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Deleted in Colorectal Cancer Inhibits Protein Synthesis, But How?
Spear, Elizabeth
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Denver, CO
Metropolitan State University of Denver
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Collected for Auraria Institutional Repository by the Self-Submittal tool. Submitted by Matthew Mariner.
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Faculty mentor: Megan Filbin
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Major: Biochemistry

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Deleted in Colorectal Cancer Inhibits Protein Synthesis, But How? Introduction Elizabeth Spear and Megan E. Filbin Department of Chemistry and Biochemistry, Metropolitan State University of Denver Axonal guidance and outgrowth during development is driven by many chemotropic signals . Deleted in Colorectal Cancer (DCC) is a transmembrane receptor protein located in the growth cones of developing neurons and interacts with a chemotropic signal known as netrin 1 . When the netrin 1 signal is present extracellularly, cytoplasmic tail will homodimerize to release ribosomal subunits for local protein synthesis and presumably promote axonal outgrowth . 1 In the absence of the netrin 1 signal, DCC tethers ribosomal subunits with its cytoplasmic tail, blocking translational function . Our lab focuses on studying the cytoplasmic tail ribosomal subunit complex in the absence of netrin 1 as this complex is poorly defined . The sequence of cytoplasmic tail contains about 330 amino acids with three highly conserved motifs (P 1 3 ) and is predicted 91 % unstructured . 2 Previous research has already investigated the inhibitory function of two regions on the DCC cytoplasmic tail : the P 1 and P 3 motifs . During this experiment, the inhibition of both cap dependent and cap independent translation was studied . When deleting the P 3 motif ( P 3 ) from the cytoplasmic tail it is still shown to be 100 % inhibitory . When removing the P 1 motif ( P 1 ), translation is inhibited by 65 % , indicating that there is still another portion of the cytoplasmic tail that is responsible for inhibition of protein synthesis and that it lies towards the N terminus . Moving forward, our lab is creating four new truncation mutants ( N Term, P 1 , 3 , P 2 +N and P 2 only) and plans to test their inhibitory function in vitro using a luciferase translational assay . Our findings will further narrow down which region of the cytoplasmic tail is necessary and sufficient for inhibition of protein synthesis machinery and provide better insight for a model of the cytoplasmic tail translational machinery complex . Background Question and Research Methods Which portion of the DCC cytoplasmic tail is necessary and sufficient for inhibiting protein synthesis in the absence of the Netrin 1 Signal? Figure 1 . Deleted in Colorectal Cancer Models . A . (Left) schematic of immature neuron growth towards the chemotropic signal netrin 1 . (Right) within the cell, cytoplasmic tail is tethered to translational machinery (cyan, yellow, pink, and orange complexes) . Netrin 1 induces homodimerization of cytoplasmic tail so translational machinery can dissociate and actively translate mRNA . Figure adapted from ( 1 ) . B . 3 D model of cytoplasmic tail using hidden Markov models via the Phyre 2 web portal . 3 The tail is predicted 91 % unstructured using the DisEMBL web portal . 2 A References & Acknowledgements 1. Tcherkezian J, Brittis PA, Thomas F, Roux PP, Flanagan JG. 2010. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell 141:632 644. 2. Linding R, Jensen LJ, Diella F, Bork P, Gibson TJ, Russell RB. 2003. Protein disorder prediction: implications for structural proteomics. Structure 11(11):1453 9. 3. Kelly LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10: 845 858. Research reported in this poster was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM134393 to M. E. Filbin . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Research reported in this poster was supported by the MSU Denver Provost Office Seed Grant for Fall 2019 to M. E. Filbin . Research reported in this poster was supported by the MSU Denver College of Letters, Arts & Sciences Undergraduate Mini grant for Spring 2020 to E. Spear. We thank members of the J.S. Kieft Lab (particularly Dr. Erik Hartwick) in the Department of Biochemistry & Molecular Genetics at the University of Colorado School of Medicine for pRL plasmids used in the luciferase assays (originally gifted by A. E. Willis, Stoneley et al. 1998). We thank Dr. J. Martin in the Department of Chemistry & Biochemistry at MSU Denver for his support with the Perkin Elmer Lambda 650 UV/Vis spectrophotometer. Future Directions In vitro luciferase assays to test the inhibitory function of each of the truncation mutants to narrow down which region is responsible for regulating protein synthesis . Discussion The N Term and P 1 , 3 truncation mutants were successfully purified and quantified . P 2 only however, proved to be a more challenging protein to generate as we could never identify a clear band on our expression gels . This could be due to the E . coli degrading it with proteases during expression since this protein is largely unstructured . To overcome this issue, the process of generating the P 2 +N truncation mutant containing the P 2 motif and the N terminal linker connecting P 1 to P 2 (figure 2 C) has been started . Once this mutant is purified, we will measure its translation inhibitory function to determine if we need to further investigate the P 2 motif . Should we see inhibition of translation from the P 2 +N mutant, we will in vitro synthesize the P 2 only peptide for subsequent analysis . If we do not see inhibition of translation from this mutant, we know P 2 is not the inhibitory region and can focus on the P 1 motif and linker regions . Figure 2 . P 1 , P 2 and/or Linker Regions are Important for Translation Inhibition . A . Depiction of cap and IRES dependent initiation mechanisms used in B . B . Luciferase reporter assay of no DCC (grays), full length DCC (blues), and truncation mutants ( P 1 reds, and P 3 purples) to test inhibition on cap dependent (Cap Fluc PolyA ) and IRES dependent ( CrPV Fluc ) translation . Error bars show one standard deviation from the mean of a triplicate experiment . C . Truncation mutants of the cytoplasmic tail for this project . Translation inhibitory function of each mutant will be tested via luciferase assay to further narrow down the inhibitory region . B Figure 4 . Mechanism of Luciferase Assay . Dose dependence inhibition of luciferase mRNA (gray) in rabbit reticulocyte lysate (which provide the translation machinery colored ovals) will be measured with each purified C tail mutant (top left) . If our mutants are inhibiting protein synthesis by binding to the ribosome and preventing initiation (as indicated in figure 2 A, B), luciferase proteins will not be translated and no light should be observed . If our mutants are not binding to the ribosome, the luciferase protein (dark green) will be translated and bind to the luciferase substrate to generate light . Standard and Mutagenic PCR, Transformation, Selection, Purification, & Sequencing Transformation, Selection, Protein Expression & Purification C A B DCC CTD Model Protein 280 (M 1 cm 1 ) A 280 [Protein] ( uM ) N Term 11460 0.15490 735.29 P1,3 5960 0.15370 270.33 Figure 3 . Generating Truncation Mutants . A . (Middle) cloning schematic via traditional and site directed mutagenesis methods . (Left gel) P 2 only PCR amplification, (right gel) Control (lane 1 ) N Term (lane 2 ) and P 1 , 3 (lane 3 ) mutagenic PCR amplification . B . (Middle) protein expression schematic . (Middle left gel) P 2 only expression, (middle right gel) N Term and P 1 , 3 expressions . Uninduced lanes 1 and 4 , 2 and 4 hour post induction lanes 2 3 and 5 6 , respectively . (Bottom left) N Term and (bottom right) P 1 , 3 size exclusion chromatography purification . C . (Left) 200 350 nm absorption graph and (right) UV Vis quantification of N Term and P 1 , 3 truncation mutants using the Beer Lambert law . A B C Protein Quantification 0.1 2 0.3 kbp 3 1 P2 only PCR Product L PCR 6 1 4 3 kbp L 1 2 3 10 Expected PCR Product Supercoiled Parent Plasmid N Term 1 4 6 95 55 43 34 kDa L St 2 5 3 7 9 8 1 95 55 43 6 4 3 34 kDa L St 8 7 5 2 P1,3 34 10 43 95 55 kDa 6 1 2 4 3 5 L L N Term P1,3 DnaK 4 2 kDa L L 5 6 3 1 95 55 43 34 10 No Clear P2 Only Band