{"id":187,"date":"2024-04-15T18:25:38","date_gmt":"2024-04-15T18:25:38","guid":{"rendered":"https:\/\/pjthornalley.com\/?page_id=187"},"modified":"2024-04-15T18:25:39","modified_gmt":"2024-04-15T18:25:39","slug":"nrf2-oscillator","status":"publish","type":"page","link":"https:\/\/pjthornalley.com\/index.php\/nrf2-oscillator\/","title":{"rendered":"Regulation of transcription factor Nrf2 – Frequency modulated translocational oscillator model"},"content":{"rendered":"\n

Summary<\/strong><\/p>\n\n\n\n

Stress responsive signalling coordinated by Nrf2 provides an adaptive response for protection of cells against toxic insults, oxidative stress and metabolic dysfunction. Nrf2 regulates a battery of protective genes by binding to regulatory antioxidant response elements (AREs). We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We proposed a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects and links stress challenge to increased cytoplasmic surveillance. The serine\/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component. We conclude that frequency modulated translocational oscillations of Nrf2 mediate the ARE-linked cytoprotective transcriptional response.<\/strong><\/p>\n\n\n\n

Background<\/strong><\/p>\n\n\n\n

Nuclear factor erythroid 2-related factor 2 (Nrf2<\/strong>) regulates the cellular expression of a battery of protective genes countering oxidative stress, toxic substances, lipid peroxidation, inflammation, metabolic dysfunction and ageing. It senses challenge to homeostasis in the cell cytoplasm and activates a protective transcriptional response. The human Nrf2 system regulates basal expression of ca.<\/em> 640 genes, inducible expression of 650 genes and basal and inducible expression of 240 genes. Nrf2 activators typically affect ARE gene subsets; the selection mechanism is unknown but may be linked to the level of functionally active Nrf2 in the nucleus, accessory proteins (e.g. Maf proteins F, G & K) and pleiotropic effects of Nrf2 activators.    <\/p>\n\n\n\n

    <\/strong>Under basal conditions, Nrf2 transcriptional activity is repressed by binding to Kelch-like erythroid cell-derived protein <\/em><\/strong>with CNC homology-associated protein 1 (Keap1<\/strong>). The Keap1-Nrf2 complex is in the cytoplasm and facilitates Nrf2 degradation: Keap1 is a substrate adaptor protein for Cullin-3 (Cul-3)-dependent E2 ubiquitin ligase complex, directing Nrf2 to the 26S proteasome. Keap1-Nrf2 binds threonine phosphatase PGAM5<\/strong> tethered to the outer mitochondrial membrane. <\/p>\n\n\n\n

Regulation of Nrf2<\/strong><\/p>\n\n\n\n

The mechanisms of stress sensing and surveillance by the Nrf2 system are uncertain. Two models have been proposed.<\/p>\n\n\n\n

Stabilization model<\/em><\/strong> Under conditions of oxidative and other stresses Nrf2 is released from Keap1. An early mechanism of regulation of Nrf2 was stabilization for Nrf2 to proteolysis by release from KEAP1 and increased Nrf2 protein equilibration into the nucleus, binding to ARE sites for transcriptional regulation.<\/p>\n\n\n\n

Oscillation model<\/em><\/strong> Nrf2 is phosphorylated by casein kinase-2 (CK2) and translocates to the nucleus via importins \u03b15 and \u03b21. In the nucleus, Nrf2 activates target genes by binding to AREs. Thereafter, Nrf2 is phosphorylated by Fyn kinase and expelled from the nucleus via the nuclear membrane export channel exportin-1\/crm1. Nrf2 translocates to the nucleus driven by CK2 phosphorylation and activates the transcriptional response therein. Nrf2 is then inactivated by acetylation, expelled from the nucleus by fyn-catalysed phosphorylation and reactivated by deacetylase and phosphatase in the cytoplasm – see Figure 1. We presented an explanatory mechanism where Nrf2 functionality is linked to reactivation on return to the cytoplasm or \u201crefresh rate\u201d. Nrf2 is a constitutive oscillator where under conditions of oxidative and other stresses, the oscillation and refresh rate increase, increasing the level of active Nrf2 and thereby ARE-linked transcriptional response. The transcriptional response is linked to sensing of the cell stress status; increased stress increased stress status sampling frequency and also increases the transcriptional response. Nrf2 oscillations were found in the human endothelial cells and fibroblasts in culture.<\/p>\n\n\n\n

Oscillation model \u2013 explanatory description<\/strong><\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Figure 1. Oscillation mechanism for Nrf2 regulation<\/strong>. <\/em>Abbreviations: a, activated; Ac, acetylation; AcT, acetyltransferase; CK2, casein kinase-2; Cul-3, Cullin-3; E2, ubiquitin E2 ligase; Fyn, fyn kinase; GSK3\u03b2, Glycogen synthase kinase-3 beta; Keap1, Kelch-like erythroid cell-derived protein <\/em><\/strong>with CNC homology-associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; P, phosphorylation; PGAM5, mitochondrial serine\/threonine protein phosphatase; Ub, ubiquitin.<\/p>\n\n\n\n

Strengths of the oscillation model<\/strong><\/p>\n\n\n\n

Experimental observations explained by the Oscillation model but NOT by the Stabilization model<\/p>\n\n\n\n

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  1. Half-maximal transcriptional response is achieved without increase in Nrf2 protein<\/li>\n\n\n\n
  2. Trapping of Nrf2 in the nucleus decreases transactivational activity<\/li>\n\n\n\n
  3. Oscillation frequency responds to physiological levels or activators (10 \u2013 20 \u03bcM hydrogen peroxide; 0.5 \u2013 2 \u03bcM sulforaphane & 5 \u2013 10 \u03bcM quercetin.<\/li>\n<\/ol>\n\n\n\n

    Oscillation model shows Nrf2 regulation is exquisitely fit-for-purpose in a stress-responsive transcriptional system<\/strong><\/p>\n\n\n\n

    Because:<\/p>\n\n\n\n

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    1. Cytoplasmic stress sensing is communicated directly to the nucleus by Nrf2 translocation<\/li>\n\n\n\n
    2. Frequency of stress surveillance is intensified when the host cell is challenged<\/li>\n\n\n\n
    3. Inactivation of Nrf2 in the nucleus prevents abnormal accumulation of active Nrf2 and loss of transcriptional fidelity.<\/li>\n<\/ol>\n\n\n\n

      We noted analogy to an engineering sensor where dynamical resolution relies on a high refresh rate because the sensor must reassess the environment at a high rate. Nrf2 goes through cycles of sense> response> inactivate>reset. The nuclear translocation period of Nrf2 was 129 min in quiescent cells and decreased to 80 min during stimulus activation.<\/p>\n\n\n\n

      Oscillation model \u2013 experimental example<\/strong><\/p>\n\n\n\n