Interactive proteomics with MYTH: identifying protein puzzles and putting them into their correct cellular pathway
By: Igor Stagljar, PhD
Every process in a cell is affected by interactions between proteins, which determine everything from the cell shape to the function of a particular biochemical pathway. Just as we tailor our own conversations depending on setting, proteins exhibit many different modes of interaction. Long-term protein-protein interactions (PPIs) result in protein complexes, while briefer protein liaisons may lead to a range of possible chemical modifications. Because they are so integral to the physiological function of any organism, PPIs are essential to many lines of research, both basic and clinical.
In the past decade, scientists working in the emerging field of “interactive proteomics” have initiated numerous projects to build comprehensive maps of all PPIs (also called “interactomes”) of a given cell or organism with the ultimate goal of understanding the functions of the many proteins whose roles are not yet known (Figure 1). This is based upon the principle that if two proteins interact with each other they very likely participate in the same or related cellular functions (the principle of “guilt by association”). In other words, clues about the function of one protein whose role is not understood can be gained by observing that it interacts with another protein whose function is known. Such an approach, when applied on a large-scale with all proteins of a certain organism, can result in the identification of novel components of previously known pathways, or, vice versa, one may conclude that a protein previously known to be involved in one biological pathway also functions in another.
A special focus of our laboratory is on proteins associated with biological membranes, also called membrane proteins, which total approximately one third of all proteins in any cell (1). These proteins mediate a wide range of fundamental biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, and cell adhesion. Interestingly, many of these membrane proteins have been found to have disease-associated function, and notably most of the drugs on the market today are targeted towards membrane proteins. As such, there is a strong demand from both academic researchers and biotech/pharma companies to gain further insight into pathways and interactions involving membrane proteins. It is therefore of utmost importance to build a comprehensive interactome of this crucial class of proteins. However, despite extensive research in the past decade, there is a lack of in-depth understanding of PPIs associated with this class of proteins because of their unique biochemical features and enormous complexity. This is a major obstacle for designing improved and more targeted therapies, and importantly, understanding the biology of deregulation of these integral membrane proteins which leads to numerous human diseases.
Previously, our lab developed the Membrane Yeast Two-Hybrid (MYTH) system, a powerful proteomic tool for identifying the interactors of membrane proteins in an in vivo setting using baker’s yeast as a model organism (Figure 2). Using the MYTH system, an interaction between two proteins can be converted into an ‘observable signal’, specifically the growth, and blue coloration, of yeast on a specialized media. A unique advantage of MYTH is that it can detect proteins associated with almost any membrane protein in a high-throughput screening format, and is therefore perfectly suited for building interactomes of this difficult class of proteins. Since the development of MYTH, it has been applied successfully to identify both transient and stable interactions among various membrane proteins from yeast, plant, fly, worm, and humans (2-5). It became a valuable proteomic tool by creating a bridge between the basics of protein biochemistry and practical applications in the field of medicine and disease management. Along this notion, MYTH has recently been applied to ATP13A2, a human lysosomal membrane protein involved in Kufor-Rakeb syndrome characterized by early-onset parkinsonism, neurodegeneration and dementia. MYTH identified dozens of ATP13A2-associated proteins many of which showed to be functionally and mechanistically interconnected to ATP13A2 (6). This and other studies showed that MYTH is an interesting translational research tool, allowing researchers to study the interactions of a disease-associated protein of interest in both the presence and absence of drugs and observe how these compounds affect the protein’s interaction profile. This information, in turn, can be used to help understand how these compounds affect the physiology of diseased cells.
Our lab has currently been involved in using MYTH and variations of MYTH to investigate various key areas of research that have direct disease relevance. For example, one large on-going project in the lab is the study of ABC transporters (3), a major class of proteins responsible for the transport of a wide range of substances across various cellular membranes. ABC transporters are of intense clinical interest because of the key role they play in the multidrug resistance of pathogenic microorganisms and tumor cells, as well 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 (Figure 3) showed that a protein called HDAC6 regulates degradation of an RTK called Epidermal Growth Factor (EGFR), a receptor that is overactive in several human cancers (5). 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 technologies 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.
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.
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 α-synuclein misfolding and toxicity, Hum Mol Genet, in press (PMID: 22645275).