| 2016 |
DDI2 (aspartyl protease) is required to cleave and activate the transcription factor Nrf1 (NFE2L1) in response to proteasome dysfunction. Deletion of DDI2 reduced the cleaved form of Nrf1 and increased the full-length cytosolic form, resulting in poor upregulation of proteasomes. These defects were restored by wild-type DDI2 but not by protease-defective DDI2, establishing that DDI2's protease activity is essential for Nrf1 processing. |
DDI2 gene deletion (KO), add-back of wild-type vs. protease-dead DDI2, Western blot for Nrf1 forms, proteasome activity assays |
eLife |
High |
27528193
|
| 2020 |
DDI2 is a ubiquitin-directed endoprotease: it cleaves NRF1 in vitro only when NRF1 is highly poly-ubiquitylated. Purified DDI2 can cleave high-molecular-weight ubiquitylated proteins in cell extracts. No evidence for DDI2 acting as a de-ubiquitylating enzyme was found. |
DDI2 KO cells, in vitro protease assay with purified DDI2 and poly-ubiquitylated NRF1, mass spectrometry, cell-based accumulation of ubiquitin conjugates |
Molecular cell |
High |
32521225
|
| 2020 |
NRF1 can be completely retrotranslocated from the ER into the cytosol, where it is then cleaved and activated by DDI2. Expression of a protease-dead point mutant of DDI2 recapitulates the loss-of-function effects on NRF1 activation, confirming that DDI2's protease activity drives cytosolic NRF1 processing. |
Cell fractionation, DDI2 depletion (siRNA/KD), protease-dead DDI2 point mutant expression, NRF1 cleavage assays in MDA-MB-231 cells |
International journal of molecular sciences |
Medium |
31947743
|
| 2022 |
DDI2 functions as a ubiquitin-shuttling factor: its ubiquitin-like (UBL) domain mediates binding to ubiquitin conjugates (K11/K48 branched chains) and to the proteasome. Adding Ub conjugates to cell extracts increases Ddi2 association with proteasomes; adding Ddi2 increases Ub conjugate binding to purified proteasomes. Blocking DDI2 endoprotease activity (genetically or with nelfinavir) increases its binding to Ub conjugates but decreases its binding to proteasomes, reducing protein degradation. |
Affinity co-purification, deletion of UBL domain, nelfinavir treatment, purified proteasome binding assays, Ub conjugate accumulation assays |
The Journal of biological chemistry |
High |
35358511
|
| 2022 |
Both the protease domain and the HDD domain of DDI2 are required to activate NRF1 in multiple myeloma cells. DDI2 expression is upregulated upon prolonged bortezomib treatment, contributing to bortezomib resistance via enhanced NRF1 activation. |
DDI2 KO in MM cells, domain mutant add-back experiments, NRF1 cleavage assays, nelfinavir (partial DDI2 protease inhibitor) treatment |
Cell death & disease |
Medium |
35589686
|
| 2022 |
DDI2 KO in multiple myeloma cells blocks NRF1 cleavage and nuclear translocation, impairing proteasome activity recovery upon irreversible proteasome inhibition. Add-back of wild-type but not catalytically dead DDI2 fully rescues these phenotypes, confirming DDI2 catalytic activity is necessary. |
DDI2 KO (CRISPR), wild-type vs. catalytically dead DDI2 add-back, NRF1 localization by immunofluorescence, proteasome activity assays, in vitro and in vivo MM models |
Blood advances |
High |
34649278
|
| 2020 |
Nelfinavir (an HIV protease inhibitor) directly inhibits DDI2 activity, blocking NFE2L1 (NRF1) proteolysis and potentiating cytotoxicity of proteasome inhibitors in cancer cells. |
DDI2 protease activity assay with nelfinavir, NFE2L1 cleavage assays in cells, cell viability assays |
Cellular signalling |
Medium |
32916277
|
| 2023 |
E3 ubiquitin ligase UBE4A catalyzes polyubiquitination of retrotranslocated NRF1 and promotes its cleavage by DDI2. UBE4A interacts with NRF1, and in vitro recombinant UBE4A promotes ubiquitination of retrotranslocated NRF1. Depletion of UBE4A reduces ubiquitin modification on NRF1, shortens polyubiquitin chain length, decreases DDI2-mediated cleavage efficiency, and reduces proteasomal subunit transcription. |
Co-IP (UBE4A–NRF1 interaction), in vitro ubiquitination assay, UBE4A KO/depletion, ligase-dead mutant expression, NRF1 cleavage assays, RT-qPCR for proteasome subunits |
Biochimica et biophysica acta. Gene regulatory mechanisms |
Medium |
37084817
|
| 2023 |
DDI2 proteolytically cleaves angiomotin (AMOT) to generate an AMOT-CT fragment that promotes angiogenesis. AMOT cleavage by DDI2 is regulated upstream by a signaling axis: NF2 controls AMOT membrane localization, TNKS1/2 catalyzes poly-ADP ribosylation of AMOT, and RNF146 catalyzes AMOT ubiquitination — all required for DDI2-mediated AMOT cleavage. Genetic inactivation of AMOT cleavage regulators in zebrafish and mice causes defective angiogenesis rescued by AMOT-CT overexpression. |
In vitro cleavage assay (DDI2 + AMOT), genetic KO in zebrafish and mice, rescue with AMOT-CT, co-IP/biochemical epistasis for NF2-TNKS1/2-RNF146-DDI2 axis |
The EMBO journal |
High |
37350545
|
| 2024 |
DDI2-mediated NRF1 (NFE2L1) proteolytic cleavage is critical for ferroptosis-induced feedback regulation of proteasome function. Cells lacking DDI2 cannot activate NFE2L1 in response to RSL3-induced ferroptosis, showing global hyperubiquitylation and diminished proteasomal activity. Nelfinavir (DDI2 inhibitor) sensitizes cells to ferroptosis. |
DDI2 KO cells, RSL3-induced ferroptosis, ubiquitylome proteomics, NRF1 cleavage assays, proteasome activity assays, nelfinavir treatment |
Cell death and differentiation |
Medium |
39384955
|
| 2022 |
DDI2 KO mice die at embryonic day E12.5 with severe developmental failure, characterized by insufficient proteasome expression, proteotoxic stress, accumulation of high-molecular-weight ubiquitin conjugates, induction of the unfolded protein response, and activation of cell death pathways. In DDI2 surrogate KO cells, proteotoxic stress activates the integrated stress response and induces a type I interferon signature. |
Conditional/germline DDI2 KO in mice, molecular characterization of embryos (ubiquitin conjugates, UPR markers, proteasome activity), surrogate KO cell lines, transcriptomics |
iScience |
Medium |
39328932
|
| 2022 |
Liver-specific DDI2 KO mice demonstrate that DDI2 contributes to metallothionein (MT) expression in hepatocytes at baseline and upon cadmium (Cd) exposure through DDI2-mediated NRF1 proteolytic maturation. Cd exposure inhibits proteasome activity, resulting in DDI2-mediated NRF1 cleavage; DDI2 deficiency sensitizes cells to Cd toxicity. NRF2 does not contribute to MT production in this context. |
Liver-specific Ddi2 KO mice, cadmium exposure, MT expression assays, proteasome activity assays, genetic analysis comparing NRF1 vs NRF2 contribution |
iScience |
Medium |
36248746
|
| 2024 |
Early recovery of proteasome activity after pulse treatment with proteasome inhibitors is DDI2-independent: it occurs before transcription of proteasomal genes is upregulated but requires protein translation. This establishes a DDI2- and transcription-independent pathway for rapid proteasome activity recovery. |
DDI2 KO cells, time-course proteasome activity assays after pulse treatment with proteasome inhibitors, translation inhibitor experiments |
eLife |
Medium |
38619391
|
| 2026 |
Loss of DDI2 leads to proteotoxic accumulation of the secretory protein CCN1, which is normally extracted from the ER by a DDI2-p97 complex and directed to lysosomes. In the absence of DDI2, CCN1 builds up, generates reactive oxygen species, and triggers compensatory autophagy. DDI2 functions as a selective cargo receptor linking the UPS and the autophagy-lysosome pathway. |
DDI2 KO in human and murine cells, CCN1 KO rescue, DDI2-p97 co-IP complex identification, CCN1-LAMP1 colocalization (immunofluorescence), ROS measurement, autophagy flux assays |
iScience |
Medium |
41809038
|
| 2020 |
Comparative structural analysis of retroviral and retroviral-like protease domains shows that DDI2 contains a retroviral protease-like domain and that the mode of dimerization and density of intermonomeric contacts differ between DDI1/DDI2 and canonical retroviral proteases, correlating with evolutionary relationships. |
Structural bioinformatics analysis of PDB entries, multiple sequence and structure alignments |
International journal of molecular sciences |
Low |
32079302
|