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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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Schematic drawings of ubiquilin expression constructs. (I) The full-length ubiquilin polypeptide consists of 595 residues and contains an NH2-terminal UB domain (speckled), a COOH-terminal UBA domain (striped), and several regularly spaced asparagine-proline (Asn-Pro) repeats (vertical bars). (II) The probes used in human Northern blots. (III) GST-fusion constructs: A (N393–S595 aa), B (Q378–S595 aa), C (Q113–M377 aa), D (Q541–S595 aa), E (D449–S595 aa), F (D449–L540 aa), G (N393–L540 aa), H (M37–S595 aa), I (M37–L540 aa), J (Q113–L540 aa), K (Q113–S595 aa), and L (GST alone). The ubiquilin portions of constructs A and B were isolated in the original Y2H screen. Bacterially expressed GST–fusion B and C polypeptides were used as immunogens for anti-ubiquilin pAb production (*). (IV) Mammalian expression constructs: M, full-length untagged ubiquilin; N, NH2-terminal GFP-tagged ubiquilin fused at residue 20 (Ala); and O, COOH-terminal myc epitope-tagged ubiquilin.
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Figure 2: Schematic drawings of ubiquilin expression constructs. (I) The full-length ubiquilin polypeptide consists of 595 residues and contains an NH2-terminal UB domain (speckled), a COOH-terminal UBA domain (striped), and several regularly spaced asparagine-proline (Asn-Pro) repeats (vertical bars). (II) The probes used in human Northern blots. (III) GST-fusion constructs: A (N393–S595 aa), B (Q378–S595 aa), C (Q113–M377 aa), D (Q541–S595 aa), E (D449–S595 aa), F (D449–L540 aa), G (N393–L540 aa), H (M37–S595 aa), I (M37–L540 aa), J (Q113–L540 aa), K (Q113–S595 aa), and L (GST alone). The ubiquilin portions of constructs A and B were isolated in the original Y2H screen. Bacterially expressed GST–fusion B and C polypeptides were used as immunogens for anti-ubiquilin pAb production (*). (IV) Mammalian expression constructs: M, full-length untagged ubiquilin; N, NH2-terminal GFP-tagged ubiquilin fused at residue 20 (Ala); and O, COOH-terminal myc epitope-tagged ubiquilin.

Mentions: cDNA hybridization probes were 32P-radiolabeled by random primer labeling of ∼100 ng of two ubiquilin cDNA restriction fragments, X (bases 1,132–1,860) and Y (bases 284–967), shown in Fig. 2 II. Each probe was hybridized to a different human mRNA Northern blot, Human Multiple Tissue Northern Blot I, and Human Brain MTN II, respectively (CLONTECH Laboratories, Inc.). After hybridization, the blots were washed twice with 2.0× SSC and 0.05% SDS at 50°C and then twice again with 0.1× SSC and 0.1% SDS at 50°C, before exposure to autoradiography film. Both blots were subsequently stripped in near-boiling temperature 0.5% SDS and then reprobed with radiolabeled human β-actin cDNA control probe (CLONTECH Laboratories, Inc.). The band intensities were quantified using GelExpert 97 2.0 (Nucleotech Corp.), divided by the β-actin control band intensity from the same lane, and then normalized against the lowest value.


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

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

Schematic drawings of ubiquilin expression constructs. (I) The full-length ubiquilin polypeptide consists of 595 residues and contains an NH2-terminal UB domain (speckled), a COOH-terminal UBA domain (striped), and several regularly spaced asparagine-proline (Asn-Pro) repeats (vertical bars). (II) The probes used in human Northern blots. (III) GST-fusion constructs: A (N393–S595 aa), B (Q378–S595 aa), C (Q113–M377 aa), D (Q541–S595 aa), E (D449–S595 aa), F (D449–L540 aa), G (N393–L540 aa), H (M37–S595 aa), I (M37–L540 aa), J (Q113–L540 aa), K (Q113–S595 aa), and L (GST alone). The ubiquilin portions of constructs A and B were isolated in the original Y2H screen. Bacterially expressed GST–fusion B and C polypeptides were used as immunogens for anti-ubiquilin pAb production (*). (IV) Mammalian expression constructs: M, full-length untagged ubiquilin; N, NH2-terminal GFP-tagged ubiquilin fused at residue 20 (Ala); and O, COOH-terminal myc epitope-tagged ubiquilin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169435&req=5

Figure 2: Schematic drawings of ubiquilin expression constructs. (I) The full-length ubiquilin polypeptide consists of 595 residues and contains an NH2-terminal UB domain (speckled), a COOH-terminal UBA domain (striped), and several regularly spaced asparagine-proline (Asn-Pro) repeats (vertical bars). (II) The probes used in human Northern blots. (III) GST-fusion constructs: A (N393–S595 aa), B (Q378–S595 aa), C (Q113–M377 aa), D (Q541–S595 aa), E (D449–S595 aa), F (D449–L540 aa), G (N393–L540 aa), H (M37–S595 aa), I (M37–L540 aa), J (Q113–L540 aa), K (Q113–S595 aa), and L (GST alone). The ubiquilin portions of constructs A and B were isolated in the original Y2H screen. Bacterially expressed GST–fusion B and C polypeptides were used as immunogens for anti-ubiquilin pAb production (*). (IV) Mammalian expression constructs: M, full-length untagged ubiquilin; N, NH2-terminal GFP-tagged ubiquilin fused at residue 20 (Ala); and O, COOH-terminal myc epitope-tagged ubiquilin.
Mentions: cDNA hybridization probes were 32P-radiolabeled by random primer labeling of ∼100 ng of two ubiquilin cDNA restriction fragments, X (bases 1,132–1,860) and Y (bases 284–967), shown in Fig. 2 II. Each probe was hybridized to a different human mRNA Northern blot, Human Multiple Tissue Northern Blot I, and Human Brain MTN II, respectively (CLONTECH Laboratories, Inc.). After hybridization, the blots were washed twice with 2.0× SSC and 0.05% SDS at 50°C and then twice again with 0.1× SSC and 0.1% SDS at 50°C, before exposure to autoradiography film. Both blots were subsequently stripped in near-boiling temperature 0.5% SDS and then reprobed with radiolabeled human β-actin cDNA control probe (CLONTECH Laboratories, Inc.). The band intensities were quantified using GelExpert 97 2.0 (Nucleotech Corp.), divided by the β-actin control band intensity from the same lane, and then normalized against the lowest value.

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