20.109 MOD3 Research Proposal

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"KEAP1 and Nrf2 PDB image"
"Peroxiredoxin 1 PDB image"


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


  • Generating mutant Nrf2 to potentially competitively inhibit Prx1 expression

"Mutant Nrf2 is competitively inhibiting WT-Nrf2 from binding to promoter"

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.
  1. Peroxiredoxins
    • Thiol=specific antioxidant protein
    • Found in mammals, yeast, and bacteria
    • Classified by having one or two cysteine residues
  2. Prx1
    • 2-Cys member
    • Cys52 is used in detoxifying peroxides by oxidation of the residue
      • “Cys52 oxidation through intermolecular disulfide formation with the other conserved Cys173 residue”
    • highly susceptible to inactivation by oxidative stress, unknown physiological function of this
      • overoxidation is common during detoxification of peroxides
      • changed activity/structure may influence Prx1’s interactions with growth regulatory proteins
        • integral in modulating their activity
    • elevated expression in several human cancers
      • suppresses radiation-induced JNK signaling and apoptosis in lung cancer
      • cancers with increased Prx1 expression are more resistant to radiation and cancer treatments targeting JNK(c-Jun-NH2-kinase) activation
      • “They observed significant growth inhibition and radiosensitization, as well as reduced metastasis of lung cancer cells stably transfected with antisense Prx1. These studies suggest that Prx1, in addition to serving as a potential prognostic marker, may also serve as a therapeutic target and/or a target for inhibiting malignant tumore progression.”
      • Little is known about prx1 regulation in cancer cells
  3. Hypoxia
    • Key factor in influencing tumor growth and progression
    • Overall reduction in oxygen availability
    • Tumors unstable tissue oxygenation due to its structure
    • Hypoxia and unstable oxygenation may trigger prx1 transription due to redox-sensitive transcription factors and signaling molecules (like Nrf2)

“Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2” University of Arizona, Online advanced publication of abstract on April 15, 2008. To be published in ‘Carcinogensis’

  • “Here we report that inhibition of Nrf2 may be a promising strategy to combat chemoresistance.”
  • Looked at in relation to ARE pathway
  • Under normal conditions, low amt of Nrf2 is maintained by the Keap1-mediated (KEAP1 = Kelch-like ECH-associated protein 1) ubiquitination and proteasomal degradation system.
  • Chemotherapeutic agents: cisplatin, doxorubicin, and etoposide
  • Down-regulation of Nrf2-dependent response by overexpression of Keap1 or transient-transfection of Nrf2-siRNA rendered the cancer cells more susceptible to disease
  • Upregulation of Nrf2 also enhanced cancer cell resistance
  • Could potentially use small molecule inhibitors of Nrf2 to aid cancer therapies

“Since this Nrf2-dependent cellular defense response is able to protect multi-organs or multi-tissues, activation of Nrf2 has been implicated in conferring protection against many human diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, acute and chronic lung injury, autoimmune diseases, and inflammation.”

"Nrf2-Prx1 Pathway from Kim et al."
"Nrf2-Prx1 Pathway from Kim et al."
  • In cancer cells, KEAP1 is often mutated to generate constitutive expression of Nrf2
  • "Many Nrf2 activators are capable of modifying the thiol (-SH) of cysteine, thus leading to the idea that the reactive Cys residues in Keap1 may function as a redox sensor. The oxidation/modification of these reactive Cys residues in response to electrophile or oxidative stress my allow Nrf2 to dissociate from Keap1"--other studies disagreed...still unknown. Maybe phosphorylation of Nrf2 to be released from Keap1.
  • Many processes working together to increase Nrf2 concentration and promote its accumulation in the nucleus
  • Nrf2-Prx1 pathway specific regulation is still unknown
  • the likelihood of expressing a high level of prx1 in tumors containing a high level of nuclear Nrf2 was 1.51 times greater than in tumors with a low level of nuclear Nrf2. (no statistically significance however)
  • more work to be done, but could potentially use Nrf2 levels to determine the potential efficacy of a cancer therapy in an individual
  • Nrf2 binds to AREs of many antioxidants and phase 2 (carcinogen defense) enzymes. Some include:[1]
    • glutathione
    • glutathione peroxidase
    • glutathione reductase
    • glutathione S-transferase
    • NAD(P)H:Quinone Oxidoreductase 1

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.


  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

Expected Outcomes

  • We will isolate mutants of Nrf2 that show decreased transcriptional activation of Prx1 compared to wild-type.
    • If mutants show decreased Prx1 expression, we believe they do so due to decreased transcriptional activity but equal or higher binding affinity that WT.
    • We never actually calculate binding affinity, so we can’t say for certain that these mutants have the higher binding affinity that we desire. Though, because of the way we isolated these mutants, we do know that these mutants still bind to the Prx1 promoter. Therefore, any mutants that show decreased Prx1 expression do so because of a loss of function and not a lack of binding to the promoter.
    • If no mutants show decreased Prx1 expression, our random mutagenesis affected neither binding nor function of the protein. Therefore, we would do more rounds of mutagenesis to obtain more mutants.
    • Since shutting off the function of a protein may be difficult, if we find that even with many rounds of mutagenesis we can’t isolate mutants with lost transcriptional function, we could shift our attention to other targets of Nrf2. Since Nrf2 is known to bind to many antioxidant proteins, we could test if any of our mutants showed lost transcriptional activity for those genes.
  • Now, we want determine if these mutants have greater binding affinity compared to WT and therefore show the competitive inhibition phenotype we seek.
    • If we see mutants that show decreased Prx1 expression when incubated with wild-type Nrf, we believe this was due to the mutant with decreased transcriptional activity competitively inhibiting binding of WT to Prx1.
      • These mutants will have the phenotype we intended to create.
      • We would make more copies of the DNA that encoded this mutant and perform binding measurements (??) on it to make sure that its effect was due to tighter binding that Prx1.
      • We could also take these mutants and do more rounds of mutagenesis(random or site-directed) to refine its characteristics.
    • If we don’t see any mutants that show decreased Prx1 expression when incubated with WT, this could be due to several reasons:
      • Our mutant can’t effectively compete with WT Nrf due to equivalent or weaker binding (thus it can be displaced by WT).
      • Still these mutants would be useful to us since they have lost transcriptional activator function. At this point, we would probably use SDM at the binding interface in order to introduce changes to make it a better competitive inhibitor.

Societal Impact

  • If our experiment is successful, it will result in the discovery of an Nrf2 mutant that has lost its transcriptional activator function for Prx1 and can competitively inhibit binding of wild-type Nrf2 to the Prx1 promoter.
  • The discovery of such a protein may have applications in gene therapy or drug delivery techniques for cancer cell targeting.
  • Once sequenced, the DNA encoding the mutant protein can be introduced into cancer cells.
  • Since the modified protein works by inhibiting the function of the wild-type protein, replacement of the wild-type gene (by homologous recombination with the mutant gene) shouldn’t be necessary.
  • If the Nrf2 protein produced from the DNA carries out its function in vivo as it did in vitro, Prx1 expression will be shut down in the cancer cells, leading to decreased resistance to standard chemotherapeutic treatments.
  • Therefore, our modified protein can provide a way for increasing the effectiveness of cancer treatments in cancer cells where Prx1 expression is upregulated.


  1. "Up-regulation of peroxiredoxin 1 in lung cancer and its implication as a prognostic and therapeutic target."[2]
  2. "Structure, mechanism and regulation of peroxiredoxins."[3]
  3. "Oxidation of archaeal peroxiredoxin involves a hypervalent sulfur intermediate."[4]
  4. "Identification of tumor antigens that elicit a humoral immune response in breast cancer patients' sera by serological proteome analysis (SERPA)"[5]
  5. "Taenia solium: antioxidant metabolism enzymes as targets for cestocidal drugs and vaccines."[6]
  6. "Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis." [7]
  7. "Peroxiredoxins, a novel protein family in lung cancer"[8]
  8. "Involvement of peroxiredoxin IV in the 16alpha-hydroxyestrone-induced proliferation of human MCF-7 breast cancer cells."[9]
  9. "Inhibition of lung tumor growth and augmentation of radiosensitivity by decreasing peroxiredoxin I expression"[10]
  10. "Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer."[11]
  11. "Human prx1 Gene Is a Target of Nrf2 and Is Up-regulated by Hypoxia/Reoxygenation: Implication to Tumor Biology"[12]
  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: [13]
  14. GOOD IMAGES"Targeting the Nrf2-Prx1 Pathway with Selenium to Enhance the Efficacy and Selectivity of Cancer Therapy"[14]
  15. Affinity-independent elution of antibody-displaying phages using cleavable DNA linker containing streptavidin beads [15]

Web Pages

  1. Wikipedia - [16]
  2. Sigma Phagemid DNA: [17]
  3. Invitrogen Nrf DNA: [18]
  4. NE Biolabs small peptide: [19]
  5. NE Biolabs LITMUS: [20]
  6. Sequencing Mutants: [21]