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Identification of ubiquilin, a novel presenilin interactor that increases presenilin protein accumulation.

Mah AL, Perry G, Smith MA, Monteiro MJ - J. Cell Biol. (2000)

Bottom Line: However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood.Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively.Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain.

View Article: PubMed Central - PubMed

Affiliation: Medical Biotechnology Center, Department of Neurology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.

ABSTRACT
Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. We describe here the properties of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, we show that ubiquilin promotes presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life. Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies revealed prominent staining of neurons. Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively. Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain.

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Ubiquilin mRNA and protein expression. (A) Human multiple tissue were analyzed by Northern blot and probed with ubiquilin cDNA fragment X (Fig. 2 II). After stripping, the blot was reprobed with a β-actin control fragment (shown below). (B) Northern blot of specific regions of the human brain probed with ubiquilin cDNA fragment Y (Fig. 2 II), with a reprobe with β-actin, which is shown below. (C) Quantification of ubiquilin mRNA expression levels. Relative expression of ubiquilin in different tissues was determined by densitometric analysis of the autoradiographs and relating the ubiquilin band intensities to the levels of β-actin hybridization from the same lanes. The values are presented after normalization against skeletal muscle (above) and spinal cord (below). (D–F) Characterization of anti-ubiquilin antibodies. Rabbit antibodies were raised against GST–ubiquilin fusion proteins B and C, generating anti-ubiquilin-B and anti–ubiquilin-C antibodies, respectively. (D) Anti–ubiquilin-B antibody was used to detect overexpressed and endogenous ubiquilin in ubiquilin transfected, mock-transfected, and untransfected (endogenous) HeLa cells. The anti–ubiquilin-B antibody detected a 66-kD doublet band, whereas preimmune sera did not. (E) Anti–ubiquilin-C antibody also recognized the 66-kD band in untransfected HeLa lysates and to varying extents a ∼55-kD band. HeLa cells transfected with GFP–ubiquilin (Fig. 2 IV, N) contain an additional 93-kD reactive band, due to the fusion of the 27-kD moiety of GFP with ubiquilin. (F) Affinity-purified anti–ubiquilin-C antibody specifically reacts with the 66-kD band from transfected HeLa cell lysates. (G) Full-length in vitro transcribed and translated [35S]methionine-radiolabeled human ubiquilin polypeptides migrated at 66 kD, whereas radiolabeled luciferase migrated at 61 kD. (H) Uninduced and IPTG-induced lysates of bacteria transformed with untagged full-length human ubiquilin and probed with anti–ubiquilin-B antibodies. Full-length immunoreactive ubiquilin (66 kD) and a series of smaller breakdown products are only seen in the induced lysate.
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Figure 3: Ubiquilin mRNA and protein expression. (A) Human multiple tissue were analyzed by Northern blot and probed with ubiquilin cDNA fragment X (Fig. 2 II). After stripping, the blot was reprobed with a β-actin control fragment (shown below). (B) Northern blot of specific regions of the human brain probed with ubiquilin cDNA fragment Y (Fig. 2 II), with a reprobe with β-actin, which is shown below. (C) Quantification of ubiquilin mRNA expression levels. Relative expression of ubiquilin in different tissues was determined by densitometric analysis of the autoradiographs and relating the ubiquilin band intensities to the levels of β-actin hybridization from the same lanes. The values are presented after normalization against skeletal muscle (above) and spinal cord (below). (D–F) Characterization of anti-ubiquilin antibodies. Rabbit antibodies were raised against GST–ubiquilin fusion proteins B and C, generating anti-ubiquilin-B and anti–ubiquilin-C antibodies, respectively. (D) Anti–ubiquilin-B antibody was used to detect overexpressed and endogenous ubiquilin in ubiquilin transfected, mock-transfected, and untransfected (endogenous) HeLa cells. The anti–ubiquilin-B antibody detected a 66-kD doublet band, whereas preimmune sera did not. (E) Anti–ubiquilin-C antibody also recognized the 66-kD band in untransfected HeLa lysates and to varying extents a ∼55-kD band. HeLa cells transfected with GFP–ubiquilin (Fig. 2 IV, N) contain an additional 93-kD reactive band, due to the fusion of the 27-kD moiety of GFP with ubiquilin. (F) Affinity-purified anti–ubiquilin-C antibody specifically reacts with the 66-kD band from transfected HeLa cell lysates. (G) Full-length in vitro transcribed and translated [35S]methionine-radiolabeled human ubiquilin polypeptides migrated at 66 kD, whereas radiolabeled luciferase migrated at 61 kD. (H) Uninduced and IPTG-induced lysates of bacteria transformed with untagged full-length human ubiquilin and probed with anti–ubiquilin-B antibodies. Full-length immunoreactive ubiquilin (66 kD) and a series of smaller breakdown products are only seen in the induced lysate.

Mentions: A novel human protein, which we named ubiquilin, for a protein with ubiquitin-related protein domains that interacts with the presenilins, was identified in a yeast two-hybrid screen (Y2H) for proteins that interact with the PS2 COOH-terminal sequence (Fig. 1a and Fig. b). Two overlapping clones encoding the COOH-terminal 203 and 218 residues of ubiquilin were isolated in the screen. To clone the entire ubiquilin ORF, 5′ RACE was performed. This yielded 1,053 bps of additional upstream sequence, including an in-frame methionine codon located at the beginning of a ubiquitin-like (UB) domain. Conceptual translation from this methionine predicted a 559-amino acid protein. However, this methionine was unlikely to be the authentic start codon since translation from it produced a protein that was noticeably smaller, as seen by SDS-PAGE, than endogenous ubiquilin detected using pAb raised to the COOH-terminal 218 residues of ubiquilin (data not shown). Subsequent EST database searches and sequencing of putative overlapping clones yielded a clone with a longer 5′ end, which included an upstream in-frame methionine codon (corresponding to a 595-amino acid protein). Several lines of evidence indicate that this methionine codon was the authentic start methionine of ubiquilin. (a) This start methionine contained a Kozak consensus sequence and an upstream in-frame stop codon. (b) Polypeptides translated from this start codon matched the endogenous ubiquilin protein in size, which was 66 kD in HeLa and other human cell types. (c) Mammalian overexpression of the complete ubiquilin ORF (see Fig. 3, D–F), in vitro transcribed and translated ubiquilin in rabbit reticulocyte lysates (see Fig. 3 G), and human ubiquilin protein synthesized in bacteria (see Fig. 3 H) all generated 66-kD proteins. These data indicated that we had cloned the complete ubiquilin ORF and that the protein is probably not extensively modified in mammalian cells, since bacterially and eukaryotically expressed human ubiquilin proteins have similar molecular masses when separated by SDS-PAGE.


Identification of ubiquilin, a novel presenilin interactor that increases presenilin protein accumulation.

Mah AL, Perry G, Smith MA, Monteiro MJ - J. Cell Biol. (2000)

Ubiquilin mRNA and protein expression. (A) Human multiple tissue were analyzed by Northern blot and probed with ubiquilin cDNA fragment X (Fig. 2 II). After stripping, the blot was reprobed with a β-actin control fragment (shown below). (B) Northern blot of specific regions of the human brain probed with ubiquilin cDNA fragment Y (Fig. 2 II), with a reprobe with β-actin, which is shown below. (C) Quantification of ubiquilin mRNA expression levels. Relative expression of ubiquilin in different tissues was determined by densitometric analysis of the autoradiographs and relating the ubiquilin band intensities to the levels of β-actin hybridization from the same lanes. The values are presented after normalization against skeletal muscle (above) and spinal cord (below). (D–F) Characterization of anti-ubiquilin antibodies. Rabbit antibodies were raised against GST–ubiquilin fusion proteins B and C, generating anti-ubiquilin-B and anti–ubiquilin-C antibodies, respectively. (D) Anti–ubiquilin-B antibody was used to detect overexpressed and endogenous ubiquilin in ubiquilin transfected, mock-transfected, and untransfected (endogenous) HeLa cells. The anti–ubiquilin-B antibody detected a 66-kD doublet band, whereas preimmune sera did not. (E) Anti–ubiquilin-C antibody also recognized the 66-kD band in untransfected HeLa lysates and to varying extents a ∼55-kD band. HeLa cells transfected with GFP–ubiquilin (Fig. 2 IV, N) contain an additional 93-kD reactive band, due to the fusion of the 27-kD moiety of GFP with ubiquilin. (F) Affinity-purified anti–ubiquilin-C antibody specifically reacts with the 66-kD band from transfected HeLa cell lysates. (G) Full-length in vitro transcribed and translated [35S]methionine-radiolabeled human ubiquilin polypeptides migrated at 66 kD, whereas radiolabeled luciferase migrated at 61 kD. (H) Uninduced and IPTG-induced lysates of bacteria transformed with untagged full-length human ubiquilin and probed with anti–ubiquilin-B antibodies. Full-length immunoreactive ubiquilin (66 kD) and a series of smaller breakdown products are only seen in the induced lysate.
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Related In: Results  -  Collection

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Figure 3: Ubiquilin mRNA and protein expression. (A) Human multiple tissue were analyzed by Northern blot and probed with ubiquilin cDNA fragment X (Fig. 2 II). After stripping, the blot was reprobed with a β-actin control fragment (shown below). (B) Northern blot of specific regions of the human brain probed with ubiquilin cDNA fragment Y (Fig. 2 II), with a reprobe with β-actin, which is shown below. (C) Quantification of ubiquilin mRNA expression levels. Relative expression of ubiquilin in different tissues was determined by densitometric analysis of the autoradiographs and relating the ubiquilin band intensities to the levels of β-actin hybridization from the same lanes. The values are presented after normalization against skeletal muscle (above) and spinal cord (below). (D–F) Characterization of anti-ubiquilin antibodies. Rabbit antibodies were raised against GST–ubiquilin fusion proteins B and C, generating anti-ubiquilin-B and anti–ubiquilin-C antibodies, respectively. (D) Anti–ubiquilin-B antibody was used to detect overexpressed and endogenous ubiquilin in ubiquilin transfected, mock-transfected, and untransfected (endogenous) HeLa cells. The anti–ubiquilin-B antibody detected a 66-kD doublet band, whereas preimmune sera did not. (E) Anti–ubiquilin-C antibody also recognized the 66-kD band in untransfected HeLa lysates and to varying extents a ∼55-kD band. HeLa cells transfected with GFP–ubiquilin (Fig. 2 IV, N) contain an additional 93-kD reactive band, due to the fusion of the 27-kD moiety of GFP with ubiquilin. (F) Affinity-purified anti–ubiquilin-C antibody specifically reacts with the 66-kD band from transfected HeLa cell lysates. (G) Full-length in vitro transcribed and translated [35S]methionine-radiolabeled human ubiquilin polypeptides migrated at 66 kD, whereas radiolabeled luciferase migrated at 61 kD. (H) Uninduced and IPTG-induced lysates of bacteria transformed with untagged full-length human ubiquilin and probed with anti–ubiquilin-B antibodies. Full-length immunoreactive ubiquilin (66 kD) and a series of smaller breakdown products are only seen in the induced lysate.
Mentions: A novel human protein, which we named ubiquilin, for a protein with ubiquitin-related protein domains that interacts with the presenilins, was identified in a yeast two-hybrid screen (Y2H) for proteins that interact with the PS2 COOH-terminal sequence (Fig. 1a and Fig. b). Two overlapping clones encoding the COOH-terminal 203 and 218 residues of ubiquilin were isolated in the screen. To clone the entire ubiquilin ORF, 5′ RACE was performed. This yielded 1,053 bps of additional upstream sequence, including an in-frame methionine codon located at the beginning of a ubiquitin-like (UB) domain. Conceptual translation from this methionine predicted a 559-amino acid protein. However, this methionine was unlikely to be the authentic start codon since translation from it produced a protein that was noticeably smaller, as seen by SDS-PAGE, than endogenous ubiquilin detected using pAb raised to the COOH-terminal 218 residues of ubiquilin (data not shown). Subsequent EST database searches and sequencing of putative overlapping clones yielded a clone with a longer 5′ end, which included an upstream in-frame methionine codon (corresponding to a 595-amino acid protein). Several lines of evidence indicate that this methionine codon was the authentic start methionine of ubiquilin. (a) This start methionine contained a Kozak consensus sequence and an upstream in-frame stop codon. (b) Polypeptides translated from this start codon matched the endogenous ubiquilin protein in size, which was 66 kD in HeLa and other human cell types. (c) Mammalian overexpression of the complete ubiquilin ORF (see Fig. 3, D–F), in vitro transcribed and translated ubiquilin in rabbit reticulocyte lysates (see Fig. 3 G), and human ubiquilin protein synthesized in bacteria (see Fig. 3 H) all generated 66-kD proteins. These data indicated that we had cloned the complete ubiquilin ORF and that the protein is probably not extensively modified in mammalian cells, since bacterially and eukaryotically expressed human ubiquilin proteins have similar molecular masses when separated by SDS-PAGE.

Bottom Line: However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood.Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively.Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain.

View Article: PubMed Central - PubMed

Affiliation: Medical Biotechnology Center, Department of Neurology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.

ABSTRACT
Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. We describe here the properties of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, we show that ubiquilin promotes presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life. Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies revealed prominent staining of neurons. Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively. Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain.

Show MeSH
Related in: MedlinePlus