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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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PolyQ–GFP reporter constructs transiently and regulatably expressed in HEK293 cells. (a, left) Schematic of polyQ–GFP fusion proteins indicating Htt exon I–derived sequences (white), SV 40 NLS (gray), and eGFP sequences (black). (a, right) Schematic of CVBE and LPR Moloney murine leukemia virus–based retroviral vectors used in production of inducible polyQ cell lines. CVBE encodes flanking long terminal repeats (LTRs), a G418-resistance gene (G418), the immediate–early CMV promoter (CMV), and the VBE transactivator protein (VBE). LPR encodes long term repeats, a puromycin-resistance gene (Puro), a minimal CMV promoter with six tandem ecdysone response elements (RE), and a SfiI–PmeI polylinker for insertion of the polyQ transgenes. (b–f) Propidium iodide nuclear-stained (red) HEK293 cells transiently transfected with the expression construct 13Q–GFP (b), 13QN–GFP (c), 96Q–GFP (d), and 96QN–GFP (e). GFP produces the bright green fluorescence. (f) IAs in transfected cells revealing stellate fibrous appearance of IAs at high magnification. 72-h vehicle- (g) or ligand-treated (h) 13QN cells stained with the nuclear stain DAPI (red), vehicle- (i) or ligand-treated (j) 96Q cells, and vehicle- (k) or ligand-treated (l) 96QN cells. Bright GFP-positive profiles indicate IA formation. (m) Western blot analysis of induced 13QN (left) or 96QN (right) cells. Numbers at the top of each blot indicate the number of days of induction. WELL, bottom of the well; RES, bottom of the stacking gel and the beginning of the resolving gel. Bar: (b–e) 125 μm; (f) 20 μm; (g–l) 250 μm.
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Figure 1: PolyQ–GFP reporter constructs transiently and regulatably expressed in HEK293 cells. (a, left) Schematic of polyQ–GFP fusion proteins indicating Htt exon I–derived sequences (white), SV 40 NLS (gray), and eGFP sequences (black). (a, right) Schematic of CVBE and LPR Moloney murine leukemia virus–based retroviral vectors used in production of inducible polyQ cell lines. CVBE encodes flanking long terminal repeats (LTRs), a G418-resistance gene (G418), the immediate–early CMV promoter (CMV), and the VBE transactivator protein (VBE). LPR encodes long term repeats, a puromycin-resistance gene (Puro), a minimal CMV promoter with six tandem ecdysone response elements (RE), and a SfiI–PmeI polylinker for insertion of the polyQ transgenes. (b–f) Propidium iodide nuclear-stained (red) HEK293 cells transiently transfected with the expression construct 13Q–GFP (b), 13QN–GFP (c), 96Q–GFP (d), and 96QN–GFP (e). GFP produces the bright green fluorescence. (f) IAs in transfected cells revealing stellate fibrous appearance of IAs at high magnification. 72-h vehicle- (g) or ligand-treated (h) 13QN cells stained with the nuclear stain DAPI (red), vehicle- (i) or ligand-treated (j) 96Q cells, and vehicle- (k) or ligand-treated (l) 96QN cells. Bright GFP-positive profiles indicate IA formation. (m) Western blot analysis of induced 13QN (left) or 96QN (right) cells. Numbers at the top of each blot indicate the number of days of induction. WELL, bottom of the well; RES, bottom of the stacking gel and the beginning of the resolving gel. Bar: (b–e) 125 μm; (f) 20 μm; (g–l) 250 μm.

Mentions: PolyQ reporter constructs were made by PCR amplification of human Huntingtin (Htt) exon I variants with 13- and 96-CAG tracts, preserving polyproline (polyP) sequences immediately downstream of the polyQ tract (see Fig. 1) and fusing these fragments in frame to the NH2 terminus of EGFP (CLONTECH Laboratories, Inc.) in the cloning vector SKSP. Unique AscI and MluI restriction sites were inserted into the 5′ and 3′ coding regions for insertion of oligonucleotides encoding the SV 40 nuclear localization signal (NLS). For the experiments in this report, only the 3′-localized SV 40 NLS was used. The 96Q tract contains a characterized arginine residue at position 42 of the 96Q tract (Senut et al. 2000). The polyQ–eGFP coding region was excised from SKSP using unique SfiI and PmeI cloning sites for insertion into retroviral vector NIT (sequence data available from GenBank/EMBL/DDBJ under accession number AF311318) for expression in transient transfection studies, or into retroviral vector LPR for use with vector CVBE for ecdysteroid-induced cells. LPR is a Moloney murine leukemia virus–based vector with puromycin resistance and six-tandem ecdysone response elements fused to a minimal cytomegalovirus (CMV) promoter directing transgene expression when combined with CVBE (Suhr et al. 1998). Transient transfection analysis was performed by calcium phosphate precipitation by standard methods. For nuclear visualization, cells were stained with either 100 ng/ml propidium iodide or DAPI.


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)

PolyQ–GFP reporter constructs transiently and regulatably expressed in HEK293 cells. (a, left) Schematic of polyQ–GFP fusion proteins indicating Htt exon I–derived sequences (white), SV 40 NLS (gray), and eGFP sequences (black). (a, right) Schematic of CVBE and LPR Moloney murine leukemia virus–based retroviral vectors used in production of inducible polyQ cell lines. CVBE encodes flanking long terminal repeats (LTRs), a G418-resistance gene (G418), the immediate–early CMV promoter (CMV), and the VBE transactivator protein (VBE). LPR encodes long term repeats, a puromycin-resistance gene (Puro), a minimal CMV promoter with six tandem ecdysone response elements (RE), and a SfiI–PmeI polylinker for insertion of the polyQ transgenes. (b–f) Propidium iodide nuclear-stained (red) HEK293 cells transiently transfected with the expression construct 13Q–GFP (b), 13QN–GFP (c), 96Q–GFP (d), and 96QN–GFP (e). GFP produces the bright green fluorescence. (f) IAs in transfected cells revealing stellate fibrous appearance of IAs at high magnification. 72-h vehicle- (g) or ligand-treated (h) 13QN cells stained with the nuclear stain DAPI (red), vehicle- (i) or ligand-treated (j) 96Q cells, and vehicle- (k) or ligand-treated (l) 96QN cells. Bright GFP-positive profiles indicate IA formation. (m) Western blot analysis of induced 13QN (left) or 96QN (right) cells. Numbers at the top of each blot indicate the number of days of induction. WELL, bottom of the well; RES, bottom of the stacking gel and the beginning of the resolving gel. Bar: (b–e) 125 μm; (f) 20 μm; (g–l) 250 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169460&req=5

Figure 1: PolyQ–GFP reporter constructs transiently and regulatably expressed in HEK293 cells. (a, left) Schematic of polyQ–GFP fusion proteins indicating Htt exon I–derived sequences (white), SV 40 NLS (gray), and eGFP sequences (black). (a, right) Schematic of CVBE and LPR Moloney murine leukemia virus–based retroviral vectors used in production of inducible polyQ cell lines. CVBE encodes flanking long terminal repeats (LTRs), a G418-resistance gene (G418), the immediate–early CMV promoter (CMV), and the VBE transactivator protein (VBE). LPR encodes long term repeats, a puromycin-resistance gene (Puro), a minimal CMV promoter with six tandem ecdysone response elements (RE), and a SfiI–PmeI polylinker for insertion of the polyQ transgenes. (b–f) Propidium iodide nuclear-stained (red) HEK293 cells transiently transfected with the expression construct 13Q–GFP (b), 13QN–GFP (c), 96Q–GFP (d), and 96QN–GFP (e). GFP produces the bright green fluorescence. (f) IAs in transfected cells revealing stellate fibrous appearance of IAs at high magnification. 72-h vehicle- (g) or ligand-treated (h) 13QN cells stained with the nuclear stain DAPI (red), vehicle- (i) or ligand-treated (j) 96Q cells, and vehicle- (k) or ligand-treated (l) 96QN cells. Bright GFP-positive profiles indicate IA formation. (m) Western blot analysis of induced 13QN (left) or 96QN (right) cells. Numbers at the top of each blot indicate the number of days of induction. WELL, bottom of the well; RES, bottom of the stacking gel and the beginning of the resolving gel. Bar: (b–e) 125 μm; (f) 20 μm; (g–l) 250 μm.
Mentions: PolyQ reporter constructs were made by PCR amplification of human Huntingtin (Htt) exon I variants with 13- and 96-CAG tracts, preserving polyproline (polyP) sequences immediately downstream of the polyQ tract (see Fig. 1) and fusing these fragments in frame to the NH2 terminus of EGFP (CLONTECH Laboratories, Inc.) in the cloning vector SKSP. Unique AscI and MluI restriction sites were inserted into the 5′ and 3′ coding regions for insertion of oligonucleotides encoding the SV 40 nuclear localization signal (NLS). For the experiments in this report, only the 3′-localized SV 40 NLS was used. The 96Q tract contains a characterized arginine residue at position 42 of the 96Q tract (Senut et al. 2000). The polyQ–eGFP coding region was excised from SKSP using unique SfiI and PmeI cloning sites for insertion into retroviral vector NIT (sequence data available from GenBank/EMBL/DDBJ under accession number AF311318) for expression in transient transfection studies, or into retroviral vector LPR for use with vector CVBE for ecdysteroid-induced cells. LPR is a Moloney murine leukemia virus–based vector with puromycin resistance and six-tandem ecdysone response elements fused to a minimal cytomegalovirus (CMV) promoter directing transgene expression when combined with CVBE (Suhr et al. 1998). Transient transfection analysis was performed by calcium phosphate precipitation by standard methods. For nuclear visualization, cells were stained with either 100 ng/ml propidium iodide or DAPI.

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