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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 localizes to the nucleus and cytoplasm. (A, B, D, and E) Indirect immunofluorescence microscopy of endogenous ubiquilin staining in untransfected HeLa cells and (C and F) confocal microscopy of HeLa cells transfected with ubiquilin or (I) myc-tagged ubiquilin. (A and B) Preimmune and the corresponding anti–ubiquilin-B antibody staining, respectively, are shown. (C) Overexpressed ubiquilin as detected with anti–ubiquilin-B antibody. (D and E) Preimmune and the corresponding anti–ubiquilin-C antibody staining, respectively, are shown. (F) Overexpressed ubiquilin is shown, as detected with affinity-purified anti–ubiquilin-C antibody. Both anti-ubiquilin sera showed specific staining in the cytoplasm and nucleus, along with cytoplasmic punctate structures in a subset of the untransfected cells (arrows). The expression levels of ubiquilin protein within the nucleus varied with some cells containing substantially more nuclear protein (arrowheads). Transient overexpression of wild-type ubiquilin caused frequent accumulation of ubiquilin to the intracellular punctate structures. (G) Endogenous ubiquilin is shown, as detected by affinity-purified anti–ubiquilin-C antibody (confocal microscopy). (H) Overexpressed GFP-tagged ubiquilin of live HeLa cells, as seen by fluorescence microscopy, revealed accumulation of the fusion protein to the cytoplasm and to similar punctate structures. (I) Additional evidence for intracellular localization of ubiquilin, using a myc-tagged construct and stained with an anti-myc mAb (confocal microscopy), is shown. Bar, 25 μm.
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Figure 6: Ubiquilin localizes to the nucleus and cytoplasm. (A, B, D, and E) Indirect immunofluorescence microscopy of endogenous ubiquilin staining in untransfected HeLa cells and (C and F) confocal microscopy of HeLa cells transfected with ubiquilin or (I) myc-tagged ubiquilin. (A and B) Preimmune and the corresponding anti–ubiquilin-B antibody staining, respectively, are shown. (C) Overexpressed ubiquilin as detected with anti–ubiquilin-B antibody. (D and E) Preimmune and the corresponding anti–ubiquilin-C antibody staining, respectively, are shown. (F) Overexpressed ubiquilin is shown, as detected with affinity-purified anti–ubiquilin-C antibody. Both anti-ubiquilin sera showed specific staining in the cytoplasm and nucleus, along with cytoplasmic punctate structures in a subset of the untransfected cells (arrows). The expression levels of ubiquilin protein within the nucleus varied with some cells containing substantially more nuclear protein (arrowheads). Transient overexpression of wild-type ubiquilin caused frequent accumulation of ubiquilin to the intracellular punctate structures. (G) Endogenous ubiquilin is shown, as detected by affinity-purified anti–ubiquilin-C antibody (confocal microscopy). (H) Overexpressed GFP-tagged ubiquilin of live HeLa cells, as seen by fluorescence microscopy, revealed accumulation of the fusion protein to the cytoplasm and to similar punctate structures. (I) Additional evidence for intracellular localization of ubiquilin, using a myc-tagged construct and stained with an anti-myc mAb (confocal microscopy), is shown. Bar, 25 μm.

Mentions: To determine the intracellular distribution of ubiquilin we used rabbit pAb that we had generated to two different GST–ubiquilin fusion proteins (see Materials and Methods). Both antisera detected endogenous and overexpressed 66-kD ubiquilin polypeptides, which were not detected by the preimmune sera (Fig. 3 D). Although one antiserum reacted with an unknown protein of ∼55 kD (Fig. 3 E), this reactivity was subsequently removed by affinity purification of the antibody (Fig. 3 F). Immunofluorescence staining of ubiquilin within cells was performed with the specific anti-ubiquilin antibodies. Compared with the preimmune sera (Fig. 6A and Fig. D), both antibodies revealed similar staining patterns (Fig. 6B and Fig. E). Indirect immunofluorescence, as well as laser confocal microscopy, revealed endogenous intracellular localization of ubiquilin in HeLa cells to both the nucleus and cytoplasm (Fig. 6B, Fig. E, and Fig. G). In some cells, endogenous ubiquilin staining was stronger in the nucleus (Fig. 6 E, arrowheads). Furthermore, in a small subset of cells, ubiquilin within the cytoplasm was localized to punctate structures (Fig. 6B and Fig. E, arrows). The frequency of these structures dramatically increased upon overexpression of full-length untagged ubiquilin (Fig. 6C and Fig. F). To corroborate the anti-ubiquilin staining pattern, we performed additional localization studies on myc- and GFP-tagged ubiquilin proteins in HeLa cells (Fig. 2 IV, O and N, respectively). Overexpressed myc-tagged ubiquilin stained with anti-myc mAb exhibited the same intracellular structures (Fig. 6 I) as overexpressed wild-type ubiquilin (Fig. 6C and Fig. F). Finally, the same fluorescent staining pattern was reproduced upon overexpression of GFP-ubiquilin (Fig. 6 H). Since the GFP-ubiquilin image in Fig. 6 H was captured from live and unfixed cells without antibodies, this established that the anti-ubiquilin structures seen previously were not artifacts of the paraformaldehyde fixation or immunofluorescence staining procedures.


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 localizes to the nucleus and cytoplasm. (A, B, D, and E) Indirect immunofluorescence microscopy of endogenous ubiquilin staining in untransfected HeLa cells and (C and F) confocal microscopy of HeLa cells transfected with ubiquilin or (I) myc-tagged ubiquilin. (A and B) Preimmune and the corresponding anti–ubiquilin-B antibody staining, respectively, are shown. (C) Overexpressed ubiquilin as detected with anti–ubiquilin-B antibody. (D and E) Preimmune and the corresponding anti–ubiquilin-C antibody staining, respectively, are shown. (F) Overexpressed ubiquilin is shown, as detected with affinity-purified anti–ubiquilin-C antibody. Both anti-ubiquilin sera showed specific staining in the cytoplasm and nucleus, along with cytoplasmic punctate structures in a subset of the untransfected cells (arrows). The expression levels of ubiquilin protein within the nucleus varied with some cells containing substantially more nuclear protein (arrowheads). Transient overexpression of wild-type ubiquilin caused frequent accumulation of ubiquilin to the intracellular punctate structures. (G) Endogenous ubiquilin is shown, as detected by affinity-purified anti–ubiquilin-C antibody (confocal microscopy). (H) Overexpressed GFP-tagged ubiquilin of live HeLa cells, as seen by fluorescence microscopy, revealed accumulation of the fusion protein to the cytoplasm and to similar punctate structures. (I) Additional evidence for intracellular localization of ubiquilin, using a myc-tagged construct and stained with an anti-myc mAb (confocal microscopy), is shown. Bar, 25 μm.
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Figure 6: Ubiquilin localizes to the nucleus and cytoplasm. (A, B, D, and E) Indirect immunofluorescence microscopy of endogenous ubiquilin staining in untransfected HeLa cells and (C and F) confocal microscopy of HeLa cells transfected with ubiquilin or (I) myc-tagged ubiquilin. (A and B) Preimmune and the corresponding anti–ubiquilin-B antibody staining, respectively, are shown. (C) Overexpressed ubiquilin as detected with anti–ubiquilin-B antibody. (D and E) Preimmune and the corresponding anti–ubiquilin-C antibody staining, respectively, are shown. (F) Overexpressed ubiquilin is shown, as detected with affinity-purified anti–ubiquilin-C antibody. Both anti-ubiquilin sera showed specific staining in the cytoplasm and nucleus, along with cytoplasmic punctate structures in a subset of the untransfected cells (arrows). The expression levels of ubiquilin protein within the nucleus varied with some cells containing substantially more nuclear protein (arrowheads). Transient overexpression of wild-type ubiquilin caused frequent accumulation of ubiquilin to the intracellular punctate structures. (G) Endogenous ubiquilin is shown, as detected by affinity-purified anti–ubiquilin-C antibody (confocal microscopy). (H) Overexpressed GFP-tagged ubiquilin of live HeLa cells, as seen by fluorescence microscopy, revealed accumulation of the fusion protein to the cytoplasm and to similar punctate structures. (I) Additional evidence for intracellular localization of ubiquilin, using a myc-tagged construct and stained with an anti-myc mAb (confocal microscopy), is shown. Bar, 25 μm.
Mentions: To determine the intracellular distribution of ubiquilin we used rabbit pAb that we had generated to two different GST–ubiquilin fusion proteins (see Materials and Methods). Both antisera detected endogenous and overexpressed 66-kD ubiquilin polypeptides, which were not detected by the preimmune sera (Fig. 3 D). Although one antiserum reacted with an unknown protein of ∼55 kD (Fig. 3 E), this reactivity was subsequently removed by affinity purification of the antibody (Fig. 3 F). Immunofluorescence staining of ubiquilin within cells was performed with the specific anti-ubiquilin antibodies. Compared with the preimmune sera (Fig. 6A and Fig. D), both antibodies revealed similar staining patterns (Fig. 6B and Fig. E). Indirect immunofluorescence, as well as laser confocal microscopy, revealed endogenous intracellular localization of ubiquilin in HeLa cells to both the nucleus and cytoplasm (Fig. 6B, Fig. E, and Fig. G). In some cells, endogenous ubiquilin staining was stronger in the nucleus (Fig. 6 E, arrowheads). Furthermore, in a small subset of cells, ubiquilin within the cytoplasm was localized to punctate structures (Fig. 6B and Fig. E, arrows). The frequency of these structures dramatically increased upon overexpression of full-length untagged ubiquilin (Fig. 6C and Fig. F). To corroborate the anti-ubiquilin staining pattern, we performed additional localization studies on myc- and GFP-tagged ubiquilin proteins in HeLa cells (Fig. 2 IV, O and N, respectively). Overexpressed myc-tagged ubiquilin stained with anti-myc mAb exhibited the same intracellular structures (Fig. 6 I) as overexpressed wild-type ubiquilin (Fig. 6C and Fig. F). Finally, the same fluorescent staining pattern was reproduced upon overexpression of GFP-ubiquilin (Fig. 6 H). Since the GFP-ubiquilin image in Fig. 6 H was captured from live and unfixed cells without antibodies, this established that the anti-ubiquilin structures seen previously were not artifacts of the paraformaldehyde fixation or immunofluorescence staining procedures.

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