20.109 MOD3 Research Proposal

Maryelise Cieslewicz
Kyle Atmore

"Peroxiredoxin 1 PDB image"

Logistics

Prepare a 12 minute powerpoint talk that describes the research question you have identified, how you propose to study the question and what you hope to learn. A general outline your research proposal presentation is:

• a brief project overview
• sufficient background information for everyone to understand your proposal
• a statement of the research problem and goals
• project details and methods
• predicted outcomes if everything goes according to plan and if nothing does
• needed resources to complete the work
• societal impact if all goes well

Topic

• Peroxiredoxin inhibition to facilitate cancer therapy

Topic Background

• Prx's are mostly found in the cytosol (also found in mitochondria, chloroplasts, and peroxisomes).
• Prx's exhibit antioxidant behavior through peroxidase activity, reducing organic hydroperoxides.
• Many cancer cells and tissues show increased expression levels of both Prx1 and Prx2.
• Expression of both Prx's in cancer cells has been associated with increased resistance to standard cancer treatments.
  • Prx1 expression may be used as a predictive indicator of relative risk of death or patient survival among cancer patients.
• Prx1 may be useful as a new therapeutic target in cancer treatments.

Research Ideas

• For overall cancer treatment, cells with increased expression levels of Prx1 could be targeted with therapies that inhibited Prx1 expression.
• Nuclear related factor 2
  • Upregulated by hypoxia
  • Binds Prx1 promoter and induces Prx1 expression
• Our idea: we want to find Nrf2 mutants with higher binding affinity to prx1 promoter, but result in decrease expression of Prx1. The idea is that such a mutant could be used to competitively inhibit the binding of wild type Nrf2 in cancer cells to the Prx1 promoter, and result in decreased expression of Prx1.
  • create library of Nrf2 mutants
  • Use phage display to test binding affinities of the library of Nrf mutants to the Prx1 promoter
  • Isolate the mutants with relatively (to WT) higher binding affinity for Prx1 promoter
  • Test these mutants to find one or more that in the presence of the Prx1 promoter in cell, show a decrease in Prx1 expression
• We are still confused about how during phage display individual phage with different mutants attached could be isolated. Most papers just say "phage mutants were isolated" but do not explain how.

Protocol

1. Get Nrf2 gene plasmid from Invitrogen
   • Contains XhoI and EcoRI restriction sites
2. Error-prone PCR on isolated Nrf2 gene sequence
3. Ligate mutants to phagemid plasmid DNA(LITMUS 38i Vector-NEB)
4. Transform plasmid into supE E. Coli strain XL1-Blue
   • TAG supressed as a glutamine in this strain
5. Perform multiple binding affinity tests
   • Streptavidin coated beads conjugated with Prx1 promoter sequence
   • Elution with complement promoter sequence?
   • Elution using G/B Binding Buffer I?
   • Characteristics of Nrf binding to Prx1 promoter
     • binds to (-536 -> -528) proximal and to (-1429 -> -1421) distal region of Prx1 promoter
     • these comprise the electrophile/antioxidant responsive element
6. Sequence the isolated phage
   • transform into E.Coli and plate on LB XGal/AMP +IPTG plates
   • white colonies contain the insert - LacZ gene is disrupted by Nrf2 insertion
   • pick and sequence colonies
7. Miniprep DNA from candidate colonies
8. Transform into a non-supressor strain of E. Coli
9. Lyse bacterial cells and isolate Nrf using binding and elution as in step 5
10. Characterization of mutant Prx1 expression using sandwich ELISA on samples with:
    • WT Nrf2 and Prx1 promoter-gene complex
      • gives us a baseline amount of Prx1 expression
    • Mutant Nrf2 and Prx1 promoter-gene complex
      • characterize mutant phenotype - we want mutants not to stimulate Prx1 expression
    • WT Nrf2, Prx1 promoter-gene complex, and any Mutant Nrf2s that display decreased Prx1 expression
      • characterize competitive inhibition properties of mutants

Publications

1. "Up-regulation of peroxiredoxin 1 in lung cancer and its implication as a prognostic and therapeutic target."[1]
2. "Structure, mechanism and regulation of peroxiredoxins."[2]
3. "Oxidation of archaeal peroxiredoxin involves a hypervalent sulfur intermediate."[3]
4. "Identification of tumor antigens that elicit a humoral immune response in breast cancer patients' sera by serological proteome analysis (SERPA)"[4]
5. "Taenia solium: antioxidant metabolism enzymes as targets for cestocidal drugs and vaccines."[5]
6. "Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis." [6]
7. "Peroxiredoxins, a novel protein family in lung cancer"[7]
8. "Involvement of peroxiredoxin IV in the 16alpha-hydroxyestrone-induced proliferation of human MCF-7 breast cancer cells."[8]
9. "Inhibition of lung tumor growth and augmentation of radiosensitivity by decreasing peroxiredoxin I expression"[9]
10. "Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer."[10]
11. "Human prx1 Gene Is a Target of Nrf2 and Is Up-regulated by Hypoxia/Reoxygenation: Implication to Tumor Biology"[11]
12. "Bach1 Competes with Nrf 2 Leading to Negative Regulation of the Antioxidant Response Element (ARE)-mediated NAD(P)H:Quinone oxidoreductase 1 Gene Expression and Induction in Response to Antioxidants*"
13. DNA conjugated beads PPT: [12]

Web Pages

1. Wikipedia - [13]
2. Sigma Phagemid DNA: [14]
3. Invitrogen Nrf DNA: [15]
4. NE Biolabs small peptide: [16]
5. NE Biolabs LITMUS: [17]
6. Sequencing Mutants: [18]