An official website of the United States government
Abstract Antibodies raised against defined phosphorylation sites of the microtubule‐associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non‐specific binding found in these antibodies. In order to quantify and compare the specificity phospho‐tau antibodies and other post‐translational modification site‐specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non‐specific signal in antibody binding. We validate our assay using anti‐tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho‐tau antibodies (AT270, AT8, AT100, AT180, PHF‐6, TG‐3, and PHF‐1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF‐6, TG‐3, and PHF‐1 have Φ values near 1, which indicates no detectable non‐specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non‐specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non‐specific binding of AT270 to non‐tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho‐tau antibodies, and potentially for post‐translational modification targeting antibodies in general. imageCover Image for this issue: doi:10.1111/jnc.14727.
more »
« less
Yeast surface display of full‐length human microtubule‐associated protein tau
Abstract Microtubule‐associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full‐length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeastSaccharomyces cerevisiae. Yeast‐displayed tau binds to anti‐tau antibodies that cover epitopes ranging from the N‐terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface‐displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF‐1). However, yeast‐displayed tau showed clear binding to paired helical filament (PHF) tau conformation‐specific antibodies Alz‐50, MC‐1, and Tau‐2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast‐displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.
more »
« less
- Award ID(s):
- 1706743
- PAR ID:
- 10458900
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology Progress
- Volume:
- 36
- Issue:
- 1
- ISSN:
- 8756-7938
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Sleep and circadian rhythm dysfunctions are common clinical features of Alzheimer’s disease (AD). Increasing evidence suggests that in addition to being a symptom, sleep disturbances can also drive the progression of neurodegeneration. Protein aggregation is a pathological hallmark of AD; however, the molecular pathways behind how sleep affects protein homeostasis remain elusive. Here we demonstrate that sleep modulation influences proteostasis and the progression of neurodegeneration inDrosophilamodels of tauopathy. We show that sleep deprivation enhanced Tau aggregational toxicity resulting in exacerbated synaptic degeneration. In contrast, sleep induction using gaboxadol led to reduced toxic Tau accumulation in neurons as a result of modulated autophagic flux and enhanced clearance of ubiquitinated Tau, suggesting altered protein processing and clearance that resulted in improved synaptic integrity and function. These findings highlight the complex relationship between sleep and regulation of protein homeostasis and the neuroprotective potential of sleep-enhancing therapeutics to slow the progression or delay the onset of neurodegeneration.more » « less
-
HS3ST1is a genetic risk gene associated with Alzheimer’s disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3-O-sulfated HS displayed sevenfold increase in the AD group (n= 14,P< 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3-O-sulfated HS is made by 3-O-sulfotransferase isoform 1 (3-OST-1), which is encoded by theHS3ST1gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3-O-sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3-O-sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression ofHS3ST1gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.more » « less
-
Cellular internalization and the spreading of misfolded tau have become increasingly important for elucidating the mechanism of Tau pathology involved in Alzheimer’s disease (AD). The low-density lipoprotein-related receptor 1 (LRP1) has been implicated in the internalization of fibrillar tau. In this work, we utilized homology modeling to model the Cluster 2 domain of LRP1 and determined that a 23-amino-acid sequence is involved in binding to paired helical filaments (PHF) of Tau. Fourteen short peptide segments derived from this ectodomain region were then designed and docked with PHF Tau. Molecular dynamics studies of the optimal peptides bound to PHF Tau demonstrated that the peptides formed critical contacts through Lys and Gln residues with Tau. Based on the computational results, flow cytometry, AFM, SPR analysis and CD studies were conducted to examine binding and cellular internalization. The results showed that the peptide sequence TauRP (1–14) (DNSDEENCES) was not only associated with fibrillar Tau but was also able to mitigate its cellular internalization in LRP1-expressed HEK-293 cells. Preliminary docking studies with Aβ (1–42) revealed that the peptides also bound to Aβ (1–42). While this study focused on the CCR2 domain of LRP1 to design peptide sequences to mitigate Tau internalization, the work can be extended to other domains of the LRP1 receptor or other receptors to examine if the cellular internalization of fibrillar Tau can be deterred. These findings show that short peptides derived from the LRP1 receptor can alter the internalization of its ligands.more » « less
-
Tau misfolding, oligomerization, and aggregation are central to the pathology of Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), frontotemporal dementia, and other tauopathies. Increased phosphorylation of tau is associated with conformational changes that are not fully understood. Moreover, tau oligomerization and aggregation are associated with proline cis-trans isomerism, with the phosphorylation-dependent prolyl isomerase Pin1 reducing tau hyperphosphorylation and aggregation. The FTDP-17 tau mutation R406W is frequently used in animal models of Alzheimer's disease, due to earlier onset of the AD phenotype. Despite its extensive application, the mechanisms by which tau-R406W leads to enhanced aggregation and neurotoxicity are poorly understood. Peptides derived from the tau C-terminal domain were examined by NMR spectroscopy as a function of residue 406 identity (Arg versus Trp) and Ser404 phosphorylation state. The R406W modification led to an increased population of Pro405 cis amide bond, which is stabilized by cis-proline-aromatic C–H/π interactions. Ser404 phosphorylation also resulted in an increase in cis amide bond, via a proposed C–H/O interaction between the Pro Hα and the phosphate that stabilizes the cis conformation. An analogous C–H/O interaction was observed in Glu-cis-Pro sequences in the PDB, and is proposed to be the basis of the increased propensity for cis amide bonds in Glu-Pro sequences. The higher activation barriers for proline cis-trans isomerization observed at pSer-Pro and pThr-Pro sequences are proposed to be due to both (a) an intraresidue phosphate-amide bond that stabilizes the trans-proline conformation and (b) the cis-stabilizing proline-phosphate C–H/O interaction identified herein. The combination of both pSer404 and R406W resulted in a further increase in the population of cis amide bond. In contrast to expectations, the R406W modification led to increased dephosphorylation of either pSer404 or pSer409 by PP2A, and had no effect on phosphorylation of Ser404 by cdk5, suggesting that R406W does not inherently increase Ser404 phosphorylation via changes in the actions of these enzymes. Modestly increased phosphorylation of Ser404 was observed by GSK-3β in tau R406W. Collectively, these data suggest a potential role for conformational change to a cis amide bond at Pro405, via Ser404 phosphorylation and/or R406W modification, as a possible mechanism involved in protein misfolding in AD, CTE, and FTDP-17. Alternatively, both Ser404 phosphorylation and the R406W modification lead to increased order, including induced turn formation, in both the trans-proline and cis-proline conformations.more » « less
