TitleInvestigation of novel NRF2 partners, RAC3 and IQGAP1
NameKim, Jung-Hwan (author), Kong, Ah-Ng Tony (chair), Chen, Suzie (internal member), Suh, Nanjoo (internal member), Chen, J. Don (outside member), Rutgers University, Graduate School - New Brunswick,
Nuclear receptors (Biochemistry)
DescriptionNuclear factor-erythroid-related factor 2 (Nrf2) is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes, which detoxify carcinogens and protect against oxidative stress. In the current study, we investigated the novel Nrf2 partners RAC3/AIB1/SRC-3 and IQGAP1 on Nrf2 signaling. Here, we found that overexpression of RAC3, a nuclear co-regulator, induced Heme oxygenase-1 through Nrf2 transactivation in HeLa cells. Next, we conducted the interaction study between the RAC3 and Nrf2 proteins using co-immunoprecipitation assay (Co-IP) and Fluorescence Resonance Energy Transfer (FRET) analysis. The results showed that RAC3 bound to Nrf2 protein directly in the nucleus. Subsequently, we identified the domains of Nrf2 and RAC3 of their interaction using GST-pull down assay. The results showed that both N-terminal RAC-pas B and C-terminal RAC3-R3B3 were tightly bound to Neh4 and Neh5 transactivation domains. Furthermore, chromatin immunoprecipitation (ChIP) showed that RAC3 tightly bound to the ARE enhancer region of the HO-1 promoter via Nrf2 binding. The data suggested that Nrf2 activation can be modulated and directly controlled via interacts with RAC3 protein in the HeLa cells. Next, we investigated IQGAP1, a calmodulin binding protein, as a novel Nrf2 partner isolated using One-strep tag pull-down method in HeLa cells. Initially, we tested the effect of calcium on Nrf2/ARE-lucifease activity and heme oxygenase-1 (HO-1) protein induction in HeLa cells. The results showed that Nrf2/ARE-lucifease activity and expression of HO-1 were increased by treatment of 3.8 mM of CaCl2, suggesting calcium may be one of the important factors in Nrf2 signaling. Next, we investigated a function of IQGAP1 on Nrf2 signaling by co-transfection with EGFP-Nrf2 and Dsredm-IQGAP1 constructs in HeLa cells. The results showed that Dsredm-IQGAP1 transfection strongly increased the HO-1 expression and the stability of EGFP-Nrf2, which was abolished by siIQGAP1. Furthermore, IP study showed that the Nrf2-IQGAP1 complex was disrupted by CaCl2, treatment suggesting Nrf2 might be liberated from IQGAP1 protein by calcium to be an active form. These results inferred that IQGAP1 may play a pivotal role in Nrf2 signaling in conjunction with intracellular calcium level. Taken together, our results suggest that both RAC3 and IQGAP1 may play an important role in Nrf2 transactivation and its signaling pathway, respectively.
NoteIncludes bibliographical references (p. 135-149)
Noteby Jung-Hwan Kim
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.