Nrf1 is a master regulator of Proteasomal function

Info

Gene Code: NFE2L1 Alternative code: TCF11 Full name: Nuclear factor-erythroid 2 p45 subunit-related factor 1

Localisations

Endoplasmic Reticulum (ER)

The full-length p120 precursor form of NRF1 is cotranslationally inserted into the ER via the classical Sec61-dependent pathway (R1)

The p120 NRF1 precursor is cotranslationally inserted into the ER through Sec61. It adopts a type II membrane orientation with most of its mass in the ER lumen and a small N-terminal portion in the cytosol, guided by a hydrophobic TMD. The protein undergoes extensive N-glycosylation in its luminal domains, adding 10-15 kDa.

During normal conditions with active proteasomes, NRF1 undergoes ERAD (ER-associated degradation) - it is ubiquitinated by HRD1, extracted from the ER membrane by p97 (VCP ; valosin containing protein; AAA ATPase), and degraded by cytosolic proteasomes.

Under proteotoxic stress or proteasome inhibition, the retrotranslocated NRF1 becomes activated in the cytosol. While the mechanism marking NRF1 for ERAD remains unclear, this rapid synthesis-destruction cycle maintains a short half-life (~12 min) and prevents unnecessary proteasome production. (R1)

USP19 activates ER-tethered Nrf1

USP19 directly interacts with Nrf1 in proximity to the ER and topologically acts as a deubiquitinating enzyme to remove ubiquitin moieties from this protein, which allow it to circumvent potential proteasomal degradation.

This USP19-mediated effect takes place only after Nrf1 is retro-translocated by p97 out of the ER membrane to dislocate the cytoplasmic side.

Conversely, knockout of USP19 causes significant decreases in the abundance of Nrf1 and the entrance of its active isoform into the nucleus, which result in the downregulation of its target proteasomal subunits (R6)

USP19 is controlled by Nrf2:

our additional experimental evidence has revealed that transcriptional expression of endogenous USP19 and its promoter-driven reporter genes is differentially regulated by Nrf2, as well by Nrf1, at distinct layers within a complex hierarchical regulatory network. (R6)

USP19 is required to stabilize HIF-1A:

USP19 interacts with HIF-1α and describe a non-catalytic role for this enzyme in stabilizing HIF-1α after cellular exposure to hypoxia. The presence of USP19 is required to mount a proper hypoxic response, and we therefore suggest that USP19 is a previously unknown regulator of HIF-1α stability. (R7)

Cytosol

Cytosolic NRF1 p120 has two potential fates: degradation when proteasome function is normal, or activation and nuclear transport during proteasome insufficiency.

For degradation, p97 transfers NRF1 to the proteasome, though intermediate ubiquitin shuttle proteins may be involved. Activation requires two key steps:

1. NGLY1 deglycosylates NRF1, converting glycosylated asparagines to aspartic acids - a modification that enhances transactivation potential.

2. DDI2 protease cleaves p120 between Trp-103 and Leu-104 to create active p110, removing the N-terminal TMD.

Recent findings show NRF1 is fully extracted into the cytosol before processing, suggesting unidentified chaperones may protect its TMD from aggregation. Research on DDI2’s yeast homolog Ddi1 indicates it recognizes substrates with long ubiquitin chains (>8 units), suggesting NRF1 requires extensive ubiquitination for DDI2 recognition and cleavage during proteasome insufficiency.

Nucleus

Nrf1 competes with Nrf2

Calpain-1 involved in degradation of Nrf1

It was previously shown that the calcium-dependent protease calpain-1 interacts with TCF11/Nrf1 and the TCF11/Nrf1 cleavage site is a potential calpain target.

Here, we tested the hypothesis that calpain-1 or -2 cleave TCF11/Nrf1. However, we did not find a role for calpain-1 or -2 in the activation of TCF11/Nrf1 after proteasome inhibition neither by using chemical inhibitors nor siRNA-mediated knockdown or overexpression of calpain subunits. Instead, we found that TCF11/Nrf1 is digested by calpain-1 in vitro and that calpain-1 inhibition slows down the degradation of membrane-bound TCF11/Nrf1 by the proteasome in cultured cells.

Thus, we provide evidence that calpain-1 is involved in the degradation of TCF11/Nrf1. (R5)