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THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF BIOLOGY THE IDENTIFICATION AND CHARACTERIZATION OF SUFU INTERACTING PROTEINS EMILY VALERIO SPRING 2013 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biology with honors in Biology Reviewed and approved* by the following: Aimin Liu Associate Professor of Biology Thesis Supervisor Gong Chen Associate Professor of Biology Honors Adviser * Signatures are on file in the Schreyer Honors College. i ABSTRACT The Hedgehog (Hh) pathway, activated by a special family of proteins, is a prominent pathway in mammalian development and also in the formation of various cancers. When interacting with cells, Hh ligands are responsible for enhancing target Hh gene expression through the activation of Gli-transcriptional activators. Suppressor of Fused (Sufu) is a specific Gli-interacting protein that functions in negatively regulating Gli activity and by doing so, suppressing Gli-activated tumor formation. The extent of how Sufu functions is not yet understood in mammals. In an effort to identify proteins that may interact with Sufu in this pathway, over 50 candidate proteins were identified through a yeast-two hybrid screen. Through much background research, four particular proteins were selected due to their known function and location in the cell: ran-binding protein 9 (RanBP9), transmembrane 131-like precursor (T131L), COP9 signalosome complex subunit 1 isoform (Gps1), and hypothetical protein LOC67513. After performing co-immunoprecipitation assays, we confirmed interactions with T131L, Gps1 and LOC67513. These proteins have also been recognized to interact with both structural domains of Sufu, the N-terminal domain and C-terminal domain. Luciferase reporter assays have indicated that these three proteins have a role in mammalian Hh signaling through promotion of Gli activity. These experiments will hopefully help us gain better understanding of the function of Sufu and the Hedgehog pathway in the human body as well as possibly giving insight in the future as to how it may affect disease development. ii TABLE OF CONTENTS List of Figures..................................................................................................................... iii List of Tables ...................................................................................................................... iv Acknowledgements ............................................................................................................. v 1. Introduction .................................................................................................................... 1 1.1 Hh signaling in development and disease ............................................................... 1 1.2 Our current understanding of Hh signaling in mammals ......................................... 5 1.3 The roles and remaining questions of Sufu ............................................................. 8 2. Materials & Methods....................................................................................................... 11 2.1 Construction of Plasmids ....................................................................................... 11 2.2 Co-Immunoprecipitation Assays ............................................................................ 17 2.3 Dual-Luciferase Reporter Assays ........................................................................... 19 3. Results ............................................................................................................................ 21 3.1 Identification of Sufu-interacting proteins through a Yeast Two-Hyrbid Screen ...... 21 3.2 The interaction between Sufu and T131L, Gps1, Loc67513 fragments are confirmed in mammalian cells ............................................................................. 22 3.3 Full-length T131L, Gps1 and Loc67513 interact with Sufu in mammalian cells ..... 23 3.4 The three proteins interact with both the N- and C-terminal domains of Sufu ......... 25 3.4 The three proteins significantly activate Gli-dependent reporters in Sufu -/- Mef cells ..................................................................................................................... 27 3.5 T131L significantly activates Gli-dependent reporter activation in wild-type Mef cells ..................................................................................................................... 29 3.6 T131L activates Gli-dependent reporter in dose-dependent manner ........................ 32 4. Discussion ...................................................................................................................... 35 Appendix .................................................................................................................... 39 REFERENCES............................................................................................................ 41 iii LIST OF FIGURES Figure 1-1. The expression patterns of mammalian Hedgehog homologues. ........................ 1 Figure 1-2. The Drosophila Hh signaling pathway mechanism ............................................ 5 Figure 1-3 The inactive and active states of mammalian Hh sigaling in cilia. ....................... 7 Figure 2-2. Basic mechanism for co-immunoprecipitation assay . ........................................ 17 Figure 3-1. Co-Immunoprecipitation assay results with protein fragments………………….22 Figure 3-2. Co-Immunoprecipitation assay results with full-length proteins……………...…24 Figure 3-3. Co-Immunoprecipitation assay results with Sufu truncations…………………...26 Figure 3-4. Relative activity of proteins in Sufu -/- Mef cells………………………………...27 Figure 3-5. Relative activity of proteins in Sufu -/- Mef cells with over-expressed Sufu….…28 Figure 3-6. Relative activity of proteins in wild-type Mef cells………………………………30 Figure 3-7. Relative activity of proteins in wild-type Mef cells with over-expressed Sufu….31 Figure 3-8. Relative activity of doses of T131L in wild-type Mef cells………………………33 iv LIST OF TABLES Table 2-1. Phusion PCR environments ................................................................................ 12 Table 2-2. Vector and insert digestion information. ............................................................. 14 Table 5-1. Fusion PCR reaction setups. ............................................................................... 39 Table 5-2. Recipe for 6x loading buffer………………………………………………………39 Table 5-3. Transfection setup…………………………………………………………………39 Table 5-4. Recipe for 1% triton lysis buffer…………………………………………………..40 Table 5-5. Recipe for 5ml SDS Separating Gel……………………………………………….40 Table 5-6. Recipe for 2.5ml SDS Stacking Gel………………………………………………..40 v ACKNOWLEDGEMENTS I would like to sincerely thank Aimin Liu for allowing me the opportunity to work and learn in his lab for the past two years and for guiding me in the thesis writing process. I would also like to thank all of the members of the Liu lab including Huiqing Zeng, Xuan Ye, Hongchen Cai, Rachel Chang and Keren Kohath for instructing me during my time here, and especially Jinling Liu for mentoring me along the way. I would also like to thank my honors advisor, Gong Chen, for reading my thesis and providing me with helpful recommendations. Finally, I would like to thank Penn State and the Eberly College of Science for providing funding to carry out our experiments. 1 1. Introduction 1.1 Hh signaling in development and disease There is an amalgamation of developmental pathways that are at work in organisms as they grow. One pathway in particular, the Hedgehog (Hh) signaling pathway, is prominent in organisms ranging from Drosophila, or fruit flies, to mammals. It not only is significant in developmental patterning, but also has major implications in human disease (Varjosalo and Taipale, 2008). Primarily, this pathway is active during early embryogenesis, but it is also circumstantially active in adults (Fig. 1-1). Figure 1-1. The expression patterns of the mammalian Hedgehog signaling homologues in Mus Musculus (Varjosalo and Taipale, 2008). 2 There are three Hh signaling molecules in mammals. As shown in figure 1-1, these include Sonic Hedgehog (Shh), Desert Hedgehog (Dhh) and Indian Hedgehog (Shh) (Varjosalo and Taipale, 2008). Development of many body areas are dependent upon these morphogenic ligands, and in their absence, defects occur. Of the three ligands, Shh has the most broad expression pattern. It is involved in the determination of the dorso-ventral axis and the left-right axis due to its presence in midline tissues; these tissues include the notochord, floor plate, prechordal plate of the axial mesoderm and ventral forebrain, among others (Varjosalo and Taipale, 2008). It is also present in the zone of polarizing activity, making it a major regulator of limb development and distal limb element patterning, such as in the digits. The development of less critical features, such as teeth and hair growth are also dependent on Shh (Varjasalo and Taipale, 2008). Due to some of its vital functions, the absence of Shh leads to major malformations. Cyclopia, limb defects and even lethal malformations involving the ventral neural tube, ribs, somites, brain and vertebrae occur in Shh null mutants (Beachy et al, 2010). The expression of Dhh, unlike Shh, is confined to a small area. As seen in Figure 1-1, it is mainly involved in testis and ovarian development and functions in concordance with Ihh expression (Varjosalo and Taipale,