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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 binds presenilins in vitro. (A and B) GST pull-down experiments. Full-length in vitro synthesized 35S-labeled PS2 and PS1 (first lanes) migrated in SDS-PAGE gels with broad bands of 54 and 48 kD (arrowheads), respectively, along with a smear of slower migrating forms, presumably due to the highly hydrophobic nature of the proteins. [35S]PS complexes (especially the slower migrating forms) were retained by GST–ubiquilin constructs containing the UBA domain (lane letters correspond to constructs shown in Fig. 2 III), but not by those lacking the domain, or by GST alone. (C) Immunoprecipitation experiments. PS2-transfected HeLa cell lysates were immunoprecipitated with preimmune sera or corresponding anti–PS2-Loop antibody and anti-PS2 NH2 terminus antibody. After separation by SDS-PAGE, coprecipitating ubiquilin (arrowhead) was detected by immunoblotting with anti–ubiquilin-B antibody. (D–F) Cell fractionation experiments. Parallel immunoblots of equal portions of soluble supernatant (S) and insoluble pellet (P) HeLa cellular fractions prepared without the use of detergent (−) or in the presence of 1% Triton X-100 (+) with (D and F) anti-ubiquilin or (E) anti-PS2 antibodies. The HeLa cells used for cell fractionation in D were untransfected, whereas, in E and F, the cells were transfected with a full-length wild-type PS2 construct. The relative ratio of ubiquilin protein in the P− compared with the S− fractions was determined by densitometric analysis of the autoradiographs. This analysis indicated that transfection of presenilin caused 30% more (F) ubiquilin protein to partition in the pellet fraction in the absence of detergent compared with (D) untransfected cells. The partitioning of lamin A/C proteins after cell fractionation from untransfected and PS2-transfected cells was monitored with anti-lamin A/C antibodies and shown below in D and F, respectively.
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Figure 5: Ubiquilin binds presenilins in vitro. (A and B) GST pull-down experiments. Full-length in vitro synthesized 35S-labeled PS2 and PS1 (first lanes) migrated in SDS-PAGE gels with broad bands of 54 and 48 kD (arrowheads), respectively, along with a smear of slower migrating forms, presumably due to the highly hydrophobic nature of the proteins. [35S]PS complexes (especially the slower migrating forms) were retained by GST–ubiquilin constructs containing the UBA domain (lane letters correspond to constructs shown in Fig. 2 III), but not by those lacking the domain, or by GST alone. (C) Immunoprecipitation experiments. PS2-transfected HeLa cell lysates were immunoprecipitated with preimmune sera or corresponding anti–PS2-Loop antibody and anti-PS2 NH2 terminus antibody. After separation by SDS-PAGE, coprecipitating ubiquilin (arrowhead) was detected by immunoblotting with anti–ubiquilin-B antibody. (D–F) Cell fractionation experiments. Parallel immunoblots of equal portions of soluble supernatant (S) and insoluble pellet (P) HeLa cellular fractions prepared without the use of detergent (−) or in the presence of 1% Triton X-100 (+) with (D and F) anti-ubiquilin or (E) anti-PS2 antibodies. The HeLa cells used for cell fractionation in D were untransfected, whereas, in E and F, the cells were transfected with a full-length wild-type PS2 construct. The relative ratio of ubiquilin protein in the P− compared with the S− fractions was determined by densitometric analysis of the autoradiographs. This analysis indicated that transfection of presenilin caused 30% more (F) ubiquilin protein to partition in the pellet fraction in the absence of detergent compared with (D) untransfected cells. The partitioning of lamin A/C proteins after cell fractionation from untransfected and PS2-transfected cells was monitored with anti-lamin A/C antibodies and shown below in D and F, respectively.

Mentions: GST-fusion proteins containing the UBA domain and nine COOH-terminal residues (QHHSSISVS) of ubiquilin were necessary and sufficient to bind [35S]methionine-radiolabeled PS2 and PS1 in a GST pull-down assay (Fig. 5A and Fig. B, respectively). Interestingly, slower migrating forms of both presenilins were much more tightly bound than the full-length forms. Whether the slower migrating forms are more ubiquitinated, extensively modified, or merely hydrophobic aggregates is not known. As expected, GST alone (Fig. 5A and Fig. B, lane L) did not bind either presenilin. No other region of ubiquilin tested was able to pull-down either of the presenilins, indicating that binding of presenilin to ubiquilin is mediated by sequences spanning the UBA domain and the COOH-terminal tail. These in vitro binding assay results were consistent with the interaction data of ubiquilin recovered in the Y2H screen.


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 binds presenilins in vitro. (A and B) GST pull-down experiments. Full-length in vitro synthesized 35S-labeled PS2 and PS1 (first lanes) migrated in SDS-PAGE gels with broad bands of 54 and 48 kD (arrowheads), respectively, along with a smear of slower migrating forms, presumably due to the highly hydrophobic nature of the proteins. [35S]PS complexes (especially the slower migrating forms) were retained by GST–ubiquilin constructs containing the UBA domain (lane letters correspond to constructs shown in Fig. 2 III), but not by those lacking the domain, or by GST alone. (C) Immunoprecipitation experiments. PS2-transfected HeLa cell lysates were immunoprecipitated with preimmune sera or corresponding anti–PS2-Loop antibody and anti-PS2 NH2 terminus antibody. After separation by SDS-PAGE, coprecipitating ubiquilin (arrowhead) was detected by immunoblotting with anti–ubiquilin-B antibody. (D–F) Cell fractionation experiments. Parallel immunoblots of equal portions of soluble supernatant (S) and insoluble pellet (P) HeLa cellular fractions prepared without the use of detergent (−) or in the presence of 1% Triton X-100 (+) with (D and F) anti-ubiquilin or (E) anti-PS2 antibodies. The HeLa cells used for cell fractionation in D were untransfected, whereas, in E and F, the cells were transfected with a full-length wild-type PS2 construct. The relative ratio of ubiquilin protein in the P− compared with the S− fractions was determined by densitometric analysis of the autoradiographs. This analysis indicated that transfection of presenilin caused 30% more (F) ubiquilin protein to partition in the pellet fraction in the absence of detergent compared with (D) untransfected cells. The partitioning of lamin A/C proteins after cell fractionation from untransfected and PS2-transfected cells was monitored with anti-lamin A/C antibodies and shown below in D and F, respectively.
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Figure 5: Ubiquilin binds presenilins in vitro. (A and B) GST pull-down experiments. Full-length in vitro synthesized 35S-labeled PS2 and PS1 (first lanes) migrated in SDS-PAGE gels with broad bands of 54 and 48 kD (arrowheads), respectively, along with a smear of slower migrating forms, presumably due to the highly hydrophobic nature of the proteins. [35S]PS complexes (especially the slower migrating forms) were retained by GST–ubiquilin constructs containing the UBA domain (lane letters correspond to constructs shown in Fig. 2 III), but not by those lacking the domain, or by GST alone. (C) Immunoprecipitation experiments. PS2-transfected HeLa cell lysates were immunoprecipitated with preimmune sera or corresponding anti–PS2-Loop antibody and anti-PS2 NH2 terminus antibody. After separation by SDS-PAGE, coprecipitating ubiquilin (arrowhead) was detected by immunoblotting with anti–ubiquilin-B antibody. (D–F) Cell fractionation experiments. Parallel immunoblots of equal portions of soluble supernatant (S) and insoluble pellet (P) HeLa cellular fractions prepared without the use of detergent (−) or in the presence of 1% Triton X-100 (+) with (D and F) anti-ubiquilin or (E) anti-PS2 antibodies. The HeLa cells used for cell fractionation in D were untransfected, whereas, in E and F, the cells were transfected with a full-length wild-type PS2 construct. The relative ratio of ubiquilin protein in the P− compared with the S− fractions was determined by densitometric analysis of the autoradiographs. This analysis indicated that transfection of presenilin caused 30% more (F) ubiquilin protein to partition in the pellet fraction in the absence of detergent compared with (D) untransfected cells. The partitioning of lamin A/C proteins after cell fractionation from untransfected and PS2-transfected cells was monitored with anti-lamin A/C antibodies and shown below in D and F, respectively.
Mentions: GST-fusion proteins containing the UBA domain and nine COOH-terminal residues (QHHSSISVS) of ubiquilin were necessary and sufficient to bind [35S]methionine-radiolabeled PS2 and PS1 in a GST pull-down assay (Fig. 5A and Fig. B, respectively). Interestingly, slower migrating forms of both presenilins were much more tightly bound than the full-length forms. Whether the slower migrating forms are more ubiquitinated, extensively modified, or merely hydrophobic aggregates is not known. As expected, GST alone (Fig. 5A and Fig. B, lane L) did not bind either presenilin. No other region of ubiquilin tested was able to pull-down either of the presenilins, indicating that binding of presenilin to ubiquilin is mediated by sequences spanning the UBA domain and the COOH-terminal tail. These in vitro binding assay results were consistent with the interaction data of ubiquilin recovered in the Y2H screen.

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