Determination Of The Nrf1D’ Subcellular Localization And Degradation Processing

BackgroundIn the recent years, more and more researchers divert attention from Nrf2 to Nrf1,because Nrf1 has been considered as an essential transcription factor in regulating both homeostatic and developmental genes. However, it should be noted that Nrf1 has multiple spliced isoforms, one of which is called Nrf1 D. The latter factor enables our interest to be here ignited, which is based on a fact that Nrf1 D is an alternatively-spliced mutant arising from removal of the functional translation terminator from within the intact wild-type Nrf1-encoding transcript. In fact, the Nrf1 D mutant does not only lack the translation terminator residing at 2267-2270 bp, and also leads to an altered sequence in the last portion of the leucine zipper domain. The remaining N-terminal 669 residues of Nrf1 D are the same as that in Nrf1, and however,the C-terminal 72 residues of Nrf1 are replaced by additional 80-aa residues in Nrf1 D. The 80-aa region negatively regulates Nrf1 D, which contains a 42-aa residues branch, which has a significant homology with the equivalent of another b ZIP protein(called fra2). Furthermore, Nrf1 D has been shown to be actually expressed, but the research of the aspect on its activity has not yet been initiated. Herein, live-cell imaging was used for researching ERAD of Nrf1, such that we employed this method to observe the degradation process of different Nrf1 isoforms.ObjectiveNrf1D should be found before the Dual-Luciferase reporter assays and western blotting were applied for detecting the activity of Nrf1 D. Finally, live-cell imaging could be sued for determinating the dynamic processing of different Nrf1 isoforms.MethodsThe semi-quantitative RT-PCR was employed to examine which tissues express Nrf1 D along with its prototypic Nrf1. An expression construct for GFP fusion proteins with Nrf1 or its mutants Nrf1 D and Nrf1Δ670-741, together with the ER/Ds Red marker,were co-transfected into COS-1 cells, followed by real-time live-cell imaging to observe the difference in the degradation process of these proteins within distinct subcellular locations, particularly after treatment with each of the chemicals, such as DTT(at 0,0.5 m M, 1 m M), vitamin C(at 0, 100 μM, 200 μM), t BHQ(at 0, 25 μM, 50 μM),or TPA(at 0, 100 n M, 200 n M) for an indicated time of 4-24 h. These samples were also collectedfor measurement of the Nrf1 D activity by the Dual-Luciferase reporter assays and its protein expression was visualized by western blotting experiments.Results①It is found that Nrf1 D is present in the mouse marrow of bone, which is supported by PCR products of the bone marrow, rather than other tissues examined,representing two bands were separated on the agarose gel.②The degradation of Nrf1 D is slower than Nrf1 by live-cell imaging.When equal amount of Nrf1, Nrf1 D or Nrf1Δ670-741 are co-transfected with ER/Ds Red into COS-1 cells, the subcellular positioning of each of these three proteins is defined in the endoplasmic reticulum of transfected cells. During the 10 min treatment with Digitonin(20 μg/ml), Nrf1 D degrades more rapidly than do Nrf1 D and Nrf1Δ670-741(both proteins seem to have a similar degradation pattern). Subsequent co-treatment with protinase K(50 μg/ml) for 30 min enables Nrf1 to be completely degraded, whilst considerable residues of Nrf1 D and Nrf1Δ670-741 are observed.③Dual-Luciferase reporter assay experiments and western blotting find that DTT,vitamin C and t BHQ play a positive regulatory role in stimulation to induce the activity of Nrf1 D, whereas TPA acts as a negative regulatory reagent. These effects on the activity of Nrf1 D, particularly its glycosylated form, were elicited in a concentration-dependent fashion.④Dymanic processing of Nrf1 isoforms are different between each other, which was visualized by live-cell imaging.ConclusionWhen compared with the C-terminal 72-aa residues of Nrf1, the C-terminal 80-aa residues of Nrf1 D play a negative role in regulating its activity. The 80-aa sequence is predicted to encompass an additional bona fide transmembrane region that enables Nrf1 D to anchor within the endoplasmic reticulum, such that it is to a certain extent protected by the membrane against putative proteolytic degradation. Conversely, the ER-anchoring of Nrf1 D restricts its translocation into the nucleus so that its transactivation activity appears to be repressed to a lower level than that of the wild-type Nrf1. As such, it is found that the chemical effects on the activity of Nrf1 D are exerted in a concentration-dependent manner. Overall, our results reveal differences in the way by which distinct isoforms of Nrf1 are differentially integrated within and around the ER membrane.