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U of Leicester s Nasim on a New Assay to Measure Protein-Protein Interactions in Mammalian Cells

Talat Nasim
Research Fellow
Department of Genetics, University
of Leicester, UK

At A Glance

Name: Talat Nasim

Position: Research Fellow, Department of Genetics, University of Leicester, UK

Background: Research Associate, University of Leicester, 2000-2004; PhD, biochemistry and applied molecular biology, University of Manchester Institute of Science and Technology, UK, 2000.

Talat Nasim has been working in the area of regulation of gene expression since 1994. Recently, he co-developed a technique for measuring protein-protein interactions in mammalian cells, work that was published in the most recent issue of Nucleic Acids Research [Vol. 33, No. 7, 2005]. Although the technique was meant to provide a means to better understand the interaction between gene and protein in gene regulation, the assay may also have application in the area of drug discovery. Last week, Nasim discussed his work with Cell-Based Assay News.

You've been involved primarily in gene-expression research for most of your career. How did you become interested in developing a method for measuring protein-protein interactions in live cells?

Given the vast number of genes, even in simple organisms gene expression is complicated. Work over the last two decades has simplified our understanding about the mechanisms that regulate gene expression. Regulation of gene expression occurs at three levels: transcriptional, post-transcriptional, and translational. A network of interactions between nucleic acid and protein regulate each of these steps. Since I have been working in the area of gene expression, I experienced these interactions and became interested in developing assays by which the steps involved in regulating gene expression can easily be studied. In early 2001, I designed reporter assays based on enzymatic and fluorescence activities to determine the efficiency of RNA processing activities in mammalian cells. In fact, these assays formed the basis of the present assay to determine the efficiency of protein-protein interaction in mammalian cells.

Your method is based on the classic yeast two-hybrid system, which you say in the paper has limitations. What are some of those limitations?

The yeast two-hybrid system has widely been used to screen prey expression libraries for proteins that interact with a bait protein. The major limitations of this technique are false positives, or interactions that are difficult to validate; and false negatives, or interactions that are not detected. The bait proteins that alone repress or activate the expression of the reporter gene can also be problematic. Many mammalian proteins are not folded correctly and some — for example selenoproteins — do not express in yeast.

How does your technique address these limitations?

To address these limitations we have developed this system in mammalian cells. Since this system is developed in mammalian cells, the problems for folding and expression are limited. The novelty of this system is that it utilizes two autonomous units of gene expression. The upstream unit is expressed regardless of protein-protein interactions. In the absence of interacting proteins the downstream unit is switched off. In the event of an interaction, the downstream expression unit is activated leading to dual reporter readouts. Thus, the ratio of two reporter activities provides a measure to determine the efficiency of protein-protein interaction. Although we have not encountered the problems of autoactivation of library screening, it is unlikely to undermine the usefulness of this method as long as a second method — for example immunoprecipitation — is used to validate the most interesting candidates.

Are there particular types of protein-protein interactions that you feel this would be more useful for than others?

We have tested a range of interactions and the results are promising. Since the principle of this system can easily be adapted with other systems including MAPPIT [Mammalian Protein-Protein Interaction Trap], we believe that this system could be useful to study a wide range of protein-protein interactions.

You also did some initial work showing how this could be used in high-throughput cell-based drug screening. Are there hopes to commercialize this assay for that purpose?

Our R&D has taken initiatives to explore its commercial potential.

There are a lot of other variations on the yeast two-hybrid approach being used in drug discovery. How would your be different?

Current methods based on yeast two-hybrid screen being used in drug discovery typically rely on single reporter functions. These are susceptible to numerous intrinsic variables particularly in regard to level of transfection efficiency, transcription, processing, and translation. The dual-light reporter system is designed in such a way that it bypasses the variables confounding single-reporter assays. Since this system utilizes two reporters, by taking advantage of the constitutively expressed upstream reporter, we can correct the expression of downstream reporter to this reference standard. The advantage of the dual fluorescence reporter is that the assay is measured directly on intact cells and does not require cell wall disruption or addition of a substrate, and is therefore suitable for high-throughput screening.

Have you filed for patents?

Yes, a patent application was filed.

In the paper, you used a standard luminescence reader, fluorescence microscope, and flow cytometer. Have you thought of more advanced instrumentation that might be able to provide increased throughput?

At the moment, we are using a fluorescence activated cell sorter to screen cDNA expression libraries and in collaboration with other institutions will employ advanced instrumentation suitable for high-throughput screening of chemical libraries.

What's next for your lab regarding this research?

The main focus of my research is to understand basic mechanisms of regulation and disruption of gene expression. We are employing and developing cell-based assay systems to understand the molecular mechanisms and to implement these discoveries to aid design of new therapeutic approaches to a human disease.

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