THE REGULATION OF NUCLEAR FACTOR ERYTHROID-2 (NF-E2)-RELATED FACTOR (NRF2) IN THE PHASE RESPONSE DAPHNE WONG PEI WEN B.Sc. (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 Acknowledgements I am grateful to A/P Thilo Hagen, my supervisor, for the opportunity to conduct my research in his lab. My work would not have been possible without his guidance and patience. Thank you for patiently teaching me and being so understanding and helpful. I would like to thank Christine Hu Zhi-Wen for her emotional support and encouragement; as well as Chua Yee Liu, Hong Shin Yee, Michelle Fong, Dr. Tan Chia Yee, Regina Wong Wan Ju and Jessica Leck Yee Chin for making my lab experience an enjoyable one. I am grateful to Dr. Boh Boon Kim and Dr. Choo Yin Yin for providing the Keap1 plasmids. I am also grateful to the endophyte team: Tan Shi Hua, Lim Shu Ying, Lim Ee Chien, Seah Wen Hui, Christine Hu, Ng Mei Ying and Daphne Ng Hui Ping for their contribution in the endophyte project. I would also like to thank all other members of the lab, past and present, for their help and support. Last but not least, I am deeply grateful to my husband Moses Tan, my parents and my sister for their love and encouragement throughout the duration of my PhD.   ii     DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Daphne Wong Pei Wen 21 August 2014   iii     Table of Contents Acknowledgements ii Declaration iii Summary vii List of Tables ix List of Figures x List of Abbreviations xii 1.0 Introduction 1.1 Oxidative stress and its implications 1.1.1 The role of oxidative stress in carcinogenesis 1 1.2 Nrf2 (Nuclear factor erythroid-2 (NF-E2)-related factor 2) 1.3 The antioxidant response mechanism by Nrf2 1.4 Nrf2 Knockout Mouse Models 1.5 Regulation of Nrf2 activity 1.5.1 Post-translational modification of Nrf2 1.5.2 Degradation of Nrf2 : Keap1 and Cullin3 E3 Ubiquitin Ligase 1.5.3 Accumulation of Nrf2 : Keap1 as a sensor for electrophilic and oxidative stress 13 1.6 The role of Nrf2 inducers in cancer chemoprevention 2.0 Materials and Methods 18 2.1 Cell culture 18 2.2 DNA Transfection 18   iv     16 2.3 Plasmid constructs 19 2.4 Chemicals and inducers 19 2.5 Immunoblotting 20 2.6 Luciferase reporter assay 20 2.7 Immunoprecipitation 21 2.8 Immunoflourescence 21 2.9 Collection and processing of plants 22 2.10 Growth and isolation of endophytes 22 2.11 Molecular identification of isolated endophytes 23 2.12 Organic extraction of secondary metabolites from endophytes 23 3.0 The Induction of Phase response by Heteroaromatic Quinols 26 3.1 Introduction 26 3.2 Results 29 3.2.1 Heteroaromatic quinols increase Nrf2 protein concentrations 29 3.2.2 Heteroaromatic quinols increase Nrf2 transcriptional activation 32 3.2.3 Effect of quinol analogues on Nrf2 transcriptional activation when Nrf2 ubiquitination is prevented 35 3.2.4 PMX290 markedly increases the interaction of Keap1 with Cullin3 39 3.2.5 PMX290 increases Keap1 autoubiquitination 42 3.2.6 Effect of PMX290 on Keap1-dependent nuclear shuttling of Nrf2 44 3.2.7 Effect of PMX290 is independent of cysteine 151 in Keap1 3.3 Summary 52 4.0 Activation of Nrf2 by Andrographolide 54 4.1 Introduction 54 4.2 Results 56   v     48 4.2.1 Andrographolide induces the accumulation of Nrf2 in a Keap1 cysteine 151-dependent manner           4.2.2 Andrographolide increases Nrf2 transcriptional activation 4.2.3 Effect of andrographolide on Keap1-Cullin3 interaction 56 58 60 4.2.4 Correlation between the dependency of Nrf2 inducers on cysteine 151 of Keap1 and their effect on the Keap1Cullin3 interaction 61 4.2.5 Proposed model through which Keap1 Cys151-independent Nrf2 inducer compounds inhibit Nrf2 ubiquitination 4.3 Summary 63 67 5.0 Investigating novel Nrf2 inducer compounds in endophytes 69 5.1 Introduction 69 5.2 Results 72 5.2.1 Isolation and identification of bacterial and fungal endophytes from tropical ferns and mosses 72 5.2.2 Investigating the effect of organic extracts isolated from the endophytes on Nrf2 transcriptional activation 81 5.2.3 Investigating the effect of organic extracts isolated from the endophytes on Nrf2 protein concentration 5.3 Summary 85 87 6.0 Discussion and Conclusion 89 7.0 References 95       vi     Summary The detrimental effects of oxidative stress have been linked to major diseases such as cancer and neurodegenerative diseases. Oxidative stress can be sensed by the Keap1-Nrf2 system in the cell, which triggers cytoprotection via the phase response. Nrf2, a transcription factor, binds to the antioxidant response element (ARE) to induce the expression of phase detoxifying and antioxidant enzymes. Nrf2 is regulated at the protein level by Keap1, a substrate receptor for the Cullin3 E3 ubiquitin ligase. Binding of Keap1 to Nrf2 facilitates the Cullin3-mediated ubiquitination and subsequent degradation of Nrf2. We have identified a class of heteroaromatic quinol compounds as novel Nrf2 inducers. We also characterized the activation of Nrf2 by the diterpenoid andrographolide. The quinol compounds as well as andrographolide were shown to increase the Nrf2 protein concentration and Nrf2 dependent transcription. Nrf2 inducers are expected to covalently modify critical cysteine residues in Keap1, resulting in the inhibition of the Keap1-mediated Nrf2 ubiquitination and degradation. Our results show that andrographolide exerts its effect by targeting cysteine 151 in the BTB domain of Keap1. On the other hand, the quinol compounds function independently of cysteine 151 in Keap1. Interestingly, the quinol compounds markedly increased the binding between Keap1 and Cullin3 whereas andrographolide did not. Given these observations and reports on the mechanism of other Nrf2   vii     inducers, we suggest a correlation where Cys151-independent Nrf2 inducers cause an increase in the Keap1-Cullin3 interaction whereas Cys151-dependent Nrf2 inducers promote the dissociation of Keap1 from Cullin3. Thus, we propose that Cys151-independent Nrf2 inducers function via a novel mechanism that is distinct from Cys151-dependent Nrf2 inducers. The elucidation of the mechanism of action of Cys151independent Nrf2 inducers is expected to improve our understanding of the regulation of the Keap1-Cullin3 E3 ubiquitin ligase. Since secondary bioactive metabolites isolated from endophytes are a useful source of novel bioactive compounds in drug discovery, we also aimed to discover and investigate novel Nrf2 inducers from endophytes. Here, we demonstrated the presence of a potential novel Nrf2 inducer in the organic extract of a fungal endophyte, Phomopsis sp The understanding of novel Nrf2 inducers would provide useful insights for the development of therapeutics against oxidative stressrelated diseases.  viii     List of Tables Table 1. List of target genes of Nrf2 based on chromatin immunoprecipitation (ChIP) analysis. Table 2. Correlation between the dependency of Nrf2 inducers on cysteine 151 of Keap1 with Keap1-Cullin3 interaction. Table 3. List of isolated fungal and bacterial endophytes.   ix     List of Figures Figure 1.1 Schematic representation of the domains and conserved regions in Keap1 and Nrf2. pg 12 Figure 1.2 Schematic representation of the binding of Keap1 to Nrf2 which targets Nrf2 for ubiquitination. pg 13 Figure 3.1 Chemical structures of PMX464, PMX290 and BW114. pg 27 Figure 3.2 Chemical reaction between PMX464 and a reactive cysteine. pg 28 Figure 3.3 Western blot analyses of the effect of the quinol compounds on Nrf2 protein. pg 31 Figure 3.4 Quinol compounds increase transcriptional activation. Nrf2 pg 34 Figure 3.5 Effect of quinol compounds when Nrf2 ubiquitination is inhibited by dnUbc12. pg 36 Figure 3.6 PMX290 may have an inhibitory effect on Nrf2 transcriptional activity. pg 38 Figure 3.7 Effect of the quinol compounds on binding of Keap1 to Nrf2, Cullin3 and Keap1 homodimerization in vivo. pg 41 Figure 3.8 Effect of PMX290 and sulforaphane on Keap1 ubiquitination in vivo. pg 44 Figure 3.9 Effect of PMX290 on Keap1-dependent nuclear shuttling of Nrf2. pg 47 Figure 3.10 Effect of PMX290 on ΔDGR Keap1-Cullin3 interaction in vivo. pg 49 Figure 3.11 Effect of PMX290 on Nrf2 protein concentration and Keap1-Cullin3 interaction when Cys151 of Keap1 is mutated. pg 51   x     required to allow the recruitment of Nrf2 into the Cullin3 E3 ubiquitin ligase complex (Wang et al., 2008). Future studies to test this mechanism would include forced-dimerization of the Keap1-Cullin3 complex. This would mimic the disruption of dynamic assembly and disassembly cycles of the Keap1-Cullin3 complex. If this mechanism is correct, the forced-dimerization assay should result in the same effects as the Cys151-independent Nrf2 inducers. In this study, we propose a model that could possibly describe the mechanism through which Cys151-independent Nrf2 inducers inhibit Nrf2 ubiquitination (as described in Section 4.2.5). We suggest that the Cys151-independent Nrf2 inducers modify cysteine residues in the IVR of Keap1 resulting in a conformational change. The conformational change could cause a disruption in binding of Keap1 at both the ETGE and DLG motifs of Nrf2, resulting in a switch from the two-site ‘hinge & latch’ binding to the one-site ‘hinge’ binding. Consequently, Nrf2 is not presented in the correct orientation and cannot be ubiquitinated and degraded, which in turn results in the accumulation of the Nrf2 protein. Normal binding assays such as immunoprecipitation assays cannot be used to detect the switch from the two-site ‘hinge & latch’ binding to the one-site ‘hinge’ binding because even if binding of Keap1 to the DLG motif is disrupted, the Nrf2 protein will remain bound to Keap1 via the high affinity ETGE site. A recent study reported the use of a quantitative Förster resonance 91         energy transfer (FRET)-based technique using multiphoton fluorescence lifetime imaging microscopy, which was able to distinguish between the two-site ‘hinge & latch’ binding and the one-site ‘hinge’ binding of Keap1 and Nrf2 in single live cells (Baird et al., 2013). Contrary to our proposed mechanism, Baird and colleagues suggested that Nrf2 inducers cause a conformational change in Keap1 resulting in the accumulation of the Keap1-Nrf2 complex in the two-site ‘hinge & latch’ conformation without the release of Nrf2. The absence of free Keap1 binding to newly synthesized Nrf2 would result in the accumulation of Nrf2. However, Baird’s study did not distinguish between Cys151-dependent and Cys151-independent Nrf2 inducers and suggested that they act via similar mechanisms. Moreover, Baird’s proposed mechanism also did not address the increase in Keap1Cullin3 interaction caused by Cys151-independent inducers, which has also been reported by other groups (Wang et al., 2008; Kansanen et al., 2011). We suggest that it may also be possible that the increase in Keap1-Cullin3 interaction caused by Cys151-independent Nrf2 inducers resulted in an inactive Keap1-Cullin3 complex conformation, which may also result in the accumulation of the Keap1-Nrf2 complex in the two-site ‘hinge & latch’ conformation without the release of Nrf2. Nevertheless, it is evident that more studies need to be carried out to understand the mechanism of action of Nrf2 inducers, particularly those that act independently from Cys151. 92         It has been established that the Keap1 protein binds to Cullin3 through its BTB domain. Our results suggest the importance of the IVR of Keap1 in its interaction with Cullin3. Some research groups have also proposed the possible involvement of the IVR of Keap1 in the interaction between Keap1 and Cullin3 (Kobayashi et al., 2004; Chauhan et al., 2013). We suggest that Cys151-independent Nrf2 inducers modify cysteine residues in the IVR of Keap1, resulting in a conformational change disrupting Keap1 function. Unfortunately, structural studies have been deemed problematic as Keap1 is highly insoluble and so far only X-ray structures of the substrate binding domain and very recently of the BTB domain (without intervening region), but not of full-length recombinant Keap1 protein have been obtained (Li et al., 2004; Cleasby et al., 2014). The elucidation of the mechanism of action of Cys151independent Nrf2 inducers is expected to improve our understanding of the regulation of the Keap1-Cullin3 E3 ubiquitin ligase. Since the activation of the Nrf2 pathway confers cytoprotection against oxidative stress-associated diseases including cancer, the understanding of the involved mechanisms would aid in the design of novel chemopreventive agents and therapeutics for oxidative stress-related diseases. Our study also aimed to discover and investigate novel Nrf2 inducers from endophytes. Besides having anti-microbial properties, 93         secondary metabolites isolated from endophytes have also been shown to have antioxidant and anti-inflammatory properties (Strobel and Daisy, 2003). Hence, secondary bioactive metabolites isolated from endophytes would be a useful source of novel bioactive compounds in drug discovery. In our study, we have identified potent Nrf2-inducing properties in the dichloromethane extract (ORX 41) of Phomopsis sp., a fungal endophyte from the lamina of Dicranopteris linearis. Further testing is required to identify the exact organic component within ORX 41 that accounts for its Nrf2-inducing properties. The elucidation of the organic compounds with Nrf2inducing properties could lead to the discovery of a novel potent Nrf2 inducer. 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Zhang, Z., Cui, W., Li, G., Yuan, S., Xu, D., Hoi, M. P. M., Lin, Z., Dou, J., Han, Y., and Lee, S. M. Y. (2012). Baicalein protects against 6OHDA-induced neurotoxicity through activation of Keap1/Nrf2/HO-1 and involving PKCα and PI3K/AKT signaling pathways. J Agric Food Chem 60, 8171-8182. 106         [...]... Nterminus of the Nrf2 DNA binding domain (Figure 1.1) The name Nuclear factor erythroid- 2 (NF-E2) -related factor 2 is derived from another transcription factor p45 NFE2 (nuclear factor erythroid- derived 2) from the same CnC-bZip family Nrf2 contains a Basic Leucine Zipper Domain (bZIP domain) which mediates sequence specific DNA binding The leucine zipper is required to hold together two DNA binding... C151 E2 E2 E2 Keap1 Ub Rbx1 Cullin3 Figure 1 .2 Schematic representation of the binding of Keap1 to Nrf2 which targets Nrf2 for ubiquitination Keap1 functions as the substrate receptor of a Cullin-3-based E3 ubiquitin ligase and binds to ETGE domain and DLG domain of Nrf2 via its Kelch repeat domain The binding of Keap1 promotes transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme to Nrf2, thus... exactly these modifications can affect Nrf2 ubiquitination or Nrf2 binding to the Cullin3 ubiquitin complex formation The elucidation of the mechanism through which Nrf2 is induced is expected to improve our understanding of the regulation of the Keap1Cullin3 E3 ubiquitin ligase The understanding of the involved mechanisms would aid in the design of novel chemopreventive agents and in the development of. .. located in the intervening region (IVR) between the BTB domain and Kelch repeat domains of Keap1 (Figure 1 .2) Zhang et al later narrowed down the number of reactive cysteine residues to two crucial cysteine residues: cysteine 27 3 and cysteine 28 8 in the IVR of Keap1 (Zhang and Hannink, 20 03) These authors showed that cysteine 27 3 and cysteine 28 8 are essential for Keap1-dependent ubiquitination of Nrf2 Their... for the subsequent low affinity interaction between Keap1 and the DLG site The positioning of the ‘latch’ may promote the correct orientation of the lysine residues on Nrf2 for ubiquitin binding This ‘hinge & latch’ model (Tong, Kobayashi, et al., 20 06) is a two-site binding model of Keap1 to Nrf2 and should be distinguished from a one-site ‘hinge’ binding model The one-site binding is a mere binding... the ETGE motif due to its higher affinity and does not present Nrf2 in the correct orientation for ubiquitination It has been shown that the deletion of the low affinity DLG domain, which is involved in the ‘latch’ binding in Nrf2, prevented Nrf2 degradation (McMahon et al., 20 04) Therefore, only the two-sites ‘hinge & latch’ binding model would allow Nrf2 to be ubiquitinated In summary, since Nrf2... IVR intervening region Keap1 Kelch-like ECH-associated protein 1 MAPK Mitogen-activated protein kinases NF-E2 Nuclear factor erythroid- 2 NF- κB Nuclear Factor- kappaB Nrf2 Nuclear factor erythroid- 2 (NF-E2) -related factor 2 Nqo1 NAD(P)H:quinine oxidoreductase 1   xii     PKC Protein Kinase C Rbx RING-box protein 1 SF sulforaphane tBHQ tert-butyl hydroxyquinone Ub ubiquitin     xiii     1.0 Introduction... with these two conserved motifs with different affinities The high affinity binding of Keap1 to the ETGE motif serves as a ‘hinge’ to pin down the Neh2 domain of Nrf2 to Keap1 (Tong, Kobayashi, et al., 20 06; Li and Kong, 20 09) On the other hand, Keap1 binds to the DLG domain with lower affinity and this interaction serves as a ‘latch’ It is likely that the high affinity interaction of Keap1 with the. .. acids 29 -31) and the ETGE motif (amino acids 79- 82) which are involved in the binding of Keap1 There are seven lysine residues between the DLG motif and the ETGE motif which can be ubiquitinated (b) Three functional domains in Keap1: BTB domain, IVR (intervening region) and Kelch repeat domain Adapted from McMahon et al., 20 06   12     Ub Ub Ub Nrf2 ET GE KEL CH DLG Ub KELCH C288 IVR C288 C273 IVR C273... 1.3 The antioxidant response mechanism by Nrf2   4     Early evidences suggesting the role of Nrf2 in the antioxidant response mechanism originated from studies showing the upregulation of NAD(P)H:quinine oxidoreductase 1 (NQO1) (an enzyme involved in maintaining redox balance in the cell) by Nrf2, in response to oxidative stress from xenobiotics and electrophiles (Venugopal and Jaiswal, 1996) In this . domain at the N- terminus of the Nrf2 DNA binding domain (Figure 1.1). The name Nuclear factor erythroid- 2 (NF-E2) -related factor 2 is derived from another transcription factor p45 NFE2 (nuclear. protein 1 MAPK Mitogen-activated protein kinases NF-E2 Nuclear factor erythroid- 2 NF- κB Nuclear Factor- kappaB Nrf2 Nuclear factor erythroid- 2 (NF-E2) -related factor 2 Nqo1 NAD(P)H:quinine. THE REGULATION OF NUCLEAR FACTOR ERYTHROID- 2 (NF-E2) -RELATED FACTOR 2 (NRF2) IN THE PHASE 2 RESPONSE DAPHNE WONG PEI WEN B.Sc. (Hons), NUS A THESIS SUBMITTED