Rapid purification of truncated tau proteins: model approach to purification of functionally active fragments of disordered proteins, implication for neurodegenerative diseases

Csokova, Natalia; Škrabana, Rostislav; Liebig, Hans‐Dieter; Mederlyova, Anna; Kontsek, Peter; Novák, Michal

doi:10.1016/j.pep.2004.01.012

Cited by 38 publications

(30 citation statements)

References 21 publications

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“…The protocol that was standardized for Tau4RD purification is a slight modification of published protocols (64,65) and makes use of cation-exchange chromatography followed by size-exclusion chromatography to obtain Ͼ99% pure protein and an average yield of ϳ100 mg/liter. A more detailed protocol is described in the supplemental Methods.…”

Section: Methodsmentioning

confidence: 99%

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

Ramachandran

Udgaonkar

2011

Journal of Biological Chemistry

124

179

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Background:The kinetic role of heparin and of intermediates populated during Tau protein aggregation is not fully understood. Results: Heparin contributes to the initial steps of fibrillation, whereas protofibrillar intermediates accumulate transiently. Conclusion: Heparin is involved in nucleation, and the transient rod-like protofibrils are off-pathway species. Significance: Understanding the kinetic role of heparin and the protofibrils has implications for the development of therapies for tauopathies.

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Section: Methodsmentioning

confidence: 99%

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

Ramachandran

Udgaonkar

2011

Journal of Biological Chemistry

124

179

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“…Integrity of each construct was verified by DNA sequence analysis (ABI Prism 377DNA Sequencer, Perkin-Elmer). Tau proteins were expressed in Escherichia coli and purified from bacterial lysates by ion-exchange chromatography [14]. The protein concentration was determined by BCA kit (Pierce, USA).…”

Section: Preparation Expression and Purification Of Tau Proteinsmentioning

confidence: 99%

“…Sarcosyl insoluble tau proteins purified from brains were analyzed on 5-20% SDS-PAGE gradient gel and Western blot as described previously [14]. Enhanced chemiluminescence developed Western blot was digitalized with LAS3000 CCD imaging system (Fujifilm, Japan).…”

Section: Western Blottingmentioning

confidence: 99%

Truncated tau from sporadic Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo

Žilka¹,

Filipčík²,

Koson³

et al. 2006

FEBS Letters

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224

203

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Truncated tau protein is the characteristic feature of human sporadic Alzheimer's disease. We have identified truncated tau proteins conformationally different from normal healthy tau. Subpopulations of these structurally different tau species promoted abnormal microtubule assembly in vitro suggesting toxic gain of function. To validate pathological activity in vivo we expressed active form of human truncated tau protein as transgene, in the rat brain. Its neuronal expression led to the development of the neurofibrillary degeneration of Alzheimer's type. Furthermore, biochemical analysis of neurofibrillary changes revealed that massive sarcosyl insoluble tau complexes consisted of human Alzheimer's tau and endogenous rat tau in ratio 1:1 including characteristic Alzheimer's disease (AD)-specific proteins (A68). This work represents first insight into the possible causative role of truncated tau in AD neurofibrillary degeneration in vivo.

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“…Labeled secondary antibody was visualized with a chemiluminescence detection system (Pierce, USA), and the signal was recorded with a LAS3000 CCD imaging system (Fujifilm, Japan). Densitometric data analysis and relative quantification of Western blots were performed by AIDA Biopackage software (Raytest, Germany) as described [12]. The amount of Hsp27 protein was analyzed in the 1S preparation, and the relative level of anti-Hsp27 signal was normalized to the Gapdh (both antibodies purchased from Abcam, USA).…”

Section: Western Blottingmentioning

confidence: 99%

Intraneuronal accumulation of misfolded tau protein induces overexpression of Hsp27 in activated astrocytes

Filipčík¹,

Cente²,

Žilka³

et al. 2015

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

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Accumulation of misfolded forms of microtubule associated, neuronal protein tau causes neurofibrillary degeneration typical of Alzheimer's disease and other tauopathies. This process is accompanied by elevated cellular stress and concomitant deregulation of heat-shock proteins. We used a transgenic rat model of tauopathy to study involvement of heat shock protein 27 (Hsp27) in the process of neurofibrillary degeneration, its cell type specific expression and correlation with the amount of insoluble tau protein aggregates. The expression of Hsp27-mRNA is more than doubled and levels of Hsp27 protein tripled in aged transgenic animals with tau pathology. The data revealed a strong positive and highly significant correlation between Hsp27-mRNA and amount of sarkosyl insoluble tau. Interestingly, intracellular accumulation of insoluble misfolded tau protein in neurons was associated with overexpression of Hsp27 almost exclusively in reactive astrocytes, not in neurons. The topological dissociation of neuronally expressed pathological tau and the induction of astrocytic Hsp27, GFAP, and Vimentin along with up-regulation of microglia specific markers such as CD18, CD68 and C3 point to cooperation of astrocytes, microglia and neurons in response to intra-neuronal accumulation of insoluble tau. Our data suggest that over expression of Hsp27 represents a part of microglia-mediated astrocytic response mechanism in the process of neurofibrillary degeneration, which is not necessarily associated with neuroprotection and which in contrary may accelerate neurodegeneration in late stage of the disease. This phenomenon should be considered during development of disease modifying strategies for treatment of tauopathies and AD via regulation of activity of Hsp27.

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Rapid purification of truncated tau proteins: model approach to purification of functionally active fragments of disordered proteins, implication for neurodegenerative diseases

Cited by 38 publications

References 21 publications

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

Truncated tau from sporadic Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo

Intraneuronal accumulation of misfolded tau protein induces overexpression of Hsp27 in activated astrocytes

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