Molecular chaperones and Transthyretin Amyloid Disease

This projects was done to have a deep familiarity with how chaperones communicate with unstable, aggregation prone, misfolded proteins linked to human disease. During the past 20 years, there has been a good deal focus on misfolding diseases within the areas of biochemistry and molecular biotechnology research. It is now clear that proteins which misfold (because of a mutation or outer factors), are the reason behind several diseases. Molecular chaperones are proteins which have been classified as agents that aid other proteins to fold accurately and also avert aggregation. Their role in the misfolding disease procedure is the topic for this project report. Transthyretin (TTR) is actually a protein present in human plasma as well as in cerebrospinal fluid. It functions like a transport protein, carrying thyroxin and holo-retinol binding protein. TTR is made up of 4 identical subunits linked through hydrogen bonds and hydrophobic interactions. More than One hundred point mutations in the TTR gene are linked with amyloidosis frequently concerning peripheral neurodegeneration. Amyloidosis stands for a range of diseases resulting in extra cellular deposition of fibrillar protein called amyloid. We utilized human SH-SY5Y neuroblastoma cells as a model for neurodegeneration. Different conformers of TTR were incubated with the cells for different durations. The tests demonstrated that early prefibrillar oligomers of TTR caused apoptosis when neuroblastoma cells were exposed to these species as opposed to mature fibrils weren’t cytotoxic. Additionally we discovered improved expression of the molecular chaperone BiP in cells challenged with TTR oligomers.

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Protein production- the background story
Protein folding
Genetic mutations
Protein misfolding
Transthyretin (TTR)
The TTR D18G mutation
Molecular chaperones
Structure and mechanism of BiP
A role for BiP during translocation
The role of molecular chaperones in misfolding diseases
The ER, cellular stress and cell death
The unfolded protein response (UPR)
Cloning, mutagenesis
SDS-PAGE and Western blotting
Circular Dichroism
Fluorescence spectroscopy
Chemical cross-linking
Transmission Electron Microscopy (TEM)
Affinity chromatography and immunoprecipitation
Analytical ultracentrifugation
Paper I
Cross-linking to probe formation of aggregates
Size and morphology of aggregates and protofibrils
Characterization of TTR conformers using molecular probes
Different kinetics for different probes
Paper II
Early oligomeric species of TTR kill human cells
Early oligomeric species of TTR induce ER stress
Paper III
BiP selectively binds to destabilized variants of TTR
Composition of the BiP- TTR D18G complex
The BiP- TTR D18G interaction
BiP plays a protective role against the toxic effects of TTR D18G
Paper IV
BiP interacts with TTR D18G in the mammalian ER
The degradation rate of TTR D18G is slowed down in the presence of BiP
The BiP- TTR D18G complex was present in a wide distribution of molecular weights…..

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Source: Linköping University

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