Disabling the Protease DDI2 Attenuates the Transcriptional Activity of NRF1 and Potentiates Proteasome Inhibitor Cytotoxicity

Abstract

Proteasome inhibition is used therapeutically to induce proteotoxic stress and trigger apoptosis in cancer cells that are highly dependent on the proteasome. As a mechanism of resistance, inhibition of the cellular proteasome induces the synthesis of new, uninhibited proteasomes to restore proteasome activity and relieve proteotoxic stress in the cell, thus evading apoptosis. This evolutionarily conserved compensatory mechanism is referred to as the proteasome-bounce back response and is orchestrated in mammalian cells by nuclear factor erythroid derived 2-related factor 1 (NRF1), a transcription factor and master regulator of proteasome subunit genes. Upon synthesis, NRF1 is cotranslationally inserted into the endoplasmic reticulum (ER), then is rapidly retrotranslocated into the cytosol and degraded by the proteasome. In contrast, during conditions of proteasome inhibition or insufficiency, NRF1 escapes degradation, is proteolytically cleaved by the aspartyl protease DNA damage inducible 1 homolog 2 (DDI2) to its active form, and enters the nucleus as an active transcription factor. Despite these insights, the cellular compartment where the proteolytic processing step occurs remains unclear. Here we further probed this pathway and found that NRF1 can be completely retrotranslocated into the cytosol where it is then cleaved and activated by DDI2. Furthermore, using a triple-negative breast cancer cell line MDA-MB-231, we investigated the therapeutic utility of attenuating DDI2 function. We found that DDI2 depletion attenuated NRF1 activation and potentiated the cytotoxic effects of the proteasome inhibitor carfilzomib. More importantly, expression of a point-mutant of DDI2 that is protease-dead recapitulated these effects. Taken together, our results provide a strong rationale for a combinational therapy that utilizes inhibition of the proteasome and the protease function of DDI2. This approach could expand the repertoire of cancer types that can be successfully treated with proteasome inhibitors in the clinic.

Dimer Interface Organization is a Main Determinant of Intermonomeric Interactions and Correlates with Evolutionary Relationships of Retroviral and Retroviral-Like Ddi1 and Ddi2 Proteases

“…Our results imply that non-viral retroviral-like PRs potentially have relatively lower dimer stability compared to retroviral counterparts. We assume that getting better insight into the structural requirements for the dimer formation through the protease domain may help understanding the roles of Ddi-like proteins in proteasomal shuttles and ubiquitination pathways [6,7,63,64,67,68,84,85], even in physiological or pathological conditions. Furthermore, future studies are needed to prove our findings by determining the in vitro dimer stabilities, and correlating the features described in our study with those of additional eukaryotic retroviral-like or endogenous retrovirus PRs.…”

“…Studies on Ddi1-Sc PR provided evidence for its proteolytic activity, which was found to be required for sufficient checkpoint regulation [61] and to contribute to protein secretion [62], DNA replication stress response [63], and DNA-protein crosslink repair [64]. In Caenorhabditis elegans, Ddi1 expression was found to be induced by proteasome dysfunction; furthermore, results proved that the catalytic activity of Ddi1 PR is necessary for protein activation [65], and involvement of PR activity in Nrf1 processing was also demonstrated for human Ddi2 PR [66,67]. Protease domain of Ddi1-Hs protein was found to undergo post-translational modification, ubiquitination sites were identified in the proximities of the active site (K192) and the dimer interface (K382) [68], but effect of these modifications of protease function has not been elucidated.…”

“…To our knowledge, comparative analyses have been conducted only for retroviral PRs [9,[69][70][71] and Ddi1-like PRs [13], but retroviral and non-viral PRs have not been compared in such analyses. While comparative analyses of retroviral PRs may support understanding of mutational capacity of HIV-1 PR and resistance development [70], exploring general and specific features of retroviral and retroviral-like PRs may support characterization of retroviral-like PRs with unknown structures and the identification of efficient inhibitors e.g., against Ddi2-Hs PR [67]. Here we describe a comparison of overall structural structures and contact maps, with a special emphasis on the regions involved in dimerization and the correlation of the key features with the evolutionary relationships.…”

ER-Resident Transcription Factor Nrf1 Regulates Proteasome Expression and Beyond

“…A recent study shows that Nrf1 is cleaved by the DNA damage-inducible 1 homolog 2 (DDI2) protease, resulting in the release of a proteolytic fragment of Nrf1 from the ER into the nucleus, activating the transcription of proteasome subunit genes. Recently, it was shown that DDI2-deficient or protease-dead DDI2 mutant MDA-MB231 human breast adenocarcinoma cells are more sensitive to carfilzomib-induced apoptosis [85], suggesting that DDI2 is significant to the bounce-back response.…”

“…However, Nrf1 processing occurs in the absence of S1P, S2P, ER membrane-resident rhomboid proteases, or the proteasome. Notably, it was recently shown that Nrf1 is able to enter the nucleus without cleavage [85]. Therefore, the Nrf1 activation mechanism was considered to be resulting from a non-canonical pathway; as such, its precise mechanism remains unknown ( Figure 4).…”