DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the transcription factor TCF11/NRF1 (NFE2L1), crucial for maintaining proteostasis in mammalian cells, enabling the expression of rescue factors, including proteasome subunits. Here, we describe the consequences of DDI2 ablation in vivo and in cells. DDI2 knock-out (KO) in mice caused embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of embryos showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates and induction of the unfolded protein response (UPR) and cell death pathways. In DDI2 surrogate KO cells, proteotoxic stress activated the integrated stress response (ISR) and induced a type I interferon (IFN) signature and IFN-induced proliferative signaling, possibly ensuring survival. These results indicate an important role for DDI2 in the cell-tissue proteostasis network and in maintaining a balanced immune response.

• Keywords
• Biological sciences, Developmental biology, Immune respons,
• Publication type
• Journal Article MeSH

Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.

• MeSH
• Amino Acid Motifs MeSH
• Aspartic Acid Proteases chemistry   metabolism   MeSH
• HEK293 Cells MeSH
• Conserved Sequence MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• Protein Interaction Mapping MeSH
• Evolution, Molecular MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Polyubiquitin metabolism   MeSH
• Protein Domains MeSH
• Proteolysis MeSH
• Solutions MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, Protein MeSH
• Structural Homology, Protein * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aspartic Acid Proteases MeSH
• DDI2 protein, human MeSH Browser
• Polyubiquitin MeSH
• Solutions MeSH
• Saccharomyces cerevisiae Proteins MeSH