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Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression.

Suhr ST, Senut MC, Whitelegge JP, Faull KF, Cuizon DB, Gage FH - J. Cell Biol. (2001)

Bottom Line: One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs).Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex.These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

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Western blot analysis of protein sequestration in purified IAs and control whole cell extracts. For all protein blots: lane 1, whole cell extracts from 5-d induced 13QN cells; lane 2, uninduced 96QN cells; lane 3, 5-d induced 96QN cells; and lane 4, isolated concentrated aggregates. Arrowheads at the right of autoradiograms indicate predicted molecular weights of individual protein species unless otherwise specified. (a) Processing for polyQ–GFP immunoreactivity. 96-P indicates the position of polyQN–GFP polymers within the stacking gel and near the top of the resolving gel (lanes 3 and 4), 96-M indicates a low level of 96QN monomers (lanes 2–4), and 13-M indicates the 13QN monomer band (lane 1). This autoradiogram was overexposed to reveal the 96QN monomer bands in lanes 3 and 4. (b) Ubiquitin; (c) HSP70; (d) MEF-2a; (e) Htt; (f) TBP; (g) Nck; (h) GRB-2; (i) caspase-9; (j) caspase-8 (putative procaspase-8, upper arrowhead; putative caspase-8 cleavage products, lower arrowheads); (k) caspase-3; (l) p53 and p50 immunoreactive species (upper and lower arrowheads, respectively); (m) mdm-2 antibody-1 (120-kD variant, upper arrowhead; p60 variant, lower arrowhead); and (n) mdm-2 antibody-2 immunoreactivity.
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Figure 3: Western blot analysis of protein sequestration in purified IAs and control whole cell extracts. For all protein blots: lane 1, whole cell extracts from 5-d induced 13QN cells; lane 2, uninduced 96QN cells; lane 3, 5-d induced 96QN cells; and lane 4, isolated concentrated aggregates. Arrowheads at the right of autoradiograms indicate predicted molecular weights of individual protein species unless otherwise specified. (a) Processing for polyQ–GFP immunoreactivity. 96-P indicates the position of polyQN–GFP polymers within the stacking gel and near the top of the resolving gel (lanes 3 and 4), 96-M indicates a low level of 96QN monomers (lanes 2–4), and 13-M indicates the 13QN monomer band (lane 1). This autoradiogram was overexposed to reveal the 96QN monomer bands in lanes 3 and 4. (b) Ubiquitin; (c) HSP70; (d) MEF-2a; (e) Htt; (f) TBP; (g) Nck; (h) GRB-2; (i) caspase-9; (j) caspase-8 (putative procaspase-8, upper arrowhead; putative caspase-8 cleavage products, lower arrowheads); (k) caspase-3; (l) p53 and p50 immunoreactive species (upper and lower arrowheads, respectively); (m) mdm-2 antibody-1 (120-kD variant, upper arrowhead; p60 variant, lower arrowhead); and (n) mdm-2 antibody-2 immunoreactivity.

Mentions: For quantification of sequestered proteins, each antibody used to detect a sequestered protein species in the Western blot analyses (see Fig. 3) was titrated against increasing concentrations of induced 96QN cell whole cell extracts or purified IA samples to determine the linear range of detection (data not shown). Each antibody was reassessed against the same samples as those described in the legend to Fig. 3 using an amount of protein and dilution of antibody determined to be within the linear range of detection. From these protein blots, the integrated densities of bands within purified IA lanes and control lanes were determined and used to calculate the percentage of protein for an individual protein species sequestered into IAs relative to the amount of the same protein in whole cell extracts. Since no recruited protein species displayed different band intensities in the 5-d induced 13QN and 96QN uninduced lanes, these two values were averaged to provide the basal protein expression level.


Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression.

Suhr ST, Senut MC, Whitelegge JP, Faull KF, Cuizon DB, Gage FH - J. Cell Biol. (2001)

Western blot analysis of protein sequestration in purified IAs and control whole cell extracts. For all protein blots: lane 1, whole cell extracts from 5-d induced 13QN cells; lane 2, uninduced 96QN cells; lane 3, 5-d induced 96QN cells; and lane 4, isolated concentrated aggregates. Arrowheads at the right of autoradiograms indicate predicted molecular weights of individual protein species unless otherwise specified. (a) Processing for polyQ–GFP immunoreactivity. 96-P indicates the position of polyQN–GFP polymers within the stacking gel and near the top of the resolving gel (lanes 3 and 4), 96-M indicates a low level of 96QN monomers (lanes 2–4), and 13-M indicates the 13QN monomer band (lane 1). This autoradiogram was overexposed to reveal the 96QN monomer bands in lanes 3 and 4. (b) Ubiquitin; (c) HSP70; (d) MEF-2a; (e) Htt; (f) TBP; (g) Nck; (h) GRB-2; (i) caspase-9; (j) caspase-8 (putative procaspase-8, upper arrowhead; putative caspase-8 cleavage products, lower arrowheads); (k) caspase-3; (l) p53 and p50 immunoreactive species (upper and lower arrowheads, respectively); (m) mdm-2 antibody-1 (120-kD variant, upper arrowhead; p60 variant, lower arrowhead); and (n) mdm-2 antibody-2 immunoreactivity.
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Related In: Results  -  Collection

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Figure 3: Western blot analysis of protein sequestration in purified IAs and control whole cell extracts. For all protein blots: lane 1, whole cell extracts from 5-d induced 13QN cells; lane 2, uninduced 96QN cells; lane 3, 5-d induced 96QN cells; and lane 4, isolated concentrated aggregates. Arrowheads at the right of autoradiograms indicate predicted molecular weights of individual protein species unless otherwise specified. (a) Processing for polyQ–GFP immunoreactivity. 96-P indicates the position of polyQN–GFP polymers within the stacking gel and near the top of the resolving gel (lanes 3 and 4), 96-M indicates a low level of 96QN monomers (lanes 2–4), and 13-M indicates the 13QN monomer band (lane 1). This autoradiogram was overexposed to reveal the 96QN monomer bands in lanes 3 and 4. (b) Ubiquitin; (c) HSP70; (d) MEF-2a; (e) Htt; (f) TBP; (g) Nck; (h) GRB-2; (i) caspase-9; (j) caspase-8 (putative procaspase-8, upper arrowhead; putative caspase-8 cleavage products, lower arrowheads); (k) caspase-3; (l) p53 and p50 immunoreactive species (upper and lower arrowheads, respectively); (m) mdm-2 antibody-1 (120-kD variant, upper arrowhead; p60 variant, lower arrowhead); and (n) mdm-2 antibody-2 immunoreactivity.
Mentions: For quantification of sequestered proteins, each antibody used to detect a sequestered protein species in the Western blot analyses (see Fig. 3) was titrated against increasing concentrations of induced 96QN cell whole cell extracts or purified IA samples to determine the linear range of detection (data not shown). Each antibody was reassessed against the same samples as those described in the legend to Fig. 3 using an amount of protein and dilution of antibody determined to be within the linear range of detection. From these protein blots, the integrated densities of bands within purified IA lanes and control lanes were determined and used to calculate the percentage of protein for an individual protein species sequestered into IAs relative to the amount of the same protein in whole cell extracts. Since no recruited protein species displayed different band intensities in the 5-d induced 13QN and 96QN uninduced lanes, these two values were averaged to provide the basal protein expression level.

Bottom Line: One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs).Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex.These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Show MeSH
Related in: MedlinePlus