White, Victoria

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Clark University

Clark University Clark Digital Commons Undergraduate Student Research Festivals Academic Spree Day 2021 Apr 26th, 12:00 AM Biochemical examination of HECTW1, an enzyme implication in Familial Amyotrophic Lateral Sclerosis and Cancer Development Victoria White Clark University, vwhite@clarku.edu Follow this and additional works at: https://commons.clarku.edu/asdff White, Victoria, "Biochemical examination of HECTW1, an enzyme implication in Familial Amyotrophic Lateral Sclerosis and Cancer Development" (2021). Undergraduate Student Research Festivals. 26. https://commons.clarku.edu/asdff/asd2021/asd2021/26 This Open Access Event is brought to you for free and open access by the Conference Proceedings at Clark Digital Commons. It has been accepted for inclusion in Undergraduate Student Research Festivals by an authorized administrator of Clark Digital Commons. For more information, please contact mkrikonis@clarku.edu, jodolan@clarku.edu, dlutz@clarku.edu,larobinson@clarku.edu. Cl17Ar18K19U92Gustaf H. Carlson School of Chemistry & BiochemistrySpratt LabBiochemical examination of HECW1, an enzyme implication in Familial Amyotrophic Lateral Sclerosis and Cancer Development  Victoria White & Donald E. SprattGustaf H. Carlson School of Chemistry & Biochemistry Clark University, Worcester MA 01610HECW1 (aka (HECT, C2 And WW Domain Containing E3 Ubiquitin Protein Ligase 1) is an E3 ubiquitin      protein ligase that mediates ubiquitination and       subsequent degradation of DVL1 (Dishevelled      Segment Polarity Protein 1). DVL1 encodes a          cytoplasmic phosphoprotein that regulates cell        proliferation.     HECW1 also targets the mutant SOD1 (Superoxide Dismutase 1) protein involved in motor neuron death in Familial Amyotrophic Lateral Sclerosis (FALS).     In cancer, HECW1 promotes the proliferation,            migration and invasion of non-small cell lung cancer cells by inducing the ubiquitination and degradation of Smad4 (Smad proteins act as a tumor suppressors and inhibits epithelial cell proliferation). Figure 6: HECW1 C-lobe is active. Alexa Fluor 647 ubiquitin assay that shows wildtype HECW1 C-lobe (lane 8) can form an E3 ligase.  Samples were run on a 15% Bis-Tris polyacrcylamide gel and visual-ized using iBright imaging.  Figure 1: FALS in a patient. A main symptom of FALS is a curvature of the spine.Image obtained from: Wokke, J., et al. Cambridge University Press. doi:10.1017/CBO9780511735905.003igure 2: Ubiquitin E3 pathway. Ubiquitin (in yellow) is assed  between E1 (in navy), E2 (in purple), and E3 (RING n black; HECT in green) enzymes. It forms an isopeptide ond between the C-terminus of Ub and the e-amino group f lysine on a substrate protein.Figure 4: SDS gel filtrations from Day one and Day two of purification. (A) Day one shows a pure Flowthrough (F1) and Elute (E1). (B) Day two shows a pure Flowthrough (F2) and Elute (E2).FpiboFigure 5:  AKTA purification analysis.  (A) Blue curve shows pure protein fractions (T14-24). (B) SDS gel filtration of protein fractions T14-24 show that 18, 20, and 22 are the most pure.A BFigure 3: A diagram of the purification protocol. First, IPTG induced cells are spun down at 10,000rpm for 10 minutes, then are lysed via homogenization. The lysed cells are ultracentrifuged where they are then proteins are isolated via Nickel chromatography. This yields Flowthrough and Elution 1. The elu-tion has TEV added where it is put in dialysis overnight. The next day, the His6 tag is removed via nickel chromatography to get Flowthrough and Elu-tion 2. All four proteins are then analyzed on SDS-PAGE, and other analyses can be done.A BAcknowledgements:  This work was supported by an R15 AREA grant from the National Institutes of Health and my Capstone  research done throughout the Fall 2020 semester at Clark      University. 

Biochemical examination of HECTW1, an enzyme implication in Familial Amyotrophic Lateral Sclerosis and Cancer Development

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Biochemical examination of HECTW1, an enzyme implication in Familial Amyotrophic Lateral Sclerosis and Cancer Development

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