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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

Schematic representation of the different APP split-luciferase constructs. (A) Schematic representation of the different human APP and APP C-terminal fragments generated for fusion to the humanized Gaussia luciferase moieties (hGLuc). APPΔC corresponds to APP695 deleted from its intracellular C-terminal domain (stop after the KKKQY intracellular sequence). C99 and C83 correspond to the APP β and α C-terminal fragments, respectively. All the N-terminally truncated CTFs are fused to the APP signal peptide (SP). Abbreviations are as follows: TM, transmembrane; JM, juxtamembrane; AICD, APP intracellular domain; ext, extracellular; int, intracellular. The positions of Flemish and mutant 5 (mut5) mutations are underlined and amino acid substitutions are in red. The cleavage sites of α (α)-, β (β)- and γ (γ and ε)-secretases are indicated by arrows. (B) Schematic representation of APP constructs fused to hGLuc moieties (hGLuc1 and hGLuc2). The epitopes of the human-specific W0-2 antibody, the APP C-terminal and hGLuc antibodies are indicated.
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f0005: Schematic representation of the different APP split-luciferase constructs. (A) Schematic representation of the different human APP and APP C-terminal fragments generated for fusion to the humanized Gaussia luciferase moieties (hGLuc). APPΔC corresponds to APP695 deleted from its intracellular C-terminal domain (stop after the KKKQY intracellular sequence). C99 and C83 correspond to the APP β and α C-terminal fragments, respectively. All the N-terminally truncated CTFs are fused to the APP signal peptide (SP). Abbreviations are as follows: TM, transmembrane; JM, juxtamembrane; AICD, APP intracellular domain; ext, extracellular; int, intracellular. The positions of Flemish and mutant 5 (mut5) mutations are underlined and amino acid substitutions are in red. The cleavage sites of α (α)-, β (β)- and γ (γ and ε)-secretases are indicated by arrows. (B) Schematic representation of APP constructs fused to hGLuc moieties (hGLuc1 and hGLuc2). The epitopes of the human-specific W0-2 antibody, the APP C-terminal and hGLuc antibodies are indicated.

Mentions: To analyze human APP dimerization and the contribution of its extracellular, juxtamembrane/transmembrane (JM/TM) and intracellular (AICD) domains to this process, we generated vectors expressing the full-length and truncated APP proteins (Fig. 1A) fused to complementary humanized Gaussia luciferase (hGluc) fragments referred to as hGluc1 and hGluc2 corresponding to N-terminal and C-terminal moieties, respectively. C99 and C83 correspond to β- and α-secretase cleavage products fused to the APP signal peptide. APPΔC corresponds to the APP protein truncated after the KKKYQ sequence at the TM/intracellular interface. To analyze the role of membrane GXXXG motifs in dimerization, we generated 2 mutants modifying the interactive properties of the GXXXG interface [19,24]. The first mutation corresponds to the FAD Flemish mutation (A617G, APP695 numbering or A21G, Aβ numbering). It extends the GXXXG interface and helical structure of the surrounding residues [29]. The double GG625/629LL mutant (GG29/33LL, Aβ numbering) hereafter referred as mutant 5 (mut5) carries glycine to leucine mutations of the central GXXXG motif. These mutations have been previously reported to affect interaction of APP TM helices and strongly impair amyloidogenic processing [19]. As a positive control, we used yeast transcription factor GCN4 leucine zipper fusion proteins (Zip-hGLuc1 and Zip-hGLuc2) [37]. The leucine zipper of GCN4 is a strong dimerization domain. All the constructs generated for this study are represented in Fig. 1A and B.


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)

Schematic representation of the different APP split-luciferase constructs. (A) Schematic representation of the different human APP and APP C-terminal fragments generated for fusion to the humanized Gaussia luciferase moieties (hGLuc). APPΔC corresponds to APP695 deleted from its intracellular C-terminal domain (stop after the KKKQY intracellular sequence). C99 and C83 correspond to the APP β and α C-terminal fragments, respectively. All the N-terminally truncated CTFs are fused to the APP signal peptide (SP). Abbreviations are as follows: TM, transmembrane; JM, juxtamembrane; AICD, APP intracellular domain; ext, extracellular; int, intracellular. The positions of Flemish and mutant 5 (mut5) mutations are underlined and amino acid substitutions are in red. The cleavage sites of α (α)-, β (β)- and γ (γ and ε)-secretases are indicated by arrows. (B) Schematic representation of APP constructs fused to hGLuc moieties (hGLuc1 and hGLuc2). The epitopes of the human-specific W0-2 antibody, the APP C-terminal and hGLuc antibodies are indicated.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588712&req=5

f0005: Schematic representation of the different APP split-luciferase constructs. (A) Schematic representation of the different human APP and APP C-terminal fragments generated for fusion to the humanized Gaussia luciferase moieties (hGLuc). APPΔC corresponds to APP695 deleted from its intracellular C-terminal domain (stop after the KKKQY intracellular sequence). C99 and C83 correspond to the APP β and α C-terminal fragments, respectively. All the N-terminally truncated CTFs are fused to the APP signal peptide (SP). Abbreviations are as follows: TM, transmembrane; JM, juxtamembrane; AICD, APP intracellular domain; ext, extracellular; int, intracellular. The positions of Flemish and mutant 5 (mut5) mutations are underlined and amino acid substitutions are in red. The cleavage sites of α (α)-, β (β)- and γ (γ and ε)-secretases are indicated by arrows. (B) Schematic representation of APP constructs fused to hGLuc moieties (hGLuc1 and hGLuc2). The epitopes of the human-specific W0-2 antibody, the APP C-terminal and hGLuc antibodies are indicated.
Mentions: To analyze human APP dimerization and the contribution of its extracellular, juxtamembrane/transmembrane (JM/TM) and intracellular (AICD) domains to this process, we generated vectors expressing the full-length and truncated APP proteins (Fig. 1A) fused to complementary humanized Gaussia luciferase (hGluc) fragments referred to as hGluc1 and hGluc2 corresponding to N-terminal and C-terminal moieties, respectively. C99 and C83 correspond to β- and α-secretase cleavage products fused to the APP signal peptide. APPΔC corresponds to the APP protein truncated after the KKKYQ sequence at the TM/intracellular interface. To analyze the role of membrane GXXXG motifs in dimerization, we generated 2 mutants modifying the interactive properties of the GXXXG interface [19,24]. The first mutation corresponds to the FAD Flemish mutation (A617G, APP695 numbering or A21G, Aβ numbering). It extends the GXXXG interface and helical structure of the surrounding residues [29]. The double GG625/629LL mutant (GG29/33LL, Aβ numbering) hereafter referred as mutant 5 (mut5) carries glycine to leucine mutations of the central GXXXG motif. These mutations have been previously reported to affect interaction of APP TM helices and strongly impair amyloidogenic processing [19]. As a positive control, we used yeast transcription factor GCN4 leucine zipper fusion proteins (Zip-hGLuc1 and Zip-hGLuc2) [37]. The leucine zipper of GCN4 is a strong dimerization domain. All the constructs generated for this study are represented in Fig. 1A and B.

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