At the foundation of the remarkable complexity of all organisms, lays an intricate network of interactions between their cellular components – proteins, nucleic acids, sugars, and other molecules. Functions such as growth and development, reproduction, and responses to pathogens and environmental stresses are ultimately the result of constant assembly and disassembly of macromolecular complexes within cells. Recent advances in proteomics and the availability of a large volume of genomics data has opened the possibility to initiate comprehensive studies of cellular processes through the prism of functional networks.
We are interested to study the function of proteins and how they assemble in complexes and pathways to carry on biological processes. Specifically, we study signal transduction pathways and gene regulatory mechanisms in the context of plant biotic interactions. For this, we have developed functional protein microarrays for the model plant system Arabidopsis thaliana (Popescu , Popescu 2007, Popescu et al. 2007).
Protein microarrays represent a novel technology for the unbiased, large scale characterization of molecular interactions. They have been used for a wide variety of applications such as the study of enzyme-substrate, protein-protein, and protein-nucleic acid interactions, profiling antibody specificity, and searching for protein posttranslational modifications.
To generate functional protein microarrays (FPAs) we employ the recently developed Arabidopsis tagged ORF expression collection (ATEC). At the moment ATEC contains approximately one third of the Arabidopsis genome, in two years it will encompass more than half of the genome. To produce purified proteins for microarray printing we are using the N. benthamiana transient expression system, optimized for large-scale protein production. FPAs once generated in our lab, are used in various types of biochemical assays. Our work has been featured in the Journal of Proteome Research (Cottingham, K., 2007) and Analytical Chemistry (Griffiths, J., 2007).
Analysis of CaM and CML substrates on FPAs
almodulin (CaM) represents an essential regulatory protein conserved in all eukaryotes, which binds calcium and controls fundamental biological processes. Plants contain a large family of calmodulins and CaM-like proteins (CMLs) with functions in stress response and development.
Using FPAs with over 1,000 protein preparations, we have identified substrates of seven Arabidopsis CaM and CML protein isoforms. Several of the previously known substrates and a number of novel CaM-interacting proteins were found to interact with CaMs on FPAs. Our work generated the first experimentally-based plant protein interaction network that will aid in formulating new hypothesis on calmodulins role in the cell (Popescu, Popescu, Bachan, Zhang, Seay, Gerstein, Snyder and Dinesh-Kumar 2007). Currently we are expanding this analysis to include other plant proteins with roles in signal transduction.
Identification of signaling components in MAP kinase pathways
In a recent work, we have used FPAs containing more than 2000 proteins to study signaling through mitogen-activated protein kinases (MPKs). With over 60 MKKKs, 20 MKKs and at least 20 MPKs, the signaling transduction networks in Arabidopsis are immensely complex. To decipher them, we need to understand how signaling molecules associate with one another to transmit information and to identify the downstream substrates that are targeted by these pathways. These problems have been difficult to resolve because of cellular interactions, and the large number of genes with overlapping or redundant functions.
Our study revealed known and new signaling modules encompassing 570 MPK phosphorylation substrates for ten activated MPKs. MPK phosphorylated mostly transcription factors that regulate development, defense and stress responses (Fig. 1). We identified a subset of activated and wild type MKKs that induced HR-like death indicating a possible role for these MKKs in the regulation of cell death. The MPK phosphorylation network generated in this study is a comprehensive resource to experimentally analyze MPK signaling systems (Fig. 2) (Popescu 2009).
Popescu, G. and Popescu, S.C. 2011. Complexity and modularity of MAPK signaling networks. In Handbook of Research on Computational and Systems Biology:. Interdisciplinary Applications (Limin, D.W., Liu, A. and Li, Y. eds) 0: IGI Global, pp. 355-368
Lee, H.Y., Bowen, C.H., Popescu, G.V., Kang, H.G., Kato, N., Ma, S., Dinesh-Kumar, S., Snyder, M. and Popescu, S.C. 2011. Arabidopsis RTNLB1 and RTNLB2 Reticulon-like proteins regulate intracellular trafficking and activity of the FLS2 immune receptor. Plant Cell 23: 3374-3391
Caspi, R., Altman, T., Dale, J.M., Dreher, K., Fulcher, C.A., Gilham, F., Kaipa, P., Karthikeyan, A.S., Kothari, A., Krummenacker, M., Latendresse, M., Mueller, L.A., Paley, S., Popescu, L., Pujar, A., Shearer, A.G., Zhang, P. and Karp, P.D. 2010. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Research 38: D473-D479
Popescu, S.C., Popescu, G.V., Snyder, M. and Dinesh-Kumar, S.P. 2009. Integrated analysis of co-expressed MAP kinase substrates in Arabidopsis thaliana. Plant Signal Behav 4: 524-527
Popescu, S., Snyder Michael, Dinesh-Kumar, Savithramma. 2007. Arabidopsis Protein Microarrays for the High-Throughput Identification of Protein-Protein Interactions. Plant Signaling & Behaviour 2: 415-419
Popescu, S.C., Popescu, G.V., Bachan, S., Zhang, Z., Seay, M., Gerstein, M., Snyder, M. and Dinesh-Kumar, S.P. 2007. Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays 10.1073/pnas.0611615104. PNAS 104: 4730-4735
Popescu S. C., Tumer, N. E.. 2004. Silencing of ribosomal protein L3 genes in N. tabacum reveals coordinate expression and significant alterations in plant growth, development and ribosome biogenesis. The Plant Journal 39: 29-44
Which proteins control which biological processes in plants?
feature released -2008
Proteins are the workers that participate in every process within cells, such as biochemical reactions (metabolism), structure (building and maintaining the shape of the cell) and defense mechanisms (disease or insect resistance), among others. Identifying all the proteins in a plant cell and determining the function of each is a major area of plant biology research.
Until recently, identifying and isolating these proteins and then using them to discover their specific function has been an arduous task. But as a result of discoveries made by Sorina Popescu, the work has become easier and more efficient. Popescu enhanced a technique called the microarray, which was previously used only to study DNA, and adapted it to be used for the study of proteins.
Working with Arabidopsis proteins, Popescu discovered that she could “print” as many as 5,000 minute protein samples on one microscope slide (the microarray) and that these tiny amounts of protein could then be used in other research that would reveal the function of each. There are about 30,000 proteins in Arabidopsis, which means that the entire proteome (the complete protein library) of this plant could be stored on six standard microscope slides.
Using her technique, Popescu and other scientists will be able to more efficiently study protein function on a very large scale. Prior to the development of protein microarray technology, scientists were able to select only a few proteins at a time for study, which restricted their ability to identify which proteins are involved in which processes. Now, Popescu and others can easily study thousands of proteins — even those no one thought were involved in a particular process. As a result, her work has significantly expanded the universe of study involving proteins and their functions.
Popescu plans to use her protein microarray technology at BTI to identify proteins involved in protecting plants from various diseases. Her work could lead to the identification of proteins that were never before thought to be involved in disease resistance, which, in turn, could lead to a new understanding of how plant defense mechanisms work. It could also lead to new ways to protect plants from disease and enhance their yields.