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Obar, Robert

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Obar

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Obar, Robert
Author's Publications: search.filters.isAuthorOfPublication.963e494f-4acf-440d-893c-3033ef831bad

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    Drosophila Protein interaction Map (DPiM): A paradigm for metazoan protein complex interactions
    (Landes Bioscience, 2012) Guruharsha, K.G.; Obar, Robert; Mintseris, Julian; Aishwarya, K.; Krishnan, R.T.; VijayRaghavan, K.; Artavanis-Tsakonas, Spyros
    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein—especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex “map” provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.
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    A Conserved CCCH-type Zinc Finger Protein Regulates mRNA Nuclear Adenylation and Export
    (The Rockefeller University Press, 2009) Hurt, Jessica A.; Obar, Robert; Zhai, Bo; Farny, Natalie G.; Gygi, Steven; Silver, Pamela
    Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export.
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    Architecture of the human interactome defines protein communities and disease networks
    (2017) Huttlin, Edward; Bruckner, Raphael; Paulo, Joao; Cannon, Joe R.; Ting, Lily; Baltier, Kurt; Colby, Greg; Gebreab, Fana; Gygi, Melanie; Parzen, Hannah; Szpyt, John; Tam, Stanley; Zarraga, Gabriela; Pontano-Vaites, Laura; Swarup, Sharan; White, Anne E.; Schweppe, Devin; Rad, Ramin; Erickson, Brian; Obar, Robert; Guruharsha, K.G.; Li, Kejie; Artavanis-Tsakonas, Spyridon; Gygi, Steven; Harper, J. Wade
    The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidation of how genome variation contributes to disease1–3. Here, we present BioPlex 2.0 (Biophysical Interactions of ORFEOME-derived complexes), which employs robust affinity purification-mass spectrometry (AP-MS) methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein coding genes from the human genome, and constitutes the largest such network to date. With >56,000 candidate interactions, BioPlex 2.0 contains >29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering (MCL)5 of interacting proteins identified more than 1300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.