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Detection of ubiquitinated huntingtin species in intracellular aggregates.

Juenemann K, Wiemhoefer A, Reits EA - Front Mol Neurosci (2015)

Bottom Line: Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins.Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt.This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam Amsterdam, Netherlands.

ABSTRACT
Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington's disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

No MeSH data available.


Related in: MedlinePlus

A stepwise protocol of SDS-soluble and SDS-insoluble cell fractionation.
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Figure 2: A stepwise protocol of SDS-soluble and SDS-insoluble cell fractionation.

Mentions: Here we provide a protocol suitable for subsequent biochemical protein analysis of aggregates, consisting of mutant Htt, by solubilizing them with 100% formic acid (Figure 2). Formic acid disrupts protein interactions such as hydrogen bonds. Previous work indicated that aggregation of Htt results from the formation of hydrogen bonds between polyQ stretches forming β-sheet structures (Perutz et al., 1994). Hazeki et al. showed that only formic acid and HCl but not acedic acid, Urea, Guanidine/HCl or NaOH dissociate higher molecular weight complexes of mutant Htt (Hazeki et al., 2000).


Detection of ubiquitinated huntingtin species in intracellular aggregates.

Juenemann K, Wiemhoefer A, Reits EA - Front Mol Neurosci (2015)

A stepwise protocol of SDS-soluble and SDS-insoluble cell fractionation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4309157&req=5

Figure 2: A stepwise protocol of SDS-soluble and SDS-insoluble cell fractionation.
Mentions: Here we provide a protocol suitable for subsequent biochemical protein analysis of aggregates, consisting of mutant Htt, by solubilizing them with 100% formic acid (Figure 2). Formic acid disrupts protein interactions such as hydrogen bonds. Previous work indicated that aggregation of Htt results from the formation of hydrogen bonds between polyQ stretches forming β-sheet structures (Perutz et al., 1994). Hazeki et al. showed that only formic acid and HCl but not acedic acid, Urea, Guanidine/HCl or NaOH dissociate higher molecular weight complexes of mutant Htt (Hazeki et al., 2000).

Bottom Line: Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins.Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt.This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam Amsterdam, Netherlands.

ABSTRACT
Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington's disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

No MeSH data available.


Related in: MedlinePlus