Bimolecular Fluorescence Complementation; Lighting-Up Tau-Tau Interaction in Living Cells

Tak, HyeJin; Haque, Md. Mamunul; Kim, Min Jung; Lee, Juhyun; Baik, Ja-Hyun; Kim, Young Soo; Kim, Dong Jin; Grailhe, Régis; Kim, Yun Kyung

doi:10.1371/journal.pone.0081682

Cited by 66 publications

(93 citation statements)

References 24 publications

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“…7a, second row). These results indicate that the binding of wild-type pT231 scFv and mutant 3.24 requires phosphorylation of tau, and as reported previously, the tau expressed in HEK293 cells is phosphorylated (49,50). In order to further assess the specificity of the scFvs, we generated point mutants of human tau at the target phosphorylation site (T231) either incapable of phosphorylation (T231A) or pseudophosphorylated (T231E).…”

Section: Cell and Tissue Section Labeling Using Purified Scfvssupporting

confidence: 73%

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Wang

Maziuk

et al. 2018

Journal of Biological Chemistry

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Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTMtargeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phospho-threonine 231 (pT231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinitymatured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved > 20-fold over that of the wild-type antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity towards the nonphosphorylated target site, but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phospho-tau antibody. In cell-and tissueimaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTMspecific antibodies, and highlight the importance of assessing the specificity in the antibody engineering process.

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Section: Cell and Tissue Section Labeling Using Purified Scfvssupporting

confidence: 73%

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Wang

Maziuk

et al. 2018

Journal of Biological Chemistry

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“…Thus, the effects of exogenous proteasomes on aggregating protein levels were restricted to UPS substrates. To visualize and quantify tau oligomerization in living cells, we utilized a tau cell line with biomolecular fluorescence complementation (BiFC) 23 . Amino-terminal and carboxyl-terminal parts of Venus proteins were independently fused to htau40, which exhibited only basal fluorescence signals under normal conditions (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A HEK293-derived stable cell line (Tau-BiFC), which constitutively expresses both the carboxyl and the amino terminus of Venus proteins independently fused with htau40, was established as recently described 23 . Tau-BiFC cells were seeded in a 96-well plate at a density of 10 5 cells per well and treated with 30 nM of okadaic acid for 24 h to accelerate tau oligomerization processes.…”

Section: Discussionmentioning

confidence: 99%

Direct cellular delivery of human proteasomes to delay tau aggregation

Han

Choi

et al. 2014

Nat Commun

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The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins.

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“…BiFC was primarily applied for the investigation of heterodimeric protein interactions. Subsequently, several studies employed this technique also for the analysis of protein homodimerization and oligomerization [14,47]. Initial studies used the fluorophore GFP for BiFC.…”

Section: Introductionmentioning

confidence: 99%

Dimerization propensities of Synucleins are not predictive for Synuclein aggregation

Eckermann

Kügler

Bähr

2015

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Aggregation and fibril formation of human alpha-Synuclein (αS) are neuropathological hallmarks of Parkinson's disease and other synucleinopathies. The molecular mechanisms of αS aggregation and fibrillogenesis are largely unknown. Several studies suggested a sequence of events from αS dimerization via oligomerization and pre-fibrillar aggregation to αS fibril formation. In contrast to αS, little evidence suggests that γS can form protein aggregates in the brain, and for βS its neurotoxic properties and aggregation propensities are controversially discussed. These apparent differences in aggregation behavior prompted us to investigate the first step in Synuclein aggregation, i.e. the formation of dimers or oligomers, by Bimolecular Fluorescence Complementation in cells. This assay showed some Synuclein-specific limitations, questioning its performance on a single cell level. Nevertheless, we unequivocally demonstrate that all Synucleins can interact with each other in a very similar way. Given the divergent aggregation properties of the three Synucleins this suggests that formation of dimers is not predictive for the aggregation of αS, βS or γS in the aged or diseased brain.

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Bimolecular Fluorescence Complementation; Lighting-Up Tau-Tau Interaction in Living Cells

Cited by 66 publications

References 24 publications

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Direct cellular delivery of human proteasomes to delay tau aggregation

Dimerization propensities of Synucleins are not predictive for Synuclein aggregation

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