Strategies for examination of Alzheimer’s disease amyloid precursor protein isoforms

Newton, Jillian R. A.; Parkinson, David; Clench, Malcolm R.
July 2006
Analytical & Bioanalytical Chemistry;Jul2006, Vol. 385 Issue 4, p692
Academic Journal
We describe a proteomics procedure using bioinformatics, immunoprecipitation, two-dimensional gel electrophoresis, Western blotting, in-gel digestion, LC–MS, MALDI–MS, and MS–MS for isolation and identification of amyloid precursor protein (APP) isoforms APP695, APP751, and APP770. Retinoic acid-induced Ntera 2 cell line, derived from a human teratocarcinoma cells, was the in-vitro source of APP. Initial isolation of whole APP was performed by immunoprecipitation, using AB10, a monoclonal antibody raised to amino acids 1–17 of the β-amyloid peptide sequence, which is present in all three alpha secretase-cleaved isoforms of interest. The next stage was separation of whole APP into its isoform components by two-dimensional gel electrophoresis. Because of low APP concentrations, detection by the usual staining methods, for example Sypro Ruby, able to detect low picomole concentrations, did not enable visualisation of the isoforms. Western analysis, however, enabled primary detection of APP, because of the inherent sensitivity of antibodies raised to specific isoform regions. This initial visualization acted as a template for excision of isoforms from 2D gels, which were then subjected to peptide mass mapping. Initial theoretical digestion of each isoform revealed the presence of specific peptides, which were then used as “tags” for isoform detection.


Related Articles

  • Impairment of Adolescent Hippocampal Plasticity in a Mouse Model for Alzheimer's Disease Precedes Disease Phenotype. Hartl, Daniela; Rohe, Michael; Mao, Lei; Staufenbiel, Matthias; Zabel, Claus; Klose, Joachim // PLoS ONE;2008, Vol. 3 Issue 7, p1 

    The amyloid precursor protein (APP) was assumed to be an important neuron-morphoregulatory protein and plays a central role in Alzheimer's disease (AD) pathology. In the study presented here, we analyzed the APP-transgenic mouse model APP23 using 2-dimensional gel electrophoresis technology in...

  • Proteomic Profiling of γ-Secretase Substrates and Mapping of Substrate Requirements. Hemming, Matthew L.; Elias, Joshua E.; Gygi, Steven P.; Selkoe, Dennis J. // PLoS Biology;Oct2008, Vol. 6 Issue 10, p2314 

    The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β...

  • Changing the Course of Alzheimer's Disease: Emerging Disease Modifying Therapies. Christensen, Daniel D. // Current Psychiatry Reviews;2006, Vol. 2 Issue 2, p179 

    Drugs that prevent the onset, delay the progression, or eliminate the symptoms of Alzheimer's disease are urgently needed. Current neurotransmitter-based treatments are widely used and provide modest symptom relief. The amyloid hypothesis in which the pathological processing of amyloid precursor...

  • Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family. Cam, Judy A.; Guojun Bu // Molecular Neurodegeneration;2006, Vol. 1, p8 

    Amyloid-β peptide (Aβ) accumulation in the brain is an early, toxic event in the pathogenesis of Alzheimer's disease (AD). Aβ is produced by proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP), by β- and γ-secretases. Mounting evidence has...

  • Phagocytosis and LPS alter the maturation state of β-amyloid precursor protein and induce different Aβ peptide release signatures in human mononuclear phagocytes. Spitzer, Philipp; Herrmann, Martin; Klafki, Hans-Wolfgang; Smirnov, Alexander; Lewczuk, Piotr; Kornhuber, Johannes; Wiltfang, Jens; Maler, Juan Manuel // Journal of Neuroinflammation;2010, Vol. 7, p59 

    Background: The classic neuritic b-amyloid plaque of Alzheimer's disease (AD) is typically associated with activated microglia and neuroinflammation. Similarly, cerebrovascular β-amyloid (Aβ) deposits are surrounded by perivascular macrophages. Both observations indicate a contribution of...

  • A firm base for drug development. de Strooper, Bart; Konig, Gerhard // Nature;12/2/1999, Vol. 402 Issue 6761, p471 

    Reports on scientific findings on Alzheimer's disease. Discovery of disease-causing mutations in the amyloid precursor protein and presenilin genes; Illustration of the proposed mechanism by which the amyloid beta peptide is generated through cleavage by beta-site amyloid precursor...

  • The Resveratrol Trimer Miyabenol C Inhibits β-Secretase Activity and β-Amyloid Generation. Hu, Jin; Lin, Ting; Gao, Yuehong; Xu, Junyue; Jiang, Chao; Wang, Guanghui; Bu, Guojun; Xu, Huaxi; Chen, Haifeng; Zhang, Yun-wu // PLoS ONE;Jan2015, Vol. 10 Issue 1, p1 

    Accumulation and deposition of amyloid-β peptide (Aβ) in the brain is a primary cause of the pathogenesis of Alzheimer’s disease (AD). Aβ is generated from amyloid-β precursor protein (APP) through sequential cleavages first by β-secretase and then by γ-secretase....

  • The Ability of Tolfenamic Acid to Penetrate the Brain: A Model for Testing the Brain Disposition of Candidate Alzheimer's Drugs Using Multiple Platforms. Subaiea, Gehad M.; Alansi, Bothaina H.; Serra, David A.; Alwan, Maged; Zawia, Nasser H. // Current Alzheimer Research;Dec2011, Vol. 8 Issue 8, p860 

    Evidence from our laboratory suggests that tolfenamic acid has a potential for slowing the progression of Alzheimer's disease (AD) through lowering cortical levels of the �-amyloid precursor protein (APP) and its pathogenic amyloid beta (A�) intermediates [1]. In this study, we...

  • Bisecting GlcNAc modification stabilizes BACE1 protein under oxidative stress conditions. Yasuhiko Kizuka; Miyako Nakano; Shinobu Kitazume; Takashi Saito; Saido, Takaomi C.; Naoyuki Taniguchi // Biochemical Journal;1/1/2016, Vol. 473 Issue 1, p21 

    β-Site amyloid precursor protein-cleaving enzyme-1 (BACE1) is a protease essential for amyloid-β (Aβ) production inAlzheimer's disease (AD). BACE1 protein is known to be up-regulated by oxidative stress-inducing stimuli but the mechanism for this upregulation still needs to be...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics