Local Protein Unfolding and Pathogenesis of Polyglutamine-Expansion Disease Yu Wai Chen Centre for Protein Engineering and Cambridge University Chemical.

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Local Protein Unfolding and Pathogenesis of Polyglutamine-Expansion Disease Yu Wai Chen Centre for Protein Engineering and Cambridge University Chemical Laboratory, Cambridge, United Kingdom Proteins 2003;51:68-73 Proteins 2003;51:68-73 speaker: S.C.Fu 24/04/03 speaker: S.C.Fu 24/04/03

What is Polyglutamine-Expansion Disease? Polyglutamine-Expansion Polyglutamine-Expansion Abnormal lengthening of polyglutamine (polyQ) repeat sequence inside the protein. Abnormal lengthening of polyglutamine (polyQ) repeat sequence inside the protein. Inherited neurodegenerative disease such as Huntington ’ s disease and spinocerebellar ataxia (SCA) have been shown to be caused by polyQ repeat. Inherited neurodegenerative disease such as Huntington ’ s disease and spinocerebellar ataxia (SCA) have been shown to be caused by polyQ repeat.

Elongation of CAG repeat Petruska et. al. 1998

These Inherited neurodegenerative diseases: Causing misfolding and aggregation of disease proteins. Causing misfolding and aggregation of disease proteins. Expansion of Glutamine repeat > 35~40 residues confers on disease proteins a toxic gain of function that cause tissue-specific neuronal death. Expansion of Glutamine repeat > 35~40 residues confers on disease proteins a toxic gain of function that cause tissue-specific neuronal death. Progress via a similar pathogenic mechanism. Progress via a similar pathogenic mechanism. Apart from polyQ repeat, these proteins and their genes have nothing in common. Apart from polyQ repeat, these proteins and their genes have nothing in common.

Hypothesis PolyQ disease proteins may share a common feature of being “ natively unfolded ” and are thus susceptible to misfolding and aggregation.

Unfolded

Proteins that are “ natively unfolded ” are characterized by high net charge and low hydrophobicity. They found that for a given protein with a mean net charge of, a boundary value exists for its mean hydrophobicity,, below which the protein is likely to be natively unfolded. This empirical relationship is expressed as:

Materials and Methods Dataset ( Dataset (from the SWISS-PROT database) Huntington ’ s disease protein (or huntingtin, Htt, P42858) androgen receptor (AR, P10275) atrophin-1 (Atf1,also known as dentatorubral-pallidoluysian atrophy protein,P54259) spinocerebellar ataxin-1 (Atx1, P54253), ataxin-2 (Atx2, Q99700) ataxin-3 (Atx3, also known as Machado-Joseph disease protein, MJD, P54252) ataxin-6(Atx6, O00555) ataxin-7 (Atx7, O15265) sperm whale myoglobin (Mb, P02185) The TATA-box-binding protein (TBP or TF2D, referred to as Atx17 here, P20226)

Materials and Methods Using ProtParam program from ExPASy for net calculation. Using ProtParam program from ExPASy for net calculation.ProtParam ProtScale is used to calculate hydrophobicity. ProtScale is used to calculate hydrophobicity. ProtScale

ProtParam

Global protein folding prediction blue dots (excluding polyQ) red dots (including the polyQ)

Result of global and local protein folding prediction

Local protein folding prediction blue dots (excluding polyQ) red dots (including the polyQ)

Compared with experimental observation An artificial system to study the structural consequences of introducing repeats of 12, 28, 35, and 50 glutamines into sperm whale myoglobin (Mb). Mb-12 Mb-28 Mb-35 Mb-50

Compared with experimental observation

The studies of secondary structures of several fusion constructs of Atx3 using circular dichorism (CD) spectroscopy Atx3 and maltose-binding protein fusions (MBP- Atx3-Q27 and MBPAtx3-Q78) hexahistidine-tagged ataxin-3 (H6-Atx3-Q27)

Circular dichorism Circular dichroism is the difference in absorption between left and right handed circular polarized light. Circular dichroism is the difference in absorption between left and right handed circular polarized light. is used to gain information about the secondary structure of proteins and polypeptides in solution. is used to gain information about the secondary structure of proteins and polypeptides in solution. very sensitive to the secondary structure of polypeptides and proteins. very sensitive to the secondary structure of polypeptides and proteins.

Standard Curves

Example of CD

Compared with experimental observation

Discussion Contribution Contribution Limitation of this method: Limitation of this method: Need more information about domain boundary. Need more information about domain boundary. Ligands or ions that affect local folding should be considered. Ligands or ions that affect local folding should be considered. Limited in applying to proteins w/ multiple domain. Limited in applying to proteins w/ multiple domain.

Simulation of Unfolding

Thanks for your attention