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Increased apoptosis arising from increased expression of the Alzheimer's disease-associated presenilin-2 mutation (N141I).

Janicki S, Monteiro MJ - J. Cell Biol. (1997)

Bottom Line: As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells.Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation.We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Mutations in the genes for presenilin 1 and 2 (PS-1 and PS-2) have been linked to development of early-onset Alzheimer's disease (AD). As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells. Although HeLa cells are strongly predisposed to continued mitosis, expression of PS-2 induced programmed cell death (apoptosis). Direct evidence for apoptosis was obtained by double staining for terminal deoxynucleotide transferase nick end labeling (TUNEL) and PS-2 expression and by following green fluorescent protein-tagged PS-2 over time. Deletion analysis indicates that as little as 166 NH2-terminal residues of PS-2 are sufficient for endoplasmic reticulum (ER) localization and apoptosis. Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation. Expression of the presenilins in several other cell lines and transgenic mice has been accompanied by rapid protein cleavage without the induction of cell death. In contrast, PS-2 expressed in HeLa cells was not cleaved, and cell death occurred. We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

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Immunoblot analysis of PS-2 proteins. (A and  B) PS-2 NH2-terminal, loop,  and COOH-terminal sequences were expressed as  GST fusion proteins and separated by SDS-PAGE on  8.5% polyacrylamide gels.  The separated proteins were  either stained with Coomassie blue (A) or transferred onto nitrocellulose filters and immunoblotted  with the PS-2 NH2-terminal  antibody (B). Lanes 1–4 are  whole cell lysates of bacteria  induced for expression of  GST fusion proteins by addition of IPTG, and lanes 5–8  are the corresponding purified GST fusion proteins.  The GST fusion proteins that  were expressed are as follows: Lanes 1 and 5, GST  alone; lanes 2 and 6, GST– NH2-terminal fusion protein;  lanes 3 and 7, GST–loop fusion protein; and lanes 4 and  8, GST–COOH-terminal fusion protein. The arrowheads  indicate the position of full-length GST fusion proteins. (C–F) Protein lysates of transfected HeLa cells separated by SDS-PAGE on  8.5% gels and immunoblotted with antibodies that recognize the transfected proteins. (C) Immunoblot with the anti–PS-2–specific antibody. Lanes 1–7 are lysates of cells transfected with the following constructs: Lanes 1 and 3, mock-transfected cells; lane 2, pPS-2; lane  4, pPS-2+Myc; lane 5, pPS-2(268aa)+Myc; lane 6, pPS-2(222aa)+Myc; and lane 7, pPS-2(166aa)+Myc. (D) Immunoblot of corresponding lysates shown in C with the anti-myc antibody. Lysates in lanes 1–5 in D correspond to lysates in lanes 3–7 in C. (E) Immunoblot  with the anti–GFP-specific antibody. Lanes 1–4 are protein lysates of cells transfected with the following constructs: Lane 1, mock-transfected cells (control); lane 2, pGFP; lane 3, pGFP–PS-2; and lane 4, pGFP–PS-2(N141I). (F) Immunoblot of nonfusion PS-2 proteins with the NH2-terminal PS-2 antibody. Lanes 1–3 are 100 μg of protein lysates of cells transfected with equivalent amounts of the  following nonfusion PS-2 constructs: Lane 1, mock-transfected; lane 2, pPS-2 (wild-type); and lane 3, pPS-2(Asn141Ile) mutant. The positions of protein molecular weight markers are indicated. Full-length PS-2 containing polypeptide bands seen only in transfected cell  lysates are marked with large arrowheads. The small arrowheads indicate the positions of larger PS-2 complexes.
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Figure 2: Immunoblot analysis of PS-2 proteins. (A and B) PS-2 NH2-terminal, loop, and COOH-terminal sequences were expressed as GST fusion proteins and separated by SDS-PAGE on 8.5% polyacrylamide gels. The separated proteins were either stained with Coomassie blue (A) or transferred onto nitrocellulose filters and immunoblotted with the PS-2 NH2-terminal antibody (B). Lanes 1–4 are whole cell lysates of bacteria induced for expression of GST fusion proteins by addition of IPTG, and lanes 5–8 are the corresponding purified GST fusion proteins. The GST fusion proteins that were expressed are as follows: Lanes 1 and 5, GST alone; lanes 2 and 6, GST– NH2-terminal fusion protein; lanes 3 and 7, GST–loop fusion protein; and lanes 4 and 8, GST–COOH-terminal fusion protein. The arrowheads indicate the position of full-length GST fusion proteins. (C–F) Protein lysates of transfected HeLa cells separated by SDS-PAGE on 8.5% gels and immunoblotted with antibodies that recognize the transfected proteins. (C) Immunoblot with the anti–PS-2–specific antibody. Lanes 1–7 are lysates of cells transfected with the following constructs: Lanes 1 and 3, mock-transfected cells; lane 2, pPS-2; lane 4, pPS-2+Myc; lane 5, pPS-2(268aa)+Myc; lane 6, pPS-2(222aa)+Myc; and lane 7, pPS-2(166aa)+Myc. (D) Immunoblot of corresponding lysates shown in C with the anti-myc antibody. Lysates in lanes 1–5 in D correspond to lysates in lanes 3–7 in C. (E) Immunoblot with the anti–GFP-specific antibody. Lanes 1–4 are protein lysates of cells transfected with the following constructs: Lane 1, mock-transfected cells (control); lane 2, pGFP; lane 3, pGFP–PS-2; and lane 4, pGFP–PS-2(N141I). (F) Immunoblot of nonfusion PS-2 proteins with the NH2-terminal PS-2 antibody. Lanes 1–3 are 100 μg of protein lysates of cells transfected with equivalent amounts of the following nonfusion PS-2 constructs: Lane 1, mock-transfected; lane 2, pPS-2 (wild-type); and lane 3, pPS-2(Asn141Ile) mutant. The positions of protein molecular weight markers are indicated. Full-length PS-2 containing polypeptide bands seen only in transfected cell lysates are marked with large arrowheads. The small arrowheads indicate the positions of larger PS-2 complexes.

Mentions: To investigate PS-2 function, full-length PS-2 and progressive COOH-terminal truncations, tagged at their COOH terminus with a myc epitope and under transcriptional control of a CMV promoter (Fig. 1), were expressed in HeLa cells by transient transfection. Immunoblot analysis of protein lysates prepared from cells transfected with either myc-tagged or nontagged full-length PS-2 contained immunoreactive bands of ∼50–54 kD when probed with a polyclonal antibody specific for NH2-terminal PS-2 sequences (Fig. 2 C, lanes 2 and 4, respectively) that were not detectable in mock-transfected lysates (Fig. 2 C, lanes 1 and 3). The PS-2 antibody recognized GST fusion proteins containing NH2-terminal but not loop or COOH-terminal PS-2 sequences (Fig. 2, A and B). PS-2 deletions in which the COOH-terminal 180, 226, and 282 residues were removed (see Fig. 1 and Materials and Methods) migrated as doublet bands in SDS-PAGE gels (evident in shorter exposures of autoradiographs, data not shown) with apparent molecular sizes of ∼35–37, 33–35, and 31–33 kD, respectively (Fig. 2 C, lanes 4–7). Apart from these bands, larger immunoreactive complexes were routinely seen in PS-2–transfected cells but not in mock-transfected cells and appeared to represent dimers, trimers, and higher molecular mass complexes of PS-2 proteins (Fig. 2 C, lanes 4–7, small arrowheads). We did not observe any significant proteolytic cleavage of the PS-2–expressed products as has been previously reported (Kim et al., 1997; Tomita et al., 1997). In fact, immunoblot analyses of a parallel gel probed with the myc antibody, which recognized the COOH terminus of PS-2, contained bands identical in size to those recognized by the anti–NH2-terminal PS-2 antibody, indicating full-length accumulation of the various PS-2 polypeptides with the presence of both NH2- and COOH-terminal epitopes (compare Fig. 2 C, lanes 3–7, with Fig. 2 D, lanes 1–5). The myc-reactive PS-2 bands were clearly distinguished from the ∼35 and 65 kD endogenous bands present in all HeLa cell lysates (Fig. 2 D, small arrowhead).


Increased apoptosis arising from increased expression of the Alzheimer's disease-associated presenilin-2 mutation (N141I).

Janicki S, Monteiro MJ - J. Cell Biol. (1997)

Immunoblot analysis of PS-2 proteins. (A and  B) PS-2 NH2-terminal, loop,  and COOH-terminal sequences were expressed as  GST fusion proteins and separated by SDS-PAGE on  8.5% polyacrylamide gels.  The separated proteins were  either stained with Coomassie blue (A) or transferred onto nitrocellulose filters and immunoblotted  with the PS-2 NH2-terminal  antibody (B). Lanes 1–4 are  whole cell lysates of bacteria  induced for expression of  GST fusion proteins by addition of IPTG, and lanes 5–8  are the corresponding purified GST fusion proteins.  The GST fusion proteins that  were expressed are as follows: Lanes 1 and 5, GST  alone; lanes 2 and 6, GST– NH2-terminal fusion protein;  lanes 3 and 7, GST–loop fusion protein; and lanes 4 and  8, GST–COOH-terminal fusion protein. The arrowheads  indicate the position of full-length GST fusion proteins. (C–F) Protein lysates of transfected HeLa cells separated by SDS-PAGE on  8.5% gels and immunoblotted with antibodies that recognize the transfected proteins. (C) Immunoblot with the anti–PS-2–specific antibody. Lanes 1–7 are lysates of cells transfected with the following constructs: Lanes 1 and 3, mock-transfected cells; lane 2, pPS-2; lane  4, pPS-2+Myc; lane 5, pPS-2(268aa)+Myc; lane 6, pPS-2(222aa)+Myc; and lane 7, pPS-2(166aa)+Myc. (D) Immunoblot of corresponding lysates shown in C with the anti-myc antibody. Lysates in lanes 1–5 in D correspond to lysates in lanes 3–7 in C. (E) Immunoblot  with the anti–GFP-specific antibody. Lanes 1–4 are protein lysates of cells transfected with the following constructs: Lane 1, mock-transfected cells (control); lane 2, pGFP; lane 3, pGFP–PS-2; and lane 4, pGFP–PS-2(N141I). (F) Immunoblot of nonfusion PS-2 proteins with the NH2-terminal PS-2 antibody. Lanes 1–3 are 100 μg of protein lysates of cells transfected with equivalent amounts of the  following nonfusion PS-2 constructs: Lane 1, mock-transfected; lane 2, pPS-2 (wild-type); and lane 3, pPS-2(Asn141Ile) mutant. The positions of protein molecular weight markers are indicated. Full-length PS-2 containing polypeptide bands seen only in transfected cell  lysates are marked with large arrowheads. The small arrowheads indicate the positions of larger PS-2 complexes.
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Figure 2: Immunoblot analysis of PS-2 proteins. (A and B) PS-2 NH2-terminal, loop, and COOH-terminal sequences were expressed as GST fusion proteins and separated by SDS-PAGE on 8.5% polyacrylamide gels. The separated proteins were either stained with Coomassie blue (A) or transferred onto nitrocellulose filters and immunoblotted with the PS-2 NH2-terminal antibody (B). Lanes 1–4 are whole cell lysates of bacteria induced for expression of GST fusion proteins by addition of IPTG, and lanes 5–8 are the corresponding purified GST fusion proteins. The GST fusion proteins that were expressed are as follows: Lanes 1 and 5, GST alone; lanes 2 and 6, GST– NH2-terminal fusion protein; lanes 3 and 7, GST–loop fusion protein; and lanes 4 and 8, GST–COOH-terminal fusion protein. The arrowheads indicate the position of full-length GST fusion proteins. (C–F) Protein lysates of transfected HeLa cells separated by SDS-PAGE on 8.5% gels and immunoblotted with antibodies that recognize the transfected proteins. (C) Immunoblot with the anti–PS-2–specific antibody. Lanes 1–7 are lysates of cells transfected with the following constructs: Lanes 1 and 3, mock-transfected cells; lane 2, pPS-2; lane 4, pPS-2+Myc; lane 5, pPS-2(268aa)+Myc; lane 6, pPS-2(222aa)+Myc; and lane 7, pPS-2(166aa)+Myc. (D) Immunoblot of corresponding lysates shown in C with the anti-myc antibody. Lysates in lanes 1–5 in D correspond to lysates in lanes 3–7 in C. (E) Immunoblot with the anti–GFP-specific antibody. Lanes 1–4 are protein lysates of cells transfected with the following constructs: Lane 1, mock-transfected cells (control); lane 2, pGFP; lane 3, pGFP–PS-2; and lane 4, pGFP–PS-2(N141I). (F) Immunoblot of nonfusion PS-2 proteins with the NH2-terminal PS-2 antibody. Lanes 1–3 are 100 μg of protein lysates of cells transfected with equivalent amounts of the following nonfusion PS-2 constructs: Lane 1, mock-transfected; lane 2, pPS-2 (wild-type); and lane 3, pPS-2(Asn141Ile) mutant. The positions of protein molecular weight markers are indicated. Full-length PS-2 containing polypeptide bands seen only in transfected cell lysates are marked with large arrowheads. The small arrowheads indicate the positions of larger PS-2 complexes.
Mentions: To investigate PS-2 function, full-length PS-2 and progressive COOH-terminal truncations, tagged at their COOH terminus with a myc epitope and under transcriptional control of a CMV promoter (Fig. 1), were expressed in HeLa cells by transient transfection. Immunoblot analysis of protein lysates prepared from cells transfected with either myc-tagged or nontagged full-length PS-2 contained immunoreactive bands of ∼50–54 kD when probed with a polyclonal antibody specific for NH2-terminal PS-2 sequences (Fig. 2 C, lanes 2 and 4, respectively) that were not detectable in mock-transfected lysates (Fig. 2 C, lanes 1 and 3). The PS-2 antibody recognized GST fusion proteins containing NH2-terminal but not loop or COOH-terminal PS-2 sequences (Fig. 2, A and B). PS-2 deletions in which the COOH-terminal 180, 226, and 282 residues were removed (see Fig. 1 and Materials and Methods) migrated as doublet bands in SDS-PAGE gels (evident in shorter exposures of autoradiographs, data not shown) with apparent molecular sizes of ∼35–37, 33–35, and 31–33 kD, respectively (Fig. 2 C, lanes 4–7). Apart from these bands, larger immunoreactive complexes were routinely seen in PS-2–transfected cells but not in mock-transfected cells and appeared to represent dimers, trimers, and higher molecular mass complexes of PS-2 proteins (Fig. 2 C, lanes 4–7, small arrowheads). We did not observe any significant proteolytic cleavage of the PS-2–expressed products as has been previously reported (Kim et al., 1997; Tomita et al., 1997). In fact, immunoblot analyses of a parallel gel probed with the myc antibody, which recognized the COOH terminus of PS-2, contained bands identical in size to those recognized by the anti–NH2-terminal PS-2 antibody, indicating full-length accumulation of the various PS-2 polypeptides with the presence of both NH2- and COOH-terminal epitopes (compare Fig. 2 C, lanes 3–7, with Fig. 2 D, lanes 1–5). The myc-reactive PS-2 bands were clearly distinguished from the ∼35 and 65 kD endogenous bands present in all HeLa cell lysates (Fig. 2 D, small arrowhead).

Bottom Line: As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells.Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation.We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Mutations in the genes for presenilin 1 and 2 (PS-1 and PS-2) have been linked to development of early-onset Alzheimer's disease (AD). As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells. Although HeLa cells are strongly predisposed to continued mitosis, expression of PS-2 induced programmed cell death (apoptosis). Direct evidence for apoptosis was obtained by double staining for terminal deoxynucleotide transferase nick end labeling (TUNEL) and PS-2 expression and by following green fluorescent protein-tagged PS-2 over time. Deletion analysis indicates that as little as 166 NH2-terminal residues of PS-2 are sufficient for endoplasmic reticulum (ER) localization and apoptosis. Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation. Expression of the presenilins in several other cell lines and transgenic mice has been accompanied by rapid protein cleavage without the induction of cell death. In contrast, PS-2 expressed in HeLa cells was not cleaved, and cell death occurred. We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

Show MeSH
Related in: MedlinePlus