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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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Protein and Western blot analyses of sequestered protein in concentrated IAs. (a) Coomassie-stained protein gel of isolated IAs revealing multiple bands from ∼10 kD (dye) up to the boundary of the stacking/resolving gel (stack). The four predominant bands used for MALDI analysis are labeled at the right. Lane organization is as described in the legend to Fig. 3. (b) Protein blot analysis of 68-kD neurofilament light polypeptide; (c) protein blot analysis of actin; (d) protein blot analysis with monoclonal antibody 414–detecting putative NPCP proteins Nup62, Nup153, Nup214, and Nup358 as labeled; (e) protein blot analysis of lamin revealing no detectable sequestration in purified IAs.
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Figure 4: Protein and Western blot analyses of sequestered protein in concentrated IAs. (a) Coomassie-stained protein gel of isolated IAs revealing multiple bands from ∼10 kD (dye) up to the boundary of the stacking/resolving gel (stack). The four predominant bands used for MALDI analysis are labeled at the right. Lane organization is as described in the legend to Fig. 3. (b) Protein blot analysis of 68-kD neurofilament light polypeptide; (c) protein blot analysis of actin; (d) protein blot analysis with monoclonal antibody 414–detecting putative NPCP proteins Nup62, Nup153, Nup214, and Nup358 as labeled; (e) protein blot analysis of lamin revealing no detectable sequestration in purified IAs.

Mentions: Fig. 4 a shows Coomassie-stained protein species in a concentrated IA sample on a 1-mm 10% SDS–polyacrylamide gel. Four bands at ∼68, 54, 50, and 41 kD stood out as much more prominent than the ∼30 other bands of lesser intensity within the sharply resolved region of the gel from 100 kD to the dye front. These four bands were excised from the gel and subjected to MALDI analysis to provide tentative identification of each protein species.


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)

Protein and Western blot analyses of sequestered protein in concentrated IAs. (a) Coomassie-stained protein gel of isolated IAs revealing multiple bands from ∼10 kD (dye) up to the boundary of the stacking/resolving gel (stack). The four predominant bands used for MALDI analysis are labeled at the right. Lane organization is as described in the legend to Fig. 3. (b) Protein blot analysis of 68-kD neurofilament light polypeptide; (c) protein blot analysis of actin; (d) protein blot analysis with monoclonal antibody 414–detecting putative NPCP proteins Nup62, Nup153, Nup214, and Nup358 as labeled; (e) protein blot analysis of lamin revealing no detectable sequestration in purified IAs.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169460&req=5

Figure 4: Protein and Western blot analyses of sequestered protein in concentrated IAs. (a) Coomassie-stained protein gel of isolated IAs revealing multiple bands from ∼10 kD (dye) up to the boundary of the stacking/resolving gel (stack). The four predominant bands used for MALDI analysis are labeled at the right. Lane organization is as described in the legend to Fig. 3. (b) Protein blot analysis of 68-kD neurofilament light polypeptide; (c) protein blot analysis of actin; (d) protein blot analysis with monoclonal antibody 414–detecting putative NPCP proteins Nup62, Nup153, Nup214, and Nup358 as labeled; (e) protein blot analysis of lamin revealing no detectable sequestration in purified IAs.
Mentions: Fig. 4 a shows Coomassie-stained protein species in a concentrated IA sample on a 1-mm 10% SDS–polyacrylamide gel. Four bands at ∼68, 54, 50, and 41 kD stood out as much more prominent than the ∼30 other bands of lesser intensity within the sharply resolved region of the gel from 100 kD to the dye front. These four bands were excised from the gel and subjected to MALDI analysis to provide tentative identification of each protein species.

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