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Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

Decock M, El Haylani L, Stanga S, Dewachter I, Octave JN, Smith SO, Constantinescu SN, Kienlen-Campard P - FEBS Open Bio (2015)

Bottom Line: Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques.We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments.Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, Université catholique de Louvain, Brussels 1200, Belgium.

ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

No MeSH data available.


Related in: MedlinePlus

Dimerization of APP and APP C-terminal fragments in living cells measured by the split-luciferase complementation assay. (A) Validation of the luciferase complementation assay for measuring protein dimerization in CHO cells. Cells were transfected with the control empty vector (mock) or the GCN4 leucine zipper–coding sequences fused to hGLuc moieties (Zip-hGluc1 and 2). Expression of the fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody (top). Luciferase activity (bioluminescence) was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock). APP-hGLuc1 and 2 (B), C99-hGLuc1 and 2 (C) or C83-hGLuc1 and 2 proteins (D) were transfected in CHO cells. Protein expression was monitored in cell lysates by Western blotting with the W0-2 and hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock).
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f0010: Dimerization of APP and APP C-terminal fragments in living cells measured by the split-luciferase complementation assay. (A) Validation of the luciferase complementation assay for measuring protein dimerization in CHO cells. Cells were transfected with the control empty vector (mock) or the GCN4 leucine zipper–coding sequences fused to hGLuc moieties (Zip-hGluc1 and 2). Expression of the fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody (top). Luciferase activity (bioluminescence) was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock). APP-hGLuc1 and 2 (B), C99-hGLuc1 and 2 (C) or C83-hGLuc1 and 2 proteins (D) were transfected in CHO cells. Protein expression was monitored in cell lysates by Western blotting with the W0-2 and hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock).

Mentions: We first validated the luciferase complementation approach as a tool to measure protein dimerization in living CHO cells by measuring bioluminescence upon transfection with the GCN4 leucine zipper hGLuc constructs. Western blotting of cell lysates indicated that Zip-hGLuc1 and Zip-hGLuc2 were detected as 17 and 18 kDa bands recognized by the polyclonal anti-hGLuc antibody (Fig. 2A). Transfection of Zip-hGLuc1 and Zip-hGLuc2 alone did not generate any bioluminescent signal whereas co-transfection of both generated high levels of luciferase activity (Fig. 2A). Co-expression of the hGLuc1 and hGLuc2 moieties alone (not fused to any APP protein sequence) did not generate luciferase activity (data not shown). This clearly validated the luciferase complementation approach as a very sensitive tool to measure dimerization in cells.


Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

Decock M, El Haylani L, Stanga S, Dewachter I, Octave JN, Smith SO, Constantinescu SN, Kienlen-Campard P - FEBS Open Bio (2015)

Dimerization of APP and APP C-terminal fragments in living cells measured by the split-luciferase complementation assay. (A) Validation of the luciferase complementation assay for measuring protein dimerization in CHO cells. Cells were transfected with the control empty vector (mock) or the GCN4 leucine zipper–coding sequences fused to hGLuc moieties (Zip-hGluc1 and 2). Expression of the fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody (top). Luciferase activity (bioluminescence) was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock). APP-hGLuc1 and 2 (B), C99-hGLuc1 and 2 (C) or C83-hGLuc1 and 2 proteins (D) were transfected in CHO cells. Protein expression was monitored in cell lysates by Western blotting with the W0-2 and hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0010: Dimerization of APP and APP C-terminal fragments in living cells measured by the split-luciferase complementation assay. (A) Validation of the luciferase complementation assay for measuring protein dimerization in CHO cells. Cells were transfected with the control empty vector (mock) or the GCN4 leucine zipper–coding sequences fused to hGLuc moieties (Zip-hGluc1 and 2). Expression of the fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody (top). Luciferase activity (bioluminescence) was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock). APP-hGLuc1 and 2 (B), C99-hGLuc1 and 2 (C) or C83-hGLuc1 and 2 proteins (D) were transfected in CHO cells. Protein expression was monitored in cell lysates by Western blotting with the W0-2 and hGluc antibodies (top panels). Luciferase activity was measured and expressed as RLU (bottom). Values (means ± SEM) are representative of 3 independent experiments (n = 4 in each experiment). ***p < 0.0001, as compared to control (mock).
Mentions: We first validated the luciferase complementation approach as a tool to measure protein dimerization in living CHO cells by measuring bioluminescence upon transfection with the GCN4 leucine zipper hGLuc constructs. Western blotting of cell lysates indicated that Zip-hGLuc1 and Zip-hGLuc2 were detected as 17 and 18 kDa bands recognized by the polyclonal anti-hGLuc antibody (Fig. 2A). Transfection of Zip-hGLuc1 and Zip-hGLuc2 alone did not generate any bioluminescent signal whereas co-transfection of both generated high levels of luciferase activity (Fig. 2A). Co-expression of the hGLuc1 and hGLuc2 moieties alone (not fused to any APP protein sequence) did not generate luciferase activity (data not shown). This clearly validated the luciferase complementation approach as a very sensitive tool to measure dimerization in cells.

Bottom Line: Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques.We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments.Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, Université catholique de Louvain, Brussels 1200, Belgium.

ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

No MeSH data available.


Related in: MedlinePlus