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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 promotes increased PS2 protein accumulation. (A–D) HeLa cells, 12 h after transfection with ubiquilin (15 μg expression plasmid, lanes 1–3), PS2 (7 μg expression plasmid, lanes 4–6), or both (lanes 7–9), were either left untreated (lanes 1, 4, and 7) or treated for 5–6 h with proteasome inhibitors (20 μM synthetic lactacystin in lanes 2, 5, and 8; 40 μM MG-132 in lanes 3, 6, and 9). Equivalent amounts of protein (100 μg) from each sample were immunoblotted with (A) anti-ubiquitin, (B) anti-PS2-NH2 terminus, (C) affinity-purified anti–ubiquilin-C, or (D) anti-α-tubulin antibodies. As expected, anti-ubiquitin antibodies detected larger molecular weight proteins in cells treated with proteasome inhibitors (lanes 2 and 3, 5 and 6, and 8 and 9) compared with untreated cells (lanes 1, 4, and 7). Significantly more PS2 protein (and slower migrating forms) could be seen in cells cotransfected with ubiquilin (lanes 7–9, arrowhead) compared with those transfected with PS2 alone (lanes 4–6). (*) A doublet of weakly reactive bands was detected in all lysates, but we considered them to be nonspecific proteins. The anti–α-tubulin blot shows equal protein loading of each sample. (E) HeLa cells were transfected with PS2 alone (9 μg expression plasmid, lane 1) or cotransfected along with increasing amounts of ubiquilin (1, 2, 3, or 4 μg expression plasmid in lanes 2–5, respectively). Equivalent amounts of the transfected lysates were separated through an 8.5% polyacrylamide gel and immunoblotted with anti–PS2-NH2 terminus antibody. (F) Same as in E, except with the same increasing amounts of GFP expression plasmid (lanes 2–5) instead of ubiquilin. (G) Same as in E, but proteins were separated on a 10% polyacrylamide gel and immunoblotted with anti–PS2-loop antibody. Note the absence of any detectable PS2 cleavage products corresponding to endoproteolytic PS2 cleavage in the loop. (H) The same blot shown in G or parallel blots were immunoblotted for lamin B, calreticulin, calnexin, BiP, and α-tubulin. The relative levels of these other endogenous proteins remained relatively unchanged compared with the PS2 levels.
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Figure 8: Ubiquilin promotes increased PS2 protein accumulation. (A–D) HeLa cells, 12 h after transfection with ubiquilin (15 μg expression plasmid, lanes 1–3), PS2 (7 μg expression plasmid, lanes 4–6), or both (lanes 7–9), were either left untreated (lanes 1, 4, and 7) or treated for 5–6 h with proteasome inhibitors (20 μM synthetic lactacystin in lanes 2, 5, and 8; 40 μM MG-132 in lanes 3, 6, and 9). Equivalent amounts of protein (100 μg) from each sample were immunoblotted with (A) anti-ubiquitin, (B) anti-PS2-NH2 terminus, (C) affinity-purified anti–ubiquilin-C, or (D) anti-α-tubulin antibodies. As expected, anti-ubiquitin antibodies detected larger molecular weight proteins in cells treated with proteasome inhibitors (lanes 2 and 3, 5 and 6, and 8 and 9) compared with untreated cells (lanes 1, 4, and 7). Significantly more PS2 protein (and slower migrating forms) could be seen in cells cotransfected with ubiquilin (lanes 7–9, arrowhead) compared with those transfected with PS2 alone (lanes 4–6). (*) A doublet of weakly reactive bands was detected in all lysates, but we considered them to be nonspecific proteins. The anti–α-tubulin blot shows equal protein loading of each sample. (E) HeLa cells were transfected with PS2 alone (9 μg expression plasmid, lane 1) or cotransfected along with increasing amounts of ubiquilin (1, 2, 3, or 4 μg expression plasmid in lanes 2–5, respectively). Equivalent amounts of the transfected lysates were separated through an 8.5% polyacrylamide gel and immunoblotted with anti–PS2-NH2 terminus antibody. (F) Same as in E, except with the same increasing amounts of GFP expression plasmid (lanes 2–5) instead of ubiquilin. (G) Same as in E, but proteins were separated on a 10% polyacrylamide gel and immunoblotted with anti–PS2-loop antibody. Note the absence of any detectable PS2 cleavage products corresponding to endoproteolytic PS2 cleavage in the loop. (H) The same blot shown in G or parallel blots were immunoblotted for lamin B, calreticulin, calnexin, BiP, and α-tubulin. The relative levels of these other endogenous proteins remained relatively unchanged compared with the PS2 levels.

Mentions: Since ubiquilin contains multiple ubiquitin-related structural motifs, we investigated whether its association with presenilin would have an effect on presenilin-protein modification and/or stability. Treatment of mock-transfected and PS2-transfected HeLa cells with proteasome inhibitors lactacystin (20 μM) or MG-132 (40 μM), as expected, increased the overall amount of ubiquitinated proteins in cells (Fig. 8 A, lanes 2 and 3, 5 and 6, and 8 and 9 versus lanes 1, 4, and 7, respectively). However, these conditions did not markedly increase PS2 protein levels (Fig. 8 B, lanes 4–6). Remarkably, coexpression of ubiquilin with PS2 caused a substantial (>10-fold) increase in PS2 protein accumulation (Fig. 8 B, lanes 7–9) compared with overexpression of PS2 alone (Fig. 8 B, lanes 4–6). This effect was independent of treatment with lactacystin or MG-132, as it also occurred just as robustly in cells not treated with the proteasome inhibitors. Particularly striking was the increased accumulation in SDS-PAGE gels of full-length uncleaved PS2 protein (Fig. 8 B, arrowhead) and higher molecular weight complexes that PS2 forms. Increasing the amount of transfected ubiquilin plasmid DNA, whereas maintaining constant levels of transfected PS2 plasmid DNA, revealed a dose-dependent effect by ubiquilin on PS2 accumulation within the cells (Fig. 8 E, lanes 1–5). This effect was specific for ubiquilin expression, since cotransfection of increasing amounts of GFP plasmid DNA with PS2 did not increase PS2 protein accumulation (Fig. 8 F, lanes 1–5). The blot in Fig. 8 E was exposed for a shorter time than that in Fig. 8 F to optimally illustrate the subtle changes in PS2 protein levels. In these experiments, we routinely observed the accumulation of full-length PS2 protein, but not the endoproteolytic cleaved forms of the protein (Haass and De Strooper 1999). In fact, recent reports have indicated that cleavage of presenilins is not essential for biological function of the presenilins, namely by its ability to rescue PS activity in C. elegans (Jacobsen et al. 1999; Steiner et al. 1999). The absence of detectable endoproteolytic cleaved forms of PS2 is more clearly illustrated in Fig. 8 G, which shows that an antibody specific for PS2-loop sequences downstream of the proposed endoproteolytic cleavage site recognized the same 54 kD and higher PS2 species (as the PS2 NH2-terminal specific antibody). Interestingly, though ubiquilin caused a dose-dependent increase in presenilin accumulation, it did not alter the accumulation levels of various other endogenous HeLa proteins, such as lamin B, calreticulin, calnexin, BiP, and α-tubulin (Fig. 8 H). Similar experiments repeated with PS1 indicated that ubiquilin also promoted a dose-dependent increase in PS1 protein accumulation in HeLa cells (data not shown).


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 promotes increased PS2 protein accumulation. (A–D) HeLa cells, 12 h after transfection with ubiquilin (15 μg expression plasmid, lanes 1–3), PS2 (7 μg expression plasmid, lanes 4–6), or both (lanes 7–9), were either left untreated (lanes 1, 4, and 7) or treated for 5–6 h with proteasome inhibitors (20 μM synthetic lactacystin in lanes 2, 5, and 8; 40 μM MG-132 in lanes 3, 6, and 9). Equivalent amounts of protein (100 μg) from each sample were immunoblotted with (A) anti-ubiquitin, (B) anti-PS2-NH2 terminus, (C) affinity-purified anti–ubiquilin-C, or (D) anti-α-tubulin antibodies. As expected, anti-ubiquitin antibodies detected larger molecular weight proteins in cells treated with proteasome inhibitors (lanes 2 and 3, 5 and 6, and 8 and 9) compared with untreated cells (lanes 1, 4, and 7). Significantly more PS2 protein (and slower migrating forms) could be seen in cells cotransfected with ubiquilin (lanes 7–9, arrowhead) compared with those transfected with PS2 alone (lanes 4–6). (*) A doublet of weakly reactive bands was detected in all lysates, but we considered them to be nonspecific proteins. The anti–α-tubulin blot shows equal protein loading of each sample. (E) HeLa cells were transfected with PS2 alone (9 μg expression plasmid, lane 1) or cotransfected along with increasing amounts of ubiquilin (1, 2, 3, or 4 μg expression plasmid in lanes 2–5, respectively). Equivalent amounts of the transfected lysates were separated through an 8.5% polyacrylamide gel and immunoblotted with anti–PS2-NH2 terminus antibody. (F) Same as in E, except with the same increasing amounts of GFP expression plasmid (lanes 2–5) instead of ubiquilin. (G) Same as in E, but proteins were separated on a 10% polyacrylamide gel and immunoblotted with anti–PS2-loop antibody. Note the absence of any detectable PS2 cleavage products corresponding to endoproteolytic PS2 cleavage in the loop. (H) The same blot shown in G or parallel blots were immunoblotted for lamin B, calreticulin, calnexin, BiP, and α-tubulin. The relative levels of these other endogenous proteins remained relatively unchanged compared with the PS2 levels.
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Related In: Results  -  Collection

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Figure 8: Ubiquilin promotes increased PS2 protein accumulation. (A–D) HeLa cells, 12 h after transfection with ubiquilin (15 μg expression plasmid, lanes 1–3), PS2 (7 μg expression plasmid, lanes 4–6), or both (lanes 7–9), were either left untreated (lanes 1, 4, and 7) or treated for 5–6 h with proteasome inhibitors (20 μM synthetic lactacystin in lanes 2, 5, and 8; 40 μM MG-132 in lanes 3, 6, and 9). Equivalent amounts of protein (100 μg) from each sample were immunoblotted with (A) anti-ubiquitin, (B) anti-PS2-NH2 terminus, (C) affinity-purified anti–ubiquilin-C, or (D) anti-α-tubulin antibodies. As expected, anti-ubiquitin antibodies detected larger molecular weight proteins in cells treated with proteasome inhibitors (lanes 2 and 3, 5 and 6, and 8 and 9) compared with untreated cells (lanes 1, 4, and 7). Significantly more PS2 protein (and slower migrating forms) could be seen in cells cotransfected with ubiquilin (lanes 7–9, arrowhead) compared with those transfected with PS2 alone (lanes 4–6). (*) A doublet of weakly reactive bands was detected in all lysates, but we considered them to be nonspecific proteins. The anti–α-tubulin blot shows equal protein loading of each sample. (E) HeLa cells were transfected with PS2 alone (9 μg expression plasmid, lane 1) or cotransfected along with increasing amounts of ubiquilin (1, 2, 3, or 4 μg expression plasmid in lanes 2–5, respectively). Equivalent amounts of the transfected lysates were separated through an 8.5% polyacrylamide gel and immunoblotted with anti–PS2-NH2 terminus antibody. (F) Same as in E, except with the same increasing amounts of GFP expression plasmid (lanes 2–5) instead of ubiquilin. (G) Same as in E, but proteins were separated on a 10% polyacrylamide gel and immunoblotted with anti–PS2-loop antibody. Note the absence of any detectable PS2 cleavage products corresponding to endoproteolytic PS2 cleavage in the loop. (H) The same blot shown in G or parallel blots were immunoblotted for lamin B, calreticulin, calnexin, BiP, and α-tubulin. The relative levels of these other endogenous proteins remained relatively unchanged compared with the PS2 levels.
Mentions: Since ubiquilin contains multiple ubiquitin-related structural motifs, we investigated whether its association with presenilin would have an effect on presenilin-protein modification and/or stability. Treatment of mock-transfected and PS2-transfected HeLa cells with proteasome inhibitors lactacystin (20 μM) or MG-132 (40 μM), as expected, increased the overall amount of ubiquitinated proteins in cells (Fig. 8 A, lanes 2 and 3, 5 and 6, and 8 and 9 versus lanes 1, 4, and 7, respectively). However, these conditions did not markedly increase PS2 protein levels (Fig. 8 B, lanes 4–6). Remarkably, coexpression of ubiquilin with PS2 caused a substantial (>10-fold) increase in PS2 protein accumulation (Fig. 8 B, lanes 7–9) compared with overexpression of PS2 alone (Fig. 8 B, lanes 4–6). This effect was independent of treatment with lactacystin or MG-132, as it also occurred just as robustly in cells not treated with the proteasome inhibitors. Particularly striking was the increased accumulation in SDS-PAGE gels of full-length uncleaved PS2 protein (Fig. 8 B, arrowhead) and higher molecular weight complexes that PS2 forms. Increasing the amount of transfected ubiquilin plasmid DNA, whereas maintaining constant levels of transfected PS2 plasmid DNA, revealed a dose-dependent effect by ubiquilin on PS2 accumulation within the cells (Fig. 8 E, lanes 1–5). This effect was specific for ubiquilin expression, since cotransfection of increasing amounts of GFP plasmid DNA with PS2 did not increase PS2 protein accumulation (Fig. 8 F, lanes 1–5). The blot in Fig. 8 E was exposed for a shorter time than that in Fig. 8 F to optimally illustrate the subtle changes in PS2 protein levels. In these experiments, we routinely observed the accumulation of full-length PS2 protein, but not the endoproteolytic cleaved forms of the protein (Haass and De Strooper 1999). In fact, recent reports have indicated that cleavage of presenilins is not essential for biological function of the presenilins, namely by its ability to rescue PS activity in C. elegans (Jacobsen et al. 1999; Steiner et al. 1999). The absence of detectable endoproteolytic cleaved forms of PS2 is more clearly illustrated in Fig. 8 G, which shows that an antibody specific for PS2-loop sequences downstream of the proposed endoproteolytic cleavage site recognized the same 54 kD and higher PS2 species (as the PS2 NH2-terminal specific antibody). Interestingly, though ubiquilin caused a dose-dependent increase in presenilin accumulation, it did not alter the accumulation levels of various other endogenous HeLa proteins, such as lamin B, calreticulin, calnexin, BiP, and α-tubulin (Fig. 8 H). Similar experiments repeated with PS1 indicated that ubiquilin also promoted a dose-dependent increase in PS1 protein accumulation in HeLa cells (data not shown).

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