Molecular Mechanisms Underlying The Post-translational Processing Of Transmembrane-Bound Nrf1 Protein To Yield Multiple Proteoforms

The transmembrane-bound Nrf1 transcription factor is,a key member of the CNC-bZIP?cap'n'collar-basic leucine zipper?family,involved in a variety of cellular processes,such as antioxidant reactions and inflammatory responses and thus plays an important role in embroy development,and so on.Notably,Nrf1 is anchored in the endoplasmic reticulum?ER?,in which it is glycosylated through its NST domain.When required,portions of its ER-resident TAD elements within Nrf1 are allowed for dynamic repositioning into extra-ER cytoplasmic side of membranes,where it enables for deglycosylation,ubiquitination and phosphorylation.Subsequently,it is released from the ER membrane by cytosolic protease-mediated processing,and then transferred to the nucleus in order to regulate target genes.Nonetheless,the molecular mechanism?s?by which Nrf1 is released from the ER membranes to the nucleus has not been elucidated,although three controversial models have been proposed.In this study,some of molecular biological techniques were used in exogenous and endogenous aspects to distinguish the essential differences of the core problems debated within these three models,such as site-specific proteolysis processing,role of proteasome and ubiquitination,the molecular characteristics of Nrf1,and so on.The detailed research is mainly described as follows:1.Identified whether the N-terminal proteolytic processing of Nrf1 is site-specificBased on the results from our preliminary experiment,the internal deletion of Nrf1from within its NTD and AD1 domains were constructed to explore which regions determine the processing of its N-terminal portions.Western blot analysis showed that production of the⁸5 kDa isoform was affected by its NHB2 region in NTD.In order to further identify the location of the processed site within and around NHB2 peptide,several deletions and/or point mutation methods were employed to construct various plasmids.Immunoblotting results demonstrate that the ¹⁰³WL¹⁰⁴ site was the best,but not the only,position for its proteolytic processing,which is also affected by the membrane topology of NHB2.Moreover,three distinct tags with being zero charged?i.e.V5,VSVG and StrepII?and eGFP were fused to the N-terminal and C-terminal of NTD,N298 and Nrf1,respectively.Total cell lysates expressing these constructs were subjected to protein separation by the proper concentrations of SDS-PAGE gels applied,and then visualized by immunoblotting with distinct antibodies against V5,VSVG or StrepII,so as to detect an N-terminal 12.5 kDa processed polypeptide of Nrf1.Furthermore,a series of intramolecular mutation were created within V5-NTD-eGFP,V5-N298-eGFP,Western blot results demonstrate that production of the 12.5 kDa polypeptide was also not resulted from a specific sites.2.Effects on the proteolytic processing of V5-NTD-eGFP and V5-N298-eGFPDeletion mutants of the TM1-adjoining peptides from within V5-N298-eGFP were transfected into COS-1 cells,and subsequently immunoblotting showed a loss of the12.5 kDa polypeptide,implying that its production was definitely dependent on the putative processing of Nrf1 in proximity to the ER.However,yield of the N-terminal polypeptide arising from the proteolytic processing of V5-N298-eGFP or V5-NTD-eGFP is also affected through p97-dependent and non-dependent pathways which were demonstracted by using the NMS-873,a p97 specific inhibitor.Both knockdown of DDI-1/2 or its over-expression experiments confirmed that the aspartic proteases can promote the proteolytic processing of Nrf1 to yield its N-terminal 12.5kDa polypeptide.Unexpectedly,the state of oxidation promote the formation of high molecular weight isoform.In addition,introduce the Gal4/UAS system to the N-or C-terminal of N298 revealed that Nrf1 could be dynamic regulated by its transmembrane topobiology repositioned in the ER.3.Identification of the molecular charactertic of endogenous Nrf1Proteasome inhibitor was used to treat HepG2 and HL7702 cells in order to induce the expression of endogenous Nrf1,and the Nrf1 specific knockout cell lines,HepG2^Nrf1?-/-?HL7702^Nrf1?-/-?MEF^Nrf1-/-,were served as a reference,then deglycosylated by Endo H,before immunoblotting of Nrf1 proteins that were separated by SDS-PAGE or NuPAGE gels.The results of three distinct Nrf1 antibodies indicate that no matter what kind of gels,the endogenous Nrf1 protein has been resolved into four distinct major isoforms.Among them,their molecular sizes on the NuPAGE gels was estimated to 120 kDa?full length glycosylated isoform?,105 kDa?partly deglycosylated isoform?,95 kDa?non-glycosylated or deglycosylated full length isoform?and 85 kDa?N-terminal processed isoform?,respectively.However,these four major isoforms were also defined as protein-A?¹40/145 kDa,full length glycosylated isoform?,-B?¹30/135 kDa,non-glycosylated,deglycosylated or partly deglycosylated full length isoform?and-C/D?¹25/120 kDa,N-terminal processed isoform?,on the SDS-PAGE gels.4.Verification of the role of proteasome and ubiquitination on the proteolytic processing of Nrf1Different concentration of proteasome inhibitor,MG132,BTZ and Epox,were used to treat eight cell lines form human,mouse and rat,before immunoblotting of Nrf1proteins that were separated by SDS-PAGE or NuPAGE gels.The results showed that the isoforms ratio of?A+B?to?C+D?increases with the increase of inhibitor concentration,which implied the proteasome could perform a proteolytic processing role on the Nrf1.However,these six potential ubiquitination sites(K⁵,K⁶,K⁷⁰,K¹⁶⁹,K¹⁹⁹,K²⁰⁵)within the N-terminal region were mutanted by site-directed mutagenesis,showing that it is not a prerequisites for the proteolytic processing of DDI,but could also affect the stability of Nrf1.Of not,the ubiquitination of K^5/6 mainly regulate the post-translation processing of 120 kDa glycosylated isoform.5.Confirmed the effects on the proteolytic processing of endogenous Nrf1 proteins and its function on the different concentration of proteasome inhibitors.The siRNA oligoes against p97,Hrd1,DDI-1,DDI-2 and NGLY-1 were respectively used to determine the effects,but none of them play a decisive role,on the proteolytic processing of endogenous Nrf1,prior to these four major isoforms of endogenous Nrf1 proteins visualized by immunoblotting.In turn,expression of the above genes were also determined by qPCR after Nrf1 deleted.Under the treatment of lower or higher proteasome inhibitors,the founction of Nrf1 and Nrf2 were examed and thus it is found that both factors were differentially stimulated at low dose and high doses of proteasomal inhibitors.Among them,the transcription activity of Nrf1 was activated by lower doses of the inhibitors,but not be further enhanced at higher dose.However,Nrf2 has a contrary role at the same situation,i.e.its activated mainly by the higher concentration of proteasomal inhibitors.A depth discussion by aforementioned strategies on the debated core problems will provide a clear understanding of selective post-translational processing of the CNC-bZIP protein,which will thus lay a theoretical foundation to develope an Nrf1-targeting anticancer strategy and also provide an available model to research other membrane-proteins.