The involvement of the ceramide transporter in Alzheimer’s disease Investigating the binding properties of the ceramide transporter in the human brain by optimizing the splitubiquitin Yeast Two-Hybrid system

  • A.M.G.S. Jansen


Background: Alzheimer’s disease (AD) is the most common form of dementia, characterized by aggregates of Amyloid-ß (Aβ) peptides. The ceramide transporter (CERTL ) exists in a complex with serum amyloid P component in the Aβ plaques of AD patients. The goal is to obtain more information about the protein binding activity of CERTL in the human brain. To be able to do so, the Yeast Two-Hybrid (Y2H) technique, that was used to confirm the results from a previously executed CERTL Y2H screening, had to be optimized. Technical problems were encountered with the plasmid purification of the yeast NMY51 using the EZNA plasmid DNA minikit I and with the transformation in competent E.coli cells. Initially it was believed that there was a problem with the EZNA plasmid DNA minikit I. In this report troubleshooting was performed to identify the problem of the plasmid purification technique that has been used. Methods: To examine the protein binding activity of CERTL a split-ubiquitin Y2H system was used. In this system proteins are attached to two parts of one complete ubiquitin molecule, called the split-ubiquitin. The middle region domain of CERTL (the bait) was attached to the C-terminal half of the split-ubiquitin and the transcription factor LexA-VP16. The possible interactor, a prey library containing human brain proteins was fused to the N-terminal half of the split-ubiquitin. The presence of an interaction between bait and prey will results in the re-assembly of the split-ubiquitin, which activates ubiquitin proteases. The transcription factor is released and reporter genes are activated.The results of a previously executed Y2H screening had to be confirmed by performing another Y2H screening. The new screening had to be carried out using glycerol stocks of the yeast NMY51, containing both bait and prey, from the earlier performed Y2H screening. To optimize prey plasmid purification from the yeast using the EZNA plasmid DNA minikit I, troubleshooting was performed on the technique. Next to this, other methods to isolate the plasmid DNA, a QIAprep spin miniprep kit protocol and a phenol extraction and ethanol precipitation, were performed. The failing results were possibly due to the yeast being stored as a glycerol stock. In order to correct for this, a freshly transformed yeast sample was made. Results: Extracting the plasmid from glycerol stocks from the yeast NMY51 did not succeed. Plasmid purification and transformation of the prey plasmid from the fresh transformed yeast in E.coli competent cells was successful. Conclusion: From the results it is now believed that storing the yeast NMY51 containing the prey plasmid in glycerol stocks causes the prey plasmid to integrate into the genomic DNA of the yeast. Performing a new Y2H screening would therefore only be possible on fresh transformed yeast NMY51 cells.


Mayeux R. Epidemiology of neurodegeneration. Annu Rev Neurosci. 2003;26:81-104. 2.

Association As. Alzheimer’s Disease Facts and Figures. 2014.

Cutler RG, Kelly J, Storie K, Pedersen WA, Tammara A, Hatanpaa K, et al. Involvement of oxidative stressinduced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer’s disease. Proc Natl Acad Sci U S A. 2004;101(7):2070-5.

Puglielli L, Ellis BC, Saunders AJ, Kovacs DM. Ceramide stabilizes beta-site amyloid precursor proteincleaving enzyme 1 and promotes amyloid beta-peptide biogenesis. J Biol Chem. 2003;278(22):19777-83.

Mencarelli C, Martinez-Martinez P. Ceramide function in the brain: when a slight tilt is enough. Cell Mol Life Sci. 2013;70(2):181-203.

Mencarelli C, Losen M, Hammels C, De Vry J, Hesselink MK, Steinbusch HW, et al. The ceramide transporter and the Goodpasture antigen binding protein: one protein--one function? J Neurochem. 2010;113(6):1369-86.

Möckli N, Deplazes A, Auerbach D. Finding new protein interactions using the DUALhunter system. Nature Methods. 2008 Feb.

Biotech D. DUALhunter starter kit user manual (P01601-P01629).

Karpati A. Identification of novel protein-protein interactions of the ceramide transporter in the human brain [Junior Thesis]. Maastricht: Maastricht University; 2014.

OMEGAbio-tek. Product Manual E.Z.N.A.® HP Plasmid DNA Mini Kit I2013.

Nori DV, McCord BR. The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs. Anal Bioanal Chem. 2015.

Technologies N. 260/280 and 260/230 Ratios NanoDrop® ND-1000 and ND-8000 8-Sample Spectrophotometers2007.

Clontech Laboraties I. Yeast Protocols Handbook. 2009.

Bergman L. Two-Hybrid Systems. 2001. In: Methods and Protocols [Internet].