Affinage

DDI1

Protein DDI1 homolog 1 · UniProt Q8WTU0

Length
396 aa
Mass
44.1 kDa
Annotated
2026-06-09
32 papers in source corpus 17 papers cited in narrative 17 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

DDI1/Ddi1 is a multidomain ubiquitin receptor and ubiquitin-dependent aspartyl protease that couples ubiquitin recognition to substrate proteolysis and proteasomal delivery (PMID:17010377, PMID:32193351). Its central RVP domain adopts a retroviral protease-like fold, dimerizing with paired Asp-Thr-Gly-Ala active sites arranged like HIV protease, establishing the catalytic core flanked by UBL and UBA ubiquitin-binding modules (PMID:17010377). The RVP domain is inactive in isolation and requires the preceding helical domain (HDD) for proteolytic activity, while the atypical UBL domain—which binds ubiquitin directly through a non-canonical interface rather than acting as a conventional proteasome-targeting tag—and the UBA domain together confer high-affinity binding to long K48-linked polyubiquitin chains, restricting cleavage to substrates bearing such chains (PMID:25703377, PMID:27646017, PMID:32193351). As a proteasome shuttle factor, Ddi1 docks on the proteasome subunit Rpn1 (at residue D517) via its UBL domain and captures ubiquitylated substrates such as the Ho endonuclease and the F-box protein Ufo1 via its UBA domain, simultaneously bridging substrate and proteasome in a ternary transfer complex to drive SCF recycling and cell-cycle progression (PMID:15964793, PMID:16478980, PMID:21627799, PMID:22815701). Independently of the proteasome, Ddi1 functions in DNA-protein crosslink repair, being recruited to persistent crosslinks in S phase and degrading trapped RNA Pol II in a manner that requires its protease activity (PMID:31276951, PMID:31902667, PMID:40702097). In higher eukaryotes the protease cleaves and activates the ER-associated transcription factor SKN-1A/Nrf1 in response to proteasome dysfunction, linking Ddi1 to a feedback program that restores proteasome capacity (PMID:27528192).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 2005 High

    Established Ddi1 as a substrate-selective proteasomal shuttle, answering whether dedicated UBL-UBA proteins deliver specific ubiquitylated clients to the proteasome.

    Evidence Co-immunoprecipitation and genetic epistasis dissecting Ho endonuclease degradation in yeast

    PMID:15964793

    Open questions at the time
    • Did not resolve the structural basis of UBL-proteasome docking
    • Did not test whether Ddi1 has intrinsic catalytic activity on substrates
  2. 2006 High

    Defined the catalytic core's architecture, showing Ddi1's central domain is a dimeric retroviral protease-like aspartyl protease fold.

    Evidence X-ray crystallography of the S. cerevisiae central domain

    PMID:17010377

    Open questions at the time
    • Catalytic activity on substrates not demonstrated structurally
    • Role of flanking UBL/UBA domains in activity not addressed
  3. 2006 Medium

    Extended the shuttle role to SCF complex turnover, linking Ddi1 to cell-cycle control via Ufo1 degradation.

    Evidence Co-IP and cell cycle analysis in ddi1Δ yeast

    PMID:16478980

    Open questions at the time
    • Single-lab Co-IP plus phenotype without reciprocal validation
    • Mechanism of Ufo1 capture not biochemically reconstituted
  4. 2008 Medium

    Mapped domain-specific functions, implicating the putative protease active site in checkpoint rescue and a separate C-terminal region in t-SNARE binding.

    Evidence GFP imaging, domain-deletion mutagenesis and genetic rescue in yeast

    PMID:18562697

    Open questions at the time
    • Protease activity inferred from active-site mutant, not measured directly
    • Single lab
  5. 2011 High

    Identified the precise proteasome docking site, showing distinct receptors use distinct Rpn1 surfaces.

    Evidence Site-directed mutagenesis of Rpn1 (D517A) with binding and substrate degradation assays

    PMID:21627799

    Open questions at the time
    • Structural details of the Ddi1-UBL:Rpn1 interface not resolved here
    • Did not address catalytic versus shuttle contributions
  6. 2012 Medium

    Demonstrated simultaneous substrate-and-proteasome bridging, defining a ternary transfer complex with a substrate-shield mechanism.

    Evidence In vitro reconstitution and pull-down assays with Ho, Ufo1 and Rpn1

    PMID:22815701

    Open questions at the time
    • Single in vitro study
    • Kinetics of substrate handoff not quantified
  7. 2015 High

    Revealed the atypical UBL domain binds ubiquitin directly, establishing a dual ubiquitin-binding mode distinct from canonical UBL receptors.

    Evidence NMR structure determination and ITC of yeast UBL and UBA domains

    PMID:25703377

    Open questions at the time
    • Functional consequence of UBL-ubiquitin binding for catalysis not tested
    • Single lab
  8. 2016 High

    Extended the protease function to higher eukaryotes, showing DDI-1 cleaves and activates SKN-1A/Nrf1 during proteasome stress.

    Evidence C. elegans genetic epistasis and immunoblotting for SKN-1 cleavage products

    PMID:27528192

    Open questions at the time
    • Direct enzyme-substrate cleavage not reconstituted in vitro here
    • Cleavage site on SKN-1A not mapped
  9. 2016 High

    Provided full-length structural context showing K48-diubiquitin preference and a putative substrate-recognition loop in the RVP domain.

    Evidence X-ray crystallography, NMR, SAXS and ITC of yeast Ddi1 domains

    PMID:27646017

    Open questions at the time
    • Catalytic mechanism on physiological substrates not shown
    • HDD similarity to DNA-binding folds not functionally tested
  10. 2020 High

    Defined Ddi1 as an autonomous ubiquitin-activated protease requiring long polyubiquitin chains and the HDD for activity, resolving how chain length gates catalysis.

    Evidence In vitro protease assays with ubiquitinated substrates, domain mutagenesis and yeast loss-of-function

    PMID:32193351

    Open questions at the time
    • Endogenous physiological substrate repertoire not defined
    • Cleavage specificity rules within substrates not determined
  11. 2020 High

    Established a proteasome-independent role in DNA-protein crosslink repair, identifying RNA Pol II as an S-phase Ddi1 target.

    Evidence Genetic screen, ChIP, epistasis and Pol II degradation immunoblots in yeast

    PMID:31902667

    Open questions at the time
    • Direct cleavage of crosslinked Pol II not reconstituted
    • Recruitment mechanism to crosslink lesions unclear
  12. 2025 Medium

    Extended the protease function to integral membrane proteins, showing cleavage of K48-ubiquitinated substrates at post-ER compartments by the HDD-RVP core.

    Evidence In vitro protease, domain truncation and cell-based cleavage assays (preprint)

    PMID:bio_10.1101_2025.04.13.648637

    Open questions at the time
    • Preprint not yet peer-reviewed
    • Membrane substrate range and physiological role not established
  13. 2025 Medium

    Connected Ddi1-mediated DPC repair to oxidative-stress survival and host defense across yeast species.

    Evidence DPC precipitation, ddi1Δ/wss1Δ genetics, complementation and macrophage killing assays

    PMID:40702097

    Open questions at the time
    • Molecular targets in oxidative DPC repair not identified
    • Single study

Open questions

Synthesis pass · forward-looking unresolved questions
  • The full endogenous substrate spectrum of Ddi1's protease activity and the rules governing its cleavage-site selection within ubiquitin-tagged substrates remain unresolved.
  • No global substrate census across organisms
  • Cleavage-site determinants beyond chain-length requirement unknown
  • Relative contributions of shuttle versus protease functions in vivo unclear

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016787 hydrolase activity 3 GO:0140096 catalytic activity, acting on a protein 3 GO:0060090 molecular adaptor activity 2
Localization
GO:0005829 cytosol 2 GO:0005634 nucleus 1
Pathway
R-HSA-392499 Metabolism of proteins 3 R-HSA-73894 DNA Repair 2 R-HSA-74160 Gene expression (Transcription) 1
Partners
HORPN1SKN-1A/NRF1SSO1UBE3AUFO1WSS1
Complex memberships
26S proteasome (Rpn1-docked receptor)

Evidence

Reading pass · 17 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2006 X-ray crystal structure of the central domain of Saccharomyces cerevisiae Ddi1 revealed it forms a homodimer with a fold similar to retroviral proteases, with the double Asp-Thr-Gly-Ala active site motif in identical geometry to HIV protease, establishing Ddi1 as a dimeric aspartyl protease-like domain flanked by UBL and UBA domains. X-ray crystallography Journal of molecular biology High 17010377
2005 Ddi1 is specifically required for degradation of ubiquitylated Ho endonuclease; Ho interacts only with Ddi1 (not Rad23 or Dsk2) and must be ubiquitylated for this interaction; Ddi1 binds the proteasome via its UbL domain and interacts with ubiquitylated Ho via its UbA domain; both domains are required for proteasomal association of Ho. Co-immunoprecipitation, genetic epistasis (ddi1Δ, mec1, ufo1Δ mutants), subcellular fractionation Molecular and cellular biology High 15964793
2006 Ddi1 is required for turnover of the F-box protein Ufo1; Ufo1 interacts with Ddi1 via its ubiquitin-interacting motifs (UIMs), and loss of Ddi1 stabilizes Ufo1 and causes cell cycle arrest at G1/S, implicating Ddi1 in SCF complex recycling. Co-immunoprecipitation, genetic loss-of-function (ddi1Δ), cell cycle analysis Molecular and cellular biology Medium 16478980
2008 Domain dissection of Ddi1/Vsm1 showed: (1) the RVP domain mediates homodimerization; (2) UBL and UBA domains are required for nuclear enrichment and rescue of the pds1-128 checkpoint mutant; (3) an aspartate-220 mutation abolishing putative protease activity blocked checkpoint rescue but not dimerization; (4) a C-terminal region (residues 344–395) binds the Sso1 t-SNARE and its phosphorylation on T348 is required for exocytic function. GFP live imaging, domain-deletion mutagenesis, phosphorylation analysis, genetic rescue assays Molecular biology of the cell Medium 18562697
2011 A specific docking site on the proteasome subunit Rpn1 (residue D517) is required for recruitment of Ddi1 (and Dsk2) to the proteasome; the D517A mutation in Rpn1 impairs delivery of ubiquitin conjugates, Ddi1 docking, and degradation of the Ddi1-dependent substrate Ufo1, while Rad23 recruitment is unaffected, indicating distinct docking mechanisms for different UBA-UBL proteins. Site-directed mutagenesis of Rpn1, genetic screening, binding assays, proteasome substrate degradation assays BMC biology High 21627799
2012 In vitro reconstitution showed that Ddi1 can simultaneously bind Ho, Ufo1, and Rpn1, forming a ternary transfer complex; Ddi1-UbL binds Rpn1 while Ddi1-UbA binds ubiquitin chains on Ufo1; Ho substrate protects Ufo1 from displacement by Rpn1, establishing a substrate-shield mechanism. Complex reconstitution in vitro, pull-down assays PloS one Medium 22815701
2015 NMR and binding studies of yeast Ddi1 UBL and UBA domains showed: (1) Ddi1-UBL does not interact with typical UBL receptors but instead binds ubiquitin via hydrophobic contacts and salt bridges, a unique interface; (2) Ddi1-UBA forms a canonical UBA:ubiquitin complex; suggesting a dual ubiquitin-binding mechanism for proteasomal shuttling. NMR structure determination, isothermal titration calorimetry, binding assays Structure High 25703377
2016 In C. elegans, the aspartic protease DDI-1 is required to cleave and activate an ER-associated isoform of the transcription factor SKN-1A/Nrf1 in response to proteasome dysfunction; DDI-1 expression is itself induced by proteasome dysfunction; genetic analyses placed DDI-1 in a pathway with ER traffic regulators and a peptide N-glycanase. Genetic epistasis (C. elegans), loss-of-function, immunoblotting for SKN-1 cleavage products eLife High 27528192
2016 Multi-domain structural analysis of yeast Ddi1 by X-ray crystallography (RVP domain), NMR (helical domain preceding RVP), and SAXS showed: (1) the RVP domain has a conserved loop forming a putative substrate recognition site; (2) both UBL and UBA domains bind ubiquitin by ITC, with enhanced affinity for K48-linked diubiquitin; (3) the helical domain (HDD) has structural similarity to DNA-binding domains of transcription regulators. X-ray crystallography, NMR, SAXS, isothermal titration calorimetry Scientific reports High 27646017
2019 The helical domain (HDD) of Ddi1 preceding the RVP protease domain is required for the cellular response to DNA replication stress caused by hydroxyurea; catalytically competent Ddi1 protease is required to complement the hypersensitivity of ddi1Δ wss1Δ double-deleted yeast. Genetic complementation, domain deletion mutagenesis, yeast growth assays under hydroxyurea DNA repair Medium 31276951
2020 Ddi1 is a ubiquitin-dependent protease: it cleaves substrate proteins only when tagged with long polyubiquitin chains (>~8 ubiquitins); the RVP domain alone is inactive and requires the HDD domain for proteolytic activity; the atypical UBL domain stimulates activity by mediating high-affinity binding to polyubiquitin chains; loss of Ddi1 activity in yeast causes accumulation of polyubiquitinated proteins. In vitro protease assay with ubiquitinated substrates, domain deletion and mutagenesis, yeast loss-of-function Proceedings of the National Academy of Sciences of the United States of America High 32193351
2020 Ddi1 is recruited to a persistent DNA-protein crosslink (DPC) lesion in S phase in yeast; loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC-trapping agents independently of Wss1 and the 26S proteasome; the core component of RNA Pol II is a Ddi1 target, as its genotoxin-induced degradation is impaired in ddi1Δ cells. Genetic screen, chromatin immunoprecipitation, genetic epistasis (ddi1Δ, wss1Δ, proteasome mutants), immunoblot for Pol II degradation Molecular cell High 31902667
2018 Human DDI1 is ubiquitinated by the E3 ligase UBE3A in neuroblastoma SH-SY5Y cells without being targeted for degradation; ubiquitination of DDI1 by UBE3A was confirmed by immunoblotting. Ubiquitin proteomics (bioUb strategy), immunoblotting, cell-based overexpression Human molecular genetics Medium 29788202
2019 UBE3A-dependent ubiquitination sites and ubiquitin chain types on DDI1 were identified; a deubiquitinating enzyme capable of reversing UBE3A-mediated ubiquitination of DDI1 was identified. Ubiquitin proteomics (site mapping), deubiquitinase activity assay Frontiers in physiology Medium 31130875
2010 The retroviral proteinase active site (aspartate residue) and N-terminal region of yeast Ddi1 are both required for repression of protein secretion, demonstrating that Ddi1 functions in vivo as a catalytically active aspartic proteinase in the context of exocytosis regulation. Site-directed mutagenesis, yeast secretion assay FEBS letters Medium 21094643
2025 Ddi1 acts as a ubiquitin-activated protease that cleaves K48-ubiquitinated integral membrane proteins at post-ER compartments, generating cytosolic fragments; the HDD-RVP catalytic core is sufficient for ubiquitin-dependent proteolysis; Ddi1 binds ubiquitin directly; activity is amplified by the atypical UBL and UBA auxiliary ubiquitin-binding domains. In vitro protease assay, domain truncation mutagenesis, ubiquitin binding assays, cell-based substrate cleavage assays bioRxivpreprint Medium bio_10.1101_2025.04.13.648637
2025 In Saccharomyces cerevisiae and Candida albicans, Ddi1 (together with Wss1) is required for DNA-protein crosslink repair under oxidative stress and for survival in response to hydrogen peroxide, sodium hypochlorite, menadione, and plumbagin; CaDdi1 plays a partially redundant role with CaWss1 in resistance to macrophage killing. DPC measurement (SDS/KCl precipitation), genetic deletion (ddi1Δ, wss1Δ), complementation assays, macrophage killing assay Scientific reports Medium 40702097

Source papers

Stage 0 corpus · 32 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2016 Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1. eLife 198 27528192
2005 The DNA damage-inducible UbL-UbA protein Ddi1 participates in Mec1-mediated degradation of Ho endonuclease. Molecular and cellular biology 85 15964793
2006 Ddi1, a eukaryotic protein with the retroviral protease fold. Journal of molecular biology 65 17010377
2020 The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast. Molecular cell 64 31902667
2011 Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1. BMC biology 62 21627799
2020 Ddi1 is a ubiquitin-dependent protease. Proceedings of the National Academy of Sciences of the United States of America 61 32193351
2015 DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin. Structure (London, England : 1993) 61 25703377
2006 Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes. Molecular and cellular biology 51 16478980
2008 Different domains of the UBL-UBA ubiquitin receptor, Ddi1/Vsm1, are involved in its multiple cellular roles. Molecular biology of the cell 47 18562697
2011 HIV proteinase inhibitors target the Ddi1-like protein of Leishmania parasites. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 38 21266539
2016 Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family. Scientific reports 36 27646017
2019 The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response. DNA repair 35 31276951
1997 Bidirectional regulation of two DNA-damage-inducible genes, MAG1 and DDI1, from Saccharomyces cerevisiae. Molecular microbiology 32 9157248
2012 Ddi1-like protein from Leishmania major is an active aspartyl proteinase. Cell stress & chaperones 28 22933181
2004 Pdr3 is required for DNA damage induction of MAG1 and DDI1 via a bi-directional promoter element. Nucleic acids research 24 15452273
2018 Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome. Human molecular genetics 23 29788202
2010 The retroviral proteinase active site and the N-terminus of Ddi1 are required for repression of protein secretion. FEBS letters 20 21094643
2001 Two alternative cell cycle checkpoint pathways differentially control DNA damage-dependent induction of MAG1 and DDI1 expression in yeast. Molecular genetics and genomics : MGG 19 11713673
1997 UAS(MAG1), a yeast cis-acting element that regulates the expression of MAG1, is located within the protein coding region of DDI1. Molecular & general genetics : MGG 18 9294038
1998 Differential regulation of two closely clustered yeast genes, MAG1 and DDI1, by cell-cycle checkpoints. Nucleic acids research 17 9826765
2022 HIV protease inhibitor nelfinavir is a potent drug candidate against echinococcosis by targeting Ddi1-like protein. EBioMedicine 14 35843171
2019 Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination. Frontiers in physiology 14 31130875
2014 The tomato DWD motif-containing protein DDI1 interacts with the CUL4-DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses. Biochemical and biophysical research communications 14 25017913
2012 Transfer of Ho endonuclease and Ufo1 to the proteasome by the UbL-UbA shuttle protein, Ddi1, analysed by complex formation in vitro. PloS one 13 22815701
2020 Normal spermatogenesis and fertility in Ddi1 (DNA damage inducible 1) mutant mice. Reproductive biology 11 33092996
2020 Dimer Interface Organization is a Main Determinant of Intermonomeric Interactions and Correlates with Evolutionary Relationships of Retroviral and Retroviral-Like Ddi1 and Ddi2 Proteases. International journal of molecular sciences 10 32079302
2021 Artemisinin Binds and Inhibits the Activity of Plasmodium falciparum Ddi1, a Retroviral Aspartyl Protease. Pathogens (Basel, Switzerland) 9 34832620
2023 Plasmodium DDI1 is a potential therapeutic target and important chromatin-associated protein. International journal for parasitology 4 36657610
2023 The Effects of DDI1 on Inducing Differentiation in Ovine Preadipocytes via Oar-miR-432. International journal of molecular sciences 4 37511326
2024 Domains in Action: Understanding Ddi1's Diverse Functions in the Ubiquitin-Proteasome System. International journal of molecular sciences 3 38612889
2022 Structural and functional insights into the DNA damage-inducible protein 1 (Ddi1) from protozoa. Current research in structural biology 2 35677776
2025 Wss1 and Ddi1 DNA-Protein crosslink repair proteases protect Saccharomyces cerevisiae and Candida albicans against oxidative stress. Scientific reports 0 40702097

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