Member SpotlightsA Study of Protein-DNA Interactions via Protein Microarrays Harald Seitz, Ph.D. A Science Advisory Board Member Since 1999 Harald Seitz, Ph.D., is a Principal Investigator at the Max Planck Institute (MPI) for Molecular Genetics, Germany. After receving a Chemistry degree from the University of Applied Science, Berlin, Germany, he studied Biochemistry at the Free University of Berlin. During this time, he completed practical courses at the MPI for Molecular Genetics, finishing with a diploma in the crystallography department. For his PhD thesis at MPI, he studied the interaction between proteins and their target DNA -- showing in vivo and in vitro that the prokaryotic DnaA protein (initiator of bacterial replication) interacts with the replicative helicase DnaB via two distinct interaction domains. The results showed that for a functional interaction between two proteins, two interaction domains on each protein are required. During this time, he developed new insights and ideas about the complexity of protein-protein and protein-DNA interactions. Since then, he has worked to characterize protein-DNA interactions with a wide variety of techniques and tried to understand how protein-protein interactions influence the binding behavior of proteins. Seitz received the Young scientist award 2004, 3rd annual HUPO World Congress, Beijing, China and the Poster award of the Falk Foundation, Inflammatory Diseases of Barrier Organs, University Hospital Kiel, Germany. Research Interests In spite of major progress in the tools available for a systematic analysis and function of the genome, its genes and gene products, we still have to invest significant effort in the development of new technologies. This is reflected by my position as a group leader at the MPI for Molecular Genetics. Within my group and in close cooperation within the institute we combine technology adaptation and development with biological questions. After joining the Department of Vertebrate Genomics, headed by Prof. Hans Lehrach, my experimental skills and expertise in molecular biology and the corresponding genomic and proteomic fields, in particular, helped me to develop, establish and verify microarray-based techniques to identify and characterize protein-protein and protein-DNA interactions. Of special interest are the studies about the usage of proteins purified and immobilized under native conditions for the identification of calcium dependent interactions between S100 proteins and their target proteins and the identification of specific targets for kinases using protein microarrays. Currently we are focusing on techniques applicable for a detailed analysis of protein-DNA interactions in real-time with a special focus on label free and real time analysis. Influences & Motivations The time spent as a Ph.D. student in the lab of Walter Messer was very inspiring and it was a stimulating atmosphere. We learned how to value the exchange with scientific colleagues within the group, the department and outside the institute. Without the possibility to discuss your results and ideas with colleagues, scientific progress would be slowed down. After joining the technology driven department of Hans Lehrach, I learned the assets and drawbacks of high-throughput technologies. Working for several years in this area I realized more and more that nearly everything that was discovered and described for higher eukaryotic organisms was already known for bacteria and yeast. Unfortunately people tend to ignore this knowledge. I am also convinced that only a very limited set of tissue specific or cell type specific genes in higher eukaryotes exist. Career Expectations I was born in Berlin, studied in Berlin and now I am working more than 10 years at the same institute in Berlin. This is very unusual and was not planned. I always got offers for a new job or for a Ph.D. position by talking to people. The advantage of staying for my whole life in Berlin is a good scientific net. By knowing the people and what they are working on, it is easy to come into contact and is an ideal platform for interactions. The drawbacks are missing experiences in other labs and countries. I would like to establish my group as a well-known and respected group in the area of protein-DNA interactions. Additionally, I would like to work together with partners from the industry to get access to new techniques and to test alpha or beta versions of new kits or techniques. The following questions are specific to our current Spotlight on Protein Science Research: Explain the use of proteomics in your current research The main interest of my research is the analysis of protein-DNA interactions and the adaptation and development of new techniques. Currently we are using protein microarrays to study protein-protein as well as protein-DNA interactions. Some groups describe the possibility to use double-stranded DNA microarrays for the characterization of protein-DNA interactions. This approach is really fascinating. We are in the process of adopting this technique for our purpose. Which particular tools or methods used in your lab are specific to proteomics? Beside the applications described above, we will use protein microarrays for the quantification of proteins in biological samples. Current applications in proteomics are focusing on the identification of proteins in a huge variety of samples ending with nearly endless lists of proteins. The identification of proteins unique for only one or a few well-defined cellular states is a rare moment. The focus of the research will hopefully shift in the following years to the identification of specific groups / complexes of proteins for a cell type, the relative quantification of the proteins and the analysis of post-translational modifications. Different amounts of the proteins influence the composition of complexes. A variation of post-translation modifications adds or removes information to a protein. The change of information is highly dynamic in time and space. Working with protein microarrays allows us to take the first steps in this direction. As proteomics are applied to medical challenges with respect to the identification of new pathological markers and therapeutic targets, do you believe the current generation of proteomic tools can meet this challenge? Up to date proteomics techniques cannot meet those challenges. The normal experimental set-up is quite often very expensive e.g., mass spectrometer, or even the labeling of proteins with dyes or tags. Additionally high-throughput screens cannot be done in every lab and, last but not least, the techniques are normally not unbiased. Methods like Yeast-2-hybrid or Tap-tagging are complex, time consuming and have a high number of false positives. Slight modifications of the set-up e.g., using a different yeast strain or using a different buffer, results in a different set of proteins that are identified. How effective is today's technology to conduct large-scale, high-throughput analyses for the detection, identification, and functional investigation of low-abundant proteins? A number of techniques exist that enable people to detect, identify and characterize proteins on a single molecular level. Today researchers restrict themselves to a very limited number of techniques. Only a few of those techniques are sensitive enough. Fluoreszenzmicroscopic analysis allows the identification of single protein interactions within a single cell. One major drawback of those techniques are the number of independent repetitions of an experiment that have to be done to distinguish between false positives and real positives and the detection of rare events. Do you agree or disagree with the following statement? Please explain your rationale. The major limitation of proteomic investigations today remains with the complexity of biological structures and physiological processes. Current techniques are not limited by the complexity of biological processes. An intelligent combination of approaches can easily draw meaningful conclusions from any complex biological sample. Analyzing samples in a time and space-resolving manner reduce the complexity drastically and shift the complexity to a new area. A cell can very often tolerate the presence or absence of proteins. Biological networks are highly redundant and can overcome those limitations. The number of examples describing this phenomenon is increasing. More important is the correct timing of new protein synthesis, the modification of proteins and the proper localization of proteins. In the case that proteins have to be transported into a cellular component e.g., a kinase into the nucleus, the transport process is the time limiting step. I think those are topics that should be addressed instead of filling endless lists of proteins identified in a certain tissue or cell. Research projects that aim to identify & catalogue the entire proteome for an organism is a giant undertaking. The result has been international collaborations & associations, often with online databases, that attempt to speed up this process by data sharing. Do you belong to any of these associations, and if so, do you find them effective? How do you think research at MPI is contributing to the national & international efforts in proteomics? Bioinformatic evaluation of data generated by those projects leads to the assumption that a high number of false positives exist. Additionally, it is very difficult and nearly not doable to repeat those experiments. Independent repetition of experiments is crucial for science. Only an independent repetition can give hints to lab artifacts and similar effects. Those projects require a standardization of sample preparation and sample analysis. This clearly means that the data are not unbiased and a time resolved analysis is nearly impossible. We try to produce “high-quality” data. This means that we try to describe when a protein is expressed, where it is localized in the cell, when, where and how long it is modified, how many different modified forms of a protein exist in a cell and similar things. I believe that these kinds of data more appropriate for a system biological description of a cell. To discuss RNAi and other topics with fellow Science Advisory Board members, please visit our community forums. Weblinks Functional Protein Analysis group Publications Seitz H., Hutschenreiter S., Hultschig C., Zeilinger C., Zimmermann B., Kleinjung F., Schuchhardt J., Eickhoff H., Herberg F. Identification of new S100 proteins interaction partners combining functional protein microarrays and surface plasmon resonance, Proteomics 2006 Oct; 6(19):5132-9. Feilner T., Hultschig C., Lee J., Meyer S., Immink R.G.H., Koenig A., Possling A., Seitz H., Beveridge A., Scheel D., Cahill D.J., Lehrach H., Kreutzberger J., Kersten B. High-throughput identification of potential Arabidopsis MAP kinases substrates, Mol Cell Proteomics 2005 Oct; 4(10):1558-68. Sauer S., Lange B., Gobom J., Nyarsik L., Seitz H., and Lehrach H. Miniaturisation in functional genomics and proteomics, Nature Reviews Genetics (2005) Jun; 6(6):465-476. Kersten B., Possling A., Blaesing F., Mirgorodskaya E., Gobom J. and Seitz H. Application of protein microarray technology and UV crosslinking combined with mass spectrometry for the analysis of protein-DNA interactions, Anal Biochem. 2004 Aug 15; 331(2):303-13. ### << Previous Next >> [ View All Member Spotlights ] |
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