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Amyloid beta peptide binds a novel death-inducing protein, AB-DIP

Madepalli K. Lakshmana^*,1, Wataru Araki and Takeshi Tabira^*,1

^* Department of Vascular Dementia Research, National Institute for Longevity Sciences, Obu, Japan; and
Department of Demyelinating Disease and Aging, National Institute of Neuroscience, Kodaira, Japan

¹Correspondence: Department of Vascular Dementia, Research National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan. E-mail: laxman@nils.go.jp; tabira{at}nils.go.jp

SPECIFIC AIMS

The aims of this study were to 1) identify Aß binding proteins by the yeast two-hybrid system from human brain cDNA library using human Aß1–42 fused to Gal4-DNA binding domain as bait; and 2) understand the role of such a potential Aß binding protein in Aß-induced cell death.

PRINCIPAL FINDINGS

1. We identified a novel protein, Aß-related death-inducing protein (AB-DIP), specifically binding to Aß peptide

AB-DIP protein is characterized by the presence of caspase activation and recruitment domain (CARD) and nuclear targeting sequence in addition to other domains.

2. Binding of Aß with AB-DIP was confirmed in SH-SY5Y neuroblastoma cells by two-way coimmunoprecipitations (Fig. 1)

View larger version (36K): [in this window] [in a new window]   Figure 1. Direct interaction of Aß with AB-DIP: pull-down assay. Aß coprecipitates with AB-DIP. A) SHSY5Y cells cotransfected with pCMV-Myc-AB-DIP or pCMV-Myc empty vector along with pCMV-HA-Abeta42. After 36 h, cells were lysed and subjected to immunoprecipitation with anti-myc antibody. B) SH-SY5Y cells cotransfected with pCMV-myc-AB-DIP and pCMV-HA-Aß40. After 36 h, cells were lysed and subjected to immunoprecipitation with anti-myc antibody. Ant-Aß (6E10) antibody was used to detect HA-Aß40 by immunoblot. C) SH-SY5Y cells cotransfected with pCMV-HA-Aß42 and various myc-tagged AB-DIP deletion mutants. After 36 h, cells were lysed and subjected to overnight immunoprecipitation with anti-myc antibody. Aß binds AB-DIP at both the N- and C-terminals, but not the region between amino acids 231–589. D) Reciprocal coimmunoprecipitation. PCMV-HA-Aß42 or pCMV-HA empty vector and pCMV-myc-AB- DIP were cotransfected and subjected to immunoprecipitation with anti-HA or anti-Aß (6E10) antibodies.

Using a series of AB-DIP deletion mutants, we mapped binding of Aß with both the N- and C-terminal regions but not the middle region.

3. Overexpression of AB-DIP-induced cell death and coexpression of Aß enhanced cell death in SH-SY5Y cells (Fig. 2 D)

View larger version (32K): [in this window] [in a new window]   Figure 2. Down-regulation of AB-DIP protein levels protects SH-SY5Y cells from Aß-induced cell death. A)Live cell counts of GFP fluorescence at different days after the transient transfection of EGFP empty vector, EGFP-AB-DIP, EGFP-AB-DIP+HA-Aß42, and EGFP-p53 by fluorescence microscopy. **P< 0.01 when EGFP control was compared with any of the other groups. B) SH-SY5Y cells were transiently transfected with either EGFP control vector (pEGFPN1) or AB-DIP fused to EGFP (pEGFP- AB-DIP); after 24 or 96 h, cells were processed for immunocytochemical staining with polyclonal GFP antibody. C) siRNA- mediated knockdown of AB-DIP protein expression in SH-SY5Y cells. Cells were cotransfected with myc-AB-DIP and either AB-DIP-specific siRNA duplex or luciferase-specific siRNAs. After 48 h, cells were subjected to immunoblot analysis and myc-AB-DIP was detected with anti-myc antibody. D) Number of annexin V-positive cells after transient transfection of SH-SY5Y cells with the indicated plasmids. *P< 0.05 when EGFP-AB-DIP+ HA-Aß42 or EGFP-p53 was compared with EGFP-Aß42; $P < 0.05 when EGFP-AB-DIP+HA-Aß42 was compared with EGFP-AB-DIP by t test (n=3). E) Knockdown of AB-DIP protein expression by siRNAs protects SH-SY5Y cells from Aß toxicity. Aß42 was fused to EGFP (EGFPAß42) and cotransfected with either AB-DIP siRNA duplex, nonspecific siRNAs (siNon), or siRNAs against the luciferase gene (siLuc). Cell death was detected by annexin V staining after 24, 48, and 72 h of transfection. *P < 0.05 compared with pEGFP-42, **P < 0.01 compared with pEGFP42 or pEGFP-42+siNon by t test (n=3).

4. During apoptosis, 97 kDa full-length AB-DIP is cleaved to p62AB-DIP

Coexpression of Aß and AB-DIP also produced p62AB-DIP.

5. Experiments using purified caspases and site-directed mutagenesis of AB-DIP confirmed the cleavage of AB-DIP by caspase-9 at the sequence LEKD

6. siRNA-mediated knockdown of AB-DIP protein expression significantly protected SH-SY5Y cells from Aß-induced cell death (Fig. 2 E)

CONCLUSIONS AND SIGNIFICANCE

Alzheimer’s disease (AD) is characterized by extensive cell death in the brains of affected individuals. There is now genetic, pathological, and biochemical evidence to suggest that Aß plays the casual role in AD. We recently demonstrated immunocytochemical staining of Aß in the cytoplasm as well as the nucleus of many degenerating neurons from AD brains. Such an accumulation of intracellular Aß is relevant to neurodegeneration in AD. Although the pathway leading to Aß generation and accumulation is well defined, specific molecular pathways and obligate mechanisms for Aß-induced neuronal death remain poorly defined. Therefore, we searched for cellular proteins that specifically bind Aß and identified a novel Aß binding protein, AB-DIP. Multiple evidence suggests that AB-DIP may mediate Aß-induced cell death. This conclusion is supported by our finding that coexpression of Aß and AB-DIP produced potentially the activated form, p62AB-DIP, and enhanced the cell death. AB-DIP-specific siRNAs significantly protected neuroblastoma cells from Aß-induced cell death. However, it remains to be verified whether targeting AB-DIP in vivo may have a therapeutic value in AD.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating a suggested mechanism for intracellular Aß-induced cell death. When the cell is in the unperturbed condition, RGD motif bind with DDM motif within AB-DIP masking the caspase cleavage site, LEKD and thus AB-DIP remain in an inactive state. When the intracellular levels of Aß increases, Aß binds to AB-DIP, changing its conformation and exposing LEKD. Caspase-9 then cleaves full-length AB-DIP to p62AB-DIP, which may then enter the nucleus with its nuclear targeting sequence and induce apoptosis through an as yet unknown mechanism.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3672fje

This Article Full Text (PDF) All Versions of this Article: 19/10/1362 05-3672fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lakshmana, M. K. Articles by Tabira, T. Search for Related Content PubMed PubMed Citation Articles by Lakshmana, M. K. Articles by Tabira, T.