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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

Conventional methods to study Htt protein aggregates. (A) Scheme of the Htt-exon1 protein fragment. N-terminal region (aa 1-17) of wildtype Htt-exon1 with three lysine residues (K6, K9, K15) compared to the Htt mutant (3xR) containing three arginine residues (R6, R9, R15). (B) Aggregates of wildtype and 3xR mutant Htt-exon1. Transient co-transfection of Neuro-2a cells with the aggregation reporter Htt-exon1-25Q-GFP and wildtype or 3xR mutant Htt-exon1-97Q-H4 constructs. After 24 h cells were fixed and aggregates of Htt-exon1 co-sequestering the aggregation reporter Htt-exon1-25Q-GFP were detected by fluorescence of GFP. The nucleus was stained by 4’,6-diamidino-2-phenylindole (DAPI). Scale bar: 10 μm. (C) Western blot analysis and (D) filter trap assay (in doublets) of cell lysates from Neuro-2a cells transient transfected for 24 h with wildtype and 3xR mutant Htt-exon1-97Q-H4 constructs. Triton X-100-soluble Htt-exon1 proteins and insoluble Htt aggregates (asterisk) in the stacking gel were detected on immunoblots with HA antibody. β-actin was used as loading control. Filter trap assay was performed using an antibody against the HA-tag of Htt and an antibody against ubiquitin (Ub) recognizing ubiquitinated proteins.
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Figure 1: Conventional methods to study Htt protein aggregates. (A) Scheme of the Htt-exon1 protein fragment. N-terminal region (aa 1-17) of wildtype Htt-exon1 with three lysine residues (K6, K9, K15) compared to the Htt mutant (3xR) containing three arginine residues (R6, R9, R15). (B) Aggregates of wildtype and 3xR mutant Htt-exon1. Transient co-transfection of Neuro-2a cells with the aggregation reporter Htt-exon1-25Q-GFP and wildtype or 3xR mutant Htt-exon1-97Q-H4 constructs. After 24 h cells were fixed and aggregates of Htt-exon1 co-sequestering the aggregation reporter Htt-exon1-25Q-GFP were detected by fluorescence of GFP. The nucleus was stained by 4’,6-diamidino-2-phenylindole (DAPI). Scale bar: 10 μm. (C) Western blot analysis and (D) filter trap assay (in doublets) of cell lysates from Neuro-2a cells transient transfected for 24 h with wildtype and 3xR mutant Htt-exon1-97Q-H4 constructs. Triton X-100-soluble Htt-exon1 proteins and insoluble Htt aggregates (asterisk) in the stacking gel were detected on immunoblots with HA antibody. β-actin was used as loading control. Filter trap assay was performed using an antibody against the HA-tag of Htt and an antibody against ubiquitin (Ub) recognizing ubiquitinated proteins.

Mentions: Htt contains three lysine residues (K6, K9, K15) at the N17 region for putative ubiquitination (Figure 1A). Previous work from Steffan et al. has shown that mutation of these three lysines to arginines obviates ubiquitination and SUMOylation of soluble Htt-exon1 in cell culture (Steffan et al., 2004). An AQUA-MS approach revealed that less than 1% of soluble N-terminal Htt expressed in HEK 293 cells is ubiquitinated without considering inhibition of cellular degradation pathways (Hipp et al., 2012). Immunoprecipitation and mass spectrometry analysis are appropriate methods to identify specific soluble protein-ubiquitin conjugates. However, once misfolded proteins start to aggregate and interacting partners are co-sequestered, this represents a challenge in terms of isolation and detection of specific protein-ubiquitin conjugates.


Detection of ubiquitinated huntingtin species in intracellular aggregates.

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

Conventional methods to study Htt protein aggregates. (A) Scheme of the Htt-exon1 protein fragment. N-terminal region (aa 1-17) of wildtype Htt-exon1 with three lysine residues (K6, K9, K15) compared to the Htt mutant (3xR) containing three arginine residues (R6, R9, R15). (B) Aggregates of wildtype and 3xR mutant Htt-exon1. Transient co-transfection of Neuro-2a cells with the aggregation reporter Htt-exon1-25Q-GFP and wildtype or 3xR mutant Htt-exon1-97Q-H4 constructs. After 24 h cells were fixed and aggregates of Htt-exon1 co-sequestering the aggregation reporter Htt-exon1-25Q-GFP were detected by fluorescence of GFP. The nucleus was stained by 4’,6-diamidino-2-phenylindole (DAPI). Scale bar: 10 μm. (C) Western blot analysis and (D) filter trap assay (in doublets) of cell lysates from Neuro-2a cells transient transfected for 24 h with wildtype and 3xR mutant Htt-exon1-97Q-H4 constructs. Triton X-100-soluble Htt-exon1 proteins and insoluble Htt aggregates (asterisk) in the stacking gel were detected on immunoblots with HA antibody. β-actin was used as loading control. Filter trap assay was performed using an antibody against the HA-tag of Htt and an antibody against ubiquitin (Ub) recognizing ubiquitinated proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4309157&req=5

Figure 1: Conventional methods to study Htt protein aggregates. (A) Scheme of the Htt-exon1 protein fragment. N-terminal region (aa 1-17) of wildtype Htt-exon1 with three lysine residues (K6, K9, K15) compared to the Htt mutant (3xR) containing three arginine residues (R6, R9, R15). (B) Aggregates of wildtype and 3xR mutant Htt-exon1. Transient co-transfection of Neuro-2a cells with the aggregation reporter Htt-exon1-25Q-GFP and wildtype or 3xR mutant Htt-exon1-97Q-H4 constructs. After 24 h cells were fixed and aggregates of Htt-exon1 co-sequestering the aggregation reporter Htt-exon1-25Q-GFP were detected by fluorescence of GFP. The nucleus was stained by 4’,6-diamidino-2-phenylindole (DAPI). Scale bar: 10 μm. (C) Western blot analysis and (D) filter trap assay (in doublets) of cell lysates from Neuro-2a cells transient transfected for 24 h with wildtype and 3xR mutant Htt-exon1-97Q-H4 constructs. Triton X-100-soluble Htt-exon1 proteins and insoluble Htt aggregates (asterisk) in the stacking gel were detected on immunoblots with HA antibody. β-actin was used as loading control. Filter trap assay was performed using an antibody against the HA-tag of Htt and an antibody against ubiquitin (Ub) recognizing ubiquitinated proteins.
Mentions: Htt contains three lysine residues (K6, K9, K15) at the N17 region for putative ubiquitination (Figure 1A). Previous work from Steffan et al. has shown that mutation of these three lysines to arginines obviates ubiquitination and SUMOylation of soluble Htt-exon1 in cell culture (Steffan et al., 2004). An AQUA-MS approach revealed that less than 1% of soluble N-terminal Htt expressed in HEK 293 cells is ubiquitinated without considering inhibition of cellular degradation pathways (Hipp et al., 2012). Immunoprecipitation and mass spectrometry analysis are appropriate methods to identify specific soluble protein-ubiquitin conjugates. However, once misfolded proteins start to aggregate and interacting partners are co-sequestered, this represents a challenge in terms of isolation and detection of specific protein-ubiquitin conjugates.

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