Biology subjects

Wufur, R.

Publications and source records attributed to Wufur, R..

3 recordsLinked to original sources

bioRxiv2022

A novel crosstalk between Nrf2 and Smad2/3 bridged by two nuanced Keap1 isoforms

The Keap1-Nrf2 signalling to transcriptionally regulate antioxidant response element (ARE)-driven target genes has been accepted as key redox-sensitive pathway governing a vast variety of cellular stresses during healthy survival and disease development. Herein, we identified two nuanced isoforms and {beta} of Keap1, arising from its first and another in-frame translation starting codons, respectively. In identifying those differential expression genes monitored by Keap1 and/or Keap1{beta}, an unusual interaction of Keap1 with Smad2/3 was discovered by parsing transcriptome sequencing, Keap1-interacting protein profiling and relevant immunoprecipitation data. Further examination validated that Smad2/3 enable physical interaction with Keap1, as well as its isoforms and {beta}, by both EDGETSD and DLG motifs in the linker regions between their MH1 and MH2 domains, such that the stability of Smad2/3 and its transcriptional activity are enhanced with the prolonged half-lives and signalling responses from the cytoplasmic to nuclear compartments. The activation of Smad2/3 by Keap1, Keap1 or Keap1{beta} was likely contributable to a coordinative or another competitive effect of Nrf2, particularly in distinct Keap1-based cellular responses to its cognate growth factor or redox stress. Overall, this discovery presents a novel functional bridge crossing both the Keap1-Nrf2 redox signalling and the TGF-{beta}1-Smad2/3 pathways in healthy growth and development.

cell biology↗

bioRxiv2022

Activation of Keap1 and its isoforms (α and β) inhibits the Nrf2 pathway to affect malignant behaviour of human hepatocellular carcinoma cells

Nrf2 (nuclear factor E2-related factor 2, encoded by Nfe2l2) acts as a master transcriptional regulator in mediating antioxidant, detoxification and cytoprotective responses against oxidative, electrophilic and metabolic stress, but also plays a crucial role in cancer metabolism and multiple oncogenic pathways, whereas the redox sensor Keap1 functions as a predominant inhibitor of Nrf2 and hence changes in its expression abundance directly affect the Nrf2 stability and transcriptional activity. However, nuanced functional isoforms of Keap1 and {beta} have rarely been identified to date. Herein, we have established four distinct cell models stably expressing Keap1-/-, Keap1{beta} (Keap1{Delta}1-31), Keap1-Restored and Keap1-Restored, aiming to gain a better understanding of similarities and differences of two Keap1 isoforms between their distinct regulatory profiles. Our experimental evidence revealed that although Keap1 and its isoforms are still localized in the cytoplasmic compartments, they elicited differential inhibitory effects on Nrf2 and its target HO-1. Furtherly, transcriptome sequencing unraveled that they possess similar but different functions. Such functions were further determined by multiple experiments in vivo (i.e. subcutaneous tumour formation in nude mice) and in vitro (e.g., cell cloning, infection, migration, wound healing, cell cycle, apoptosis, CAT enzymatic activity and intracellular GSH levels). Of note, the results obtained from tumorigenesis experiments in xenograft model mice were verified based on the prominent changes in the PTEN signaling to the PI3K-AKT-mTOR pathways, in addition to substantially aberrant expression patterns of those typical genes involved in the EMT (epithelial-mesenchymal transition), cell cycle and apoptosis.

biochemistry↗

bioRxiv2022

Distinct roles of Nrf1 and Nrf2 in monitoring the reductive stress response to dithiothreitol (DTT)

Transcription factor Nrf2 (nuclear factor, erythroid 2-like 2, encoded by Nfe2l2) has been accepted as a key player in redox regulatory responses to oxidative or reductive stresses. However, it is less or not known about the potential role for Nrf1 (nuclear factor, erythroid 2-like 1, encoded by Nfe2l1) in the redox responses, particularly to reductive stress, albeit this fossil-like factor is indispensable for cell homeostasis and organ integrity during life process. Here, we examine distinct roles of Nrf1 and Nrf2 in monitoring the defense response to 1,4-dithiothreitol (DTT, serving as a reductive stressor), concomitantly with unfolded protein response being induced by this chemical (also as an endoplasmic reticulum stressor). The results revealed that intracellular reactive oxygen species (ROS) were modestly increased in DTT-treated wild-type (WT) and Nrf1-/- cell lines, but almost unaltered in Nrf2-/-{Delta}TA or caNrf2{Delta}N cell lines (with a genetic loss of its transactivation or N-terminal Keap1-binding domains, respectively). This chemical treatment also enabled the rate of oxidized to reduced glutathione (i.e., GSSG to GSH) to be amplified in WT and Nrf2-/-{Delta}TA cells, but diminished in Nrf1-/- cells, along with no changes in caNrf2{Delta}N cells. Consequently, Nrf1-/-, but not Nrf2-/-{Delta}TA or caNrf2{Delta}N, cell viability was reinforced by DTT against its cytotoxicity, as accompanied by decreased apoptosis. Further experiments unraveled that Nrf1 and Nrf2 differentially, and also synergistically, regulated DTT-inducible expression of critical genes for defending redox stress and endoplasmic reticulum stress. In addition, we have also identified that Cys342 and Cys640 of Nrf1 (as redox-sensing sites within its N-glycodomain and DNA-binding domain, respectively) are required for its protein stability and transcription activity.

cell biology↗