methods. Aggregation propensity was then compared at the level of elementary rate constants for nucleation and extension phases. We found that all three alternatively spliced segments modulated Tau aggregation but through differing kinetic mechanisms that could synergize or compete depending on sequence context. Overall, segments encoded by exons 2 and 10 promoted aggregation, whereas the segment encoded by exon 3 depressed it with its efficacy dependent on the presence or absence of a fourth microtubule binding repeat. In general, aggregation propensity correlated with genetic risk reported for multiple tauopathies, implicating aggregation as one candidate mechanism rationalizing the correlation between Tau expression patterns and disease.

In-Source Decay and Pseudo Tandem Mass Spectrometry Fragmentation Processes of Entire High Mass Proteins on a Hybrid Vacuum Matrix-Assisted Laser Desorption Ionization-Quadrupole Ion-Trap Time-of-Flight Mass Spectrometer

In-source decay (ISD), although a process known for decades in mass spectrometry, has a renewed interest due to increased theoretical knowledge in fragmentation processes of large biomolecules coupled with technological improvements. We report here an original method consisting of isolating matrix-assisted laser desorption ionization (MALDI)-generated in-source fragments of large proteins and subsequently performing selective fragmentation experiments (up to four cycles) using a hybrid MALDI quadrupole ion-trap time-of-flight mass spectrometer (MALDI-QIT-TOF). This technology takes advantage of keeping high resolution on the selection of precursors and detection of fragments. It allows exhaustive N- and C-terminal sequencing of proteins. In this work, human serum albumin (HSA), β-casein, and recombinant Tau proteins were submitted to in source decay in the MALDI source. The fragments were stored in the ion-trap and submitted to sequential collision-induced dissociation (CID). Finally, ISD and pseudo MS(n) were performed on oxidized Tau protein and acetylated bovine serum albumin to identify amino acid modifications. This work highlights the potential of the MALDI-QIT-TOF instrument for pseudo MS(n) strategies and top down proteomics.

Biochemical Characterization of Novel Lignans Isolated from the Wood of Taxus yunnanensis as Effective Stimulators for Glycogen Synthase Kinase-3β and the Phosphorylation of Basic Brain Proteins by the Kinase in Vitro

The stimulatory and inhibitory effects of several compounds and lignans isolated from the water extract of Taxus yunnanensis on the phosphorylation of three functional brain proteins (bovine myelin basic protein (bMBP), recombinant human tau protein (rhTP) and rat collapsin response mediator protein-2 (rCRMP-2)) by glycogen synthase kinase-3β (GSK-3β) were quantitatively compared in vitro, using (-)-epigallocatechin-3-gallate [(-)EGCG] as a positive control. We found that (i) three selected Taxus lignans [(3S,4R)-4'-hydroxy-6,3'-dimethoxyisoflavan-4-ol,(7R)-7-hydroxytaxiresinol and tanegool] highly stimulated the autophosphorylation of GSK-3β and the GSK-3β-mediated phosphorylation of two basic brain proteins [bMBP (pI=11.3) and rhTP (pI=8.2)], but inhibited dose-dependently the phosphorylation of an acidic protein (rCRMP-2, pI=6.0) by the kinase; (ii) these three Taxus lignans showed binding-affinities with bMBP as well as rhTP, but had low affinities with rCRMP-2; (iii) the binding of tanegool and (7R)-7-hydroxytaxiresinol to these two basic proteins induced their novel potent phosphorylation sites for GSK-3β; and (iv) these three Taxus lignans, but not EGCG, induced Tyr-phosphorylation of GSK-3β in vitro. These results provided here suggest that (i) these three Taxus lignans act as novel effective activators for GSK-3β and the GSK-3β-mediated phosphorylation of their binding basic proteins (rhTP and bMBP); and (ii) tanegool (IC(50)=1 μM) is an effective inhibitor for the phosphorylation of rCRMP-2 by the kinase in vitro.

Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced profiling of protein phosphorylation

We describe an improved Phos-tag SDS-PAGE (Zn2+-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) to detect shifts in the mobility of phosphorylated proteins. Our alternative technique (Mn2+-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn2+-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements and utilities were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant substrate protein tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole cell lysates. Additionally, the Zn2+-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn2+-Phos-tag SDS-PAGE gels. We can thus present a simple, convenient, and more reliable "in-house" gel system for phosphate-affinity SDS-PAGE.

Fibrillization of Human Tau Is Accelerated by Exposure to Lead via Interaction with His-330 and His-362

BackgroundNeurofibrillary tangles, mainly consisted of bundles of filaments formed by the microtubule-associated protein Tau, are a hallmark of Alzheimer disease. Lead is a potent neurotoxin for human being especially for the developing children, and Pb2+ at high concentrations is found in the brains of patients with Alzheimer disease. However, it has not been reported so far whether Pb2+ plays a role in the pathology of Alzheimer disease through interaction with human Tau protein and thereby mediates Tau filament formation. In this study, we have investigated the effect of Pb2+ on fibril formation of recombinant human Tau fragment Tau244–372 and its mutants at physiological pH.Methodology/Principal FindingsAs revealed by thioflavin T and 8-anilino-1-naphthalene sulfonic acid fluorescence, the addition of 5–40 µM Pb2+ significantly accelerates the exposure of hydrophobic region and filament formation of wild-type Tau244–372 on the investigated time scale. As evidenced by circular dichroism and Fourier transform infrared spectroscopy, fibrils formed by wild-type Tau244–372 in the presence of 5–40 µM Pb2+ contain more β-sheet structure than the same amount of fibrils formed by the protein in the absence of Pb2+. However, unlike wild-type Tau244–372, the presence of 5–40 µM Pb2+ has no obvious effects on fibrillization kinetics of single mutants H330A and H362A and double mutant H330A/H362A, and fibrils formed by such mutants in the absence and in the presence of Pb2+ contain similar amounts of β-sheet structure. The results from isothermal titration calorimetry show that one Pb2+ binds to one Tau monomer via interaction with His-330 and His-362, with sub-micromolar affinity.Conclusions/SignificanceWe demonstrate for the first time that the fibrillization of human Tau protein is accelerated by exposure to lead via interaction with His-330 and His-362. Our results suggest the possible involvement of Pb2+ in the pathogenesis of Alzheimer disease and provide critical insights into the mechanism of lead toxicity.