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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

Detection of aggregated Htt and its ubiquitinated species. (A) SDS-insoluble fraction of Neuro-2a cell lysate after transient transfection of cells with wildtype or 3xR mutant Htt-exon1-97Q constructs. Formic acid-dissolved aggregates of Htt were detected on western blot by the Htt-specific antibodies 1C2 and N18. Formic acid soluble Htt-exon1 monomer (arrow) and a higher wildtype Htt-specific protein ladder, which is not detectable with 3xR mutant Htt, are shown. Remaining non-dissolved Htt aggregates were trapped in the stacking gel and recognized by the N18 antibody only. (B) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently transfected with Htt-exon1-97Q-H4. In addition to the monomeric Htt-exon1-97Q-H4 protein a ubiquitin-positive Htt protein ladder (asterisks) is shown by the HA and ubiquitin (Ub) antibody, respectively. (C) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently co-transfected with Htt-exon1-97Q and the ubiquitin constructs HA-Ub-wt and its lysine-dead mutant HA-Ub-K0, respectively. In addition to the monomeric Htt-exon1-97Q protein a ubiquitin-positive Htt protein ladder is shown by the 1C2 and Ub antibody, respectively. Lysine residue-dependent integration of HA-Ub-wt into the Htt-exon1 polyubiquitination chains is shown by an upward shift of the ubiquitin-modified Htt bands at the size of the ubiquitin N-terminal HA-tag (asterisks).
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Figure 3: Detection of aggregated Htt and its ubiquitinated species. (A) SDS-insoluble fraction of Neuro-2a cell lysate after transient transfection of cells with wildtype or 3xR mutant Htt-exon1-97Q constructs. Formic acid-dissolved aggregates of Htt were detected on western blot by the Htt-specific antibodies 1C2 and N18. Formic acid soluble Htt-exon1 monomer (arrow) and a higher wildtype Htt-specific protein ladder, which is not detectable with 3xR mutant Htt, are shown. Remaining non-dissolved Htt aggregates were trapped in the stacking gel and recognized by the N18 antibody only. (B) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently transfected with Htt-exon1-97Q-H4. In addition to the monomeric Htt-exon1-97Q-H4 protein a ubiquitin-positive Htt protein ladder (asterisks) is shown by the HA and ubiquitin (Ub) antibody, respectively. (C) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently co-transfected with Htt-exon1-97Q and the ubiquitin constructs HA-Ub-wt and its lysine-dead mutant HA-Ub-K0, respectively. In addition to the monomeric Htt-exon1-97Q protein a ubiquitin-positive Htt protein ladder is shown by the 1C2 and Ub antibody, respectively. Lysine residue-dependent integration of HA-Ub-wt into the Htt-exon1 polyubiquitination chains is shown by an upward shift of the ubiquitin-modified Htt bands at the size of the ubiquitin N-terminal HA-tag (asterisks).

Mentions: In order to solubilize SDS-insoluble protein pellets containing aggregated Htt from Neuro-2a cells transfected with the constructs encoding Htt-exon1-97Q and its 3xR Htt mutant variant, pellets were treated with 100% formic acid followed by SDS-PAGE and immunoblot analysis. The formic acid treated SDS-insoluble fraction showed the presence of monomeric Htt-exon1-97Q at around 45 kDa (arrow) and at higher molecular weight post-translational modified species of Htt-exon1-97Q in form of distinct bands (Figure 3A). Htt was stained by the Htt-specific antibodies 1C2 against the polyQ tract and N18 against the N-terminal region. With the 3xR Htt mutant variant the higher molecular Htt bands disappeared, indicating that the post-translational modification of Htt is dependent on the N-terminal lysine residues. The size difference between the distinct bands suggests a ubiquitination of Htt. The observation that the aggregated 3xR Htt mutant does not show this high molecular weight bands indicates that the Htt N-terminal lysine residues are the ubiquitination sides and not, as described as more rare forms of ubiquitination, serine, threonine and cysteine residues or the N-terminal α-amino group ubiquitination in a linear fashion (McDowell and Philpott, 2013).


Detection of ubiquitinated huntingtin species in intracellular aggregates.

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

Detection of aggregated Htt and its ubiquitinated species. (A) SDS-insoluble fraction of Neuro-2a cell lysate after transient transfection of cells with wildtype or 3xR mutant Htt-exon1-97Q constructs. Formic acid-dissolved aggregates of Htt were detected on western blot by the Htt-specific antibodies 1C2 and N18. Formic acid soluble Htt-exon1 monomer (arrow) and a higher wildtype Htt-specific protein ladder, which is not detectable with 3xR mutant Htt, are shown. Remaining non-dissolved Htt aggregates were trapped in the stacking gel and recognized by the N18 antibody only. (B) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently transfected with Htt-exon1-97Q-H4. In addition to the monomeric Htt-exon1-97Q-H4 protein a ubiquitin-positive Htt protein ladder (asterisks) is shown by the HA and ubiquitin (Ub) antibody, respectively. (C) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently co-transfected with Htt-exon1-97Q and the ubiquitin constructs HA-Ub-wt and its lysine-dead mutant HA-Ub-K0, respectively. In addition to the monomeric Htt-exon1-97Q protein a ubiquitin-positive Htt protein ladder is shown by the 1C2 and Ub antibody, respectively. Lysine residue-dependent integration of HA-Ub-wt into the Htt-exon1 polyubiquitination chains is shown by an upward shift of the ubiquitin-modified Htt bands at the size of the ubiquitin N-terminal HA-tag (asterisks).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Detection of aggregated Htt and its ubiquitinated species. (A) SDS-insoluble fraction of Neuro-2a cell lysate after transient transfection of cells with wildtype or 3xR mutant Htt-exon1-97Q constructs. Formic acid-dissolved aggregates of Htt were detected on western blot by the Htt-specific antibodies 1C2 and N18. Formic acid soluble Htt-exon1 monomer (arrow) and a higher wildtype Htt-specific protein ladder, which is not detectable with 3xR mutant Htt, are shown. Remaining non-dissolved Htt aggregates were trapped in the stacking gel and recognized by the N18 antibody only. (B) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently transfected with Htt-exon1-97Q-H4. In addition to the monomeric Htt-exon1-97Q-H4 protein a ubiquitin-positive Htt protein ladder (asterisks) is shown by the HA and ubiquitin (Ub) antibody, respectively. (C) Western blot analysis of the SDS-insoluble fraction of Neuro-2a cells transiently co-transfected with Htt-exon1-97Q and the ubiquitin constructs HA-Ub-wt and its lysine-dead mutant HA-Ub-K0, respectively. In addition to the monomeric Htt-exon1-97Q protein a ubiquitin-positive Htt protein ladder is shown by the 1C2 and Ub antibody, respectively. Lysine residue-dependent integration of HA-Ub-wt into the Htt-exon1 polyubiquitination chains is shown by an upward shift of the ubiquitin-modified Htt bands at the size of the ubiquitin N-terminal HA-tag (asterisks).
Mentions: In order to solubilize SDS-insoluble protein pellets containing aggregated Htt from Neuro-2a cells transfected with the constructs encoding Htt-exon1-97Q and its 3xR Htt mutant variant, pellets were treated with 100% formic acid followed by SDS-PAGE and immunoblot analysis. The formic acid treated SDS-insoluble fraction showed the presence of monomeric Htt-exon1-97Q at around 45 kDa (arrow) and at higher molecular weight post-translational modified species of Htt-exon1-97Q in form of distinct bands (Figure 3A). Htt was stained by the Htt-specific antibodies 1C2 against the polyQ tract and N18 against the N-terminal region. With the 3xR Htt mutant variant the higher molecular Htt bands disappeared, indicating that the post-translational modification of Htt is dependent on the N-terminal lysine residues. The size difference between the distinct bands suggests a ubiquitination of Htt. The observation that the aggregated 3xR Htt mutant does not show this high molecular weight bands indicates that the Htt N-terminal lysine residues are the ubiquitination sides and not, as described as more rare forms of ubiquitination, serine, threonine and cysteine residues or the N-terminal α-amino group ubiquitination in a linear fashion (McDowell and Philpott, 2013).

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