Determining protein interaction specificity of native and designed bZIP family transcription factors

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Page 428 blank. Cataloged from PDF version of thesis.Includes bibliographical references.Protein-protein interactions are important for almost all cellular functions. Knowing which proteins interact with one another is important for understanding protein function as well as for being able to disrupt their interactions. The basic leucine-zipper transcription factors (bZIPs) are a class of eukaryotic transcription factors that form either homodimers or heterodimers that bind to DNA in a site-specific manner. bZIPs are similar in sequence and structure, yet bZIP protein-protein interactions are specific, and this specificity is important for determining which DNA sites are bound. bZIP proteins have a simple structure that makes them experimentally tractable and well suited for developing models of interaction specificity. While current models perform well at being able to distinguish interactions from non-interactions, they are not fully accurate or able to predict interaction affinity. Our current understanding of protein interaction specificity is limited by the small number of large, high-quality interaction data sets that can be analyzed. For my thesis work I took a biophysical approach to experimentally measure the interactions of many native and designed bZIP and bZIP-like proteins in a high-throughput manner. The first method I used involved protein arrays containing small spots of bZIP-derived peptides immobilized on glass slides, which were probed with fluorescently labeled candidate protein partners. To improve upon this technique, I developed a solution-based FRET assay. In this experiment, two different dye-labeled versions of each protein are purified and mixed together at multiple concentrations to generate binding curves that quantify the affinity of each pair-wise interaction. Using the array assay, I identified novel interactions between human proteins and virally encoded bZIPs, characterized peptides designed to bind specifically to native bZIPs, and measured the interactions of a large set of synthetic bZIP-like coiled coils. Using the solution-based FRET assay, I quantified the bZIP interaction networks of five metazoan species and observed conservation as well as rewiring of interactions throughout evolution. Together, these studies have identified new interactions, created peptide reagents, identified sequence determinants of interaction specificity, and generated large amounts of interaction data that will help in the further understanding of bZIP protein interaction specificity.by Aaron W. Reinke.Ph.D

Design of Peptide Inhibitors That Bind the bZIP Domain of Epstein–Barr Virus Protein BZLF1

Designing proteins or peptides that bind native protein targets can aid the development of novel reagents and/or therapeutics. Rational design also tests our understanding of the principles underlying protein recognition. This article describes several strategies used to design peptides that bind to the basic region leucine zipper (bZIP) domain of the viral transcription factor BZLF1, which is encoded by the Epstein–Barr virus. BZLF1 regulates the transition of the Epstein–Barr virus from a latent state to a lytic state. It shares some properties in common with the more studied human bZIP transcription factors, but also includes novel structural elements that pose interesting challenges to inhibitor design. In designing peptides that bind to BZLF1 by forming a coiled-coil structure, we considered both affinity for BZLF1 and undesired self-association, which can weaken the effectiveness of an inhibitor. Several designed peptides exhibited different degrees of target-binding affinity and self-association. Rationally engineered molecules were more potent inhibitors of DNA binding than a control peptide corresponding to the native BZLF1 dimerization region itself. The most potent inhibitors included both positive and negative design elements and exploited interaction with the coiled-coil and basic DNA-binding regions of BZLF1.David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship)National Institutes of Health (U.S.) (Award GM067681)National Science Foundation (U.S.) (Award 0821391

Why rankings of biomedical image analysis competitions should be interpreted with care

International challenges have become the standard for validation of biomedical image analysis methods. Given their scientific impact, it is surprising that a critical analysis of common practices related to the organization of challenges has not yet been performed. In this paper, we present a comprehensive analysis of biomedical image analysis challenges conducted up to now. We demonstrate the importance of challenges and show that the lack of quality control has critical consequences. First, reproducibility and interpretation of the results is often hampered as only a fraction of relevant information is typically provided. Second, the rank of an algorithm is generally not robust to a number of variables such as the test data used for validation, the ranking scheme applied and the observers that make the reference annotations. To overcome these problems, we recommend best practice guidelines and define open research questions to be addressed in the future

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes.

Microsporidia are obligate intracellular parasites that are known to infect most types of animals. Many species of microsporidia can infect multiple related hosts, but it is not known if microsporidia express different genes depending upon which host species is infected or if the host response to infection is specific to each microsporidia species. To address these questions, we took advantage of two species of Nematocida microsporidia, N. parisii and N. ausubeli, that infect two species of Caenorhabditis nematodes, C. elegans and C. briggsae. We performed RNA-seq at several time points for each host infected with either microsporidia species. We observed that Nematocida transcription was largely independent of its host. We also observed that the host transcriptional response was similar when infected with either microsporidia species. Finally, we analyzed if the host response to microsporidia infection was conserved across host species. We observed that although many of the genes upregulated in response to infection are not direct orthologs, the same expanded gene families are upregulated in both Caenorhabditis hosts. Together our results describe the transcriptional interactions of Nematocida infection in Caenorhabditis hosts and demonstrate that these responses are evolutionarily conserved

Issues to consider in developing DNA transformation in microsporidia.

<p>Issues to consider in developing DNA transformation in microsporidia.</p