EXPERT OPINION nologies are similar, however MaMTH will allow for testing of human membrane proteins in the context of human cell lines. Our initial proof of concept experiments were a success and we are currently in the process of upscaling MaMTH to a high-throughput screening format by building cDNA libraries that contain all 21,000 human Open Reading Frames (ORFs). The groundbreaking aspect of this technology is that it can be used to study PPIs in a drug and/or agonist dependent manner in the context of the human cell, which has the potential to change the way drug screening is performed in industry.
Figure 1. Interactive proteomics is a subdiscipline of proteomics whose goal is to map all protein-protein interactions of a given cell or organism. Here, the interactome of the 50 selected human G-protein coupled receptors is shown (recent unpublished work from the Jurisica & Stagljar lab). the observation that dysfunction of these transporters is associated with a range of human diseases. Similarly, we are also working on building a comprehensive interaction map between the human receptor tyrosine kinase (RTKs) and selected human G-protein coupled receptors (GPCRs). These two families of membrane proteins play a crucial role in cell signaling, a process by which cells respond to cues in their internal or external environment. The finished interactomes of RTKs and GPCRs represent a robust bank of knowledge which will greatly contribute to therapeutic research and shed new light on the mechanism of natural control circuits that regulate biological systems. As an example, one of our recent findings showed that a
protein called HDAC6 regulates degradation of an RTK called Epidermal Growth Factor (EGFR), a receptor that is overactive in several human cancers5. Based on this work, we are investigating the possibility of treating those cancers in which EGFR is involved by using drugs to inhibit HDAC6, thereby speeding up degradation of the oncogenic EGFR protein. In keeping with the tradition of our research group, we are still active in developing new technologies which can further the field of interactive proteomics. Recently, we have been working on developing a mammalian version of MYTH named MaMTH. The fundamentals of the yeast-based and mammalian tech-
Figure 2. An example of a positive (upper row) and negative (lower row) interaction detected by MYTH. Proteins X and Y are synthesized in yeast and tested for interaction via MYTH by assessing their growth on specific media. The blue staining and growth of yeast cells on specific media are indicative of two proteins interacting in MYTH.
In summary, although membrane proteins play a very crucial role in maintaining a healthy cell state, and dysfunction of membrane proteins has been linked to a plethora of diseases, there are still huge gaps remaining in our knowledge and understanding of this group of proteins. MYTH and MaMTH can be used to fill in this gap, which in turn will have great implications in our understanding of various diseases and how to better create effective treatments for them.
References 1. Stagljar, I. and Fields, S. (2002) Analysis of membrane protein interactions using yeast-based technologies. Trends Biochem Sci 27, 559-563. 2. Thaminy, S., Auerbach, D., Arnoldo, A., and Stagljar, I., (2003) Identification of novel ErbB3- interacting factors using the split-ubiquitin membrane yeast two-hybrid system, Genome Res 13, 1744–1753. 3. Paumi, C.M., Menendez, J., Arnoldo, A., Engels, K., Iyer, K., Thaminy, S., Georgiev, O., Barral, Y., Michaelis, S., and Stagljar, I. (2007) Mapping Protein-Protein Interactions for the Yeast ABC Transporter Ycf1p by Integrated Split-Ubiquitin Membrane Yeast Two-Hybrid (iMYTH) Analysis, Mol Cell 26, 15-25. 4. Gisler, S.M., Kittanakom, S., Fuster, D., Radanovic, T., Wong, V., Bertic, M., Hall, R.A., Engels, K., Murer, H., Biber, J., Markovic, D., Moe, O.W., and Stagljar, I. (2008) Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system, Mol Cell Proteomics 7, 1362-1377. 5. Deribe, Y. L., Wild, P., Chandrashaker, A., Curak, J., Schmidt, M. H., Kalaidzidis, Y., Milutinovic, N., Kratchmarova, I., Buerkle, L., Fetchko, M. J., Schmidt, P., Kittanakom, S., Brown, K. R., Jurisica, I., Blagoev, B., Zerial, M., Stagljar, I., and Dikic, I., (2009) Regulation of epidermal growth factor receptor trafficking by lysine deacetylase HDAC6, Sci Signal 2, p. ra84. 6. Usenovic, M., Knight, A. L., Raj, A., Wong, V., Brown, K. R., Caldwell, G. A., Caldwell,K. A., Stagljar, I., Krainc, D. (2012) Identification of novel ATP13A2 interactors and their role in ι-synuclein misfolding and toxicity, Hum Mol Genet, in press (PMID: 22645275).
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