DNAJC24

DNAJC24 (DPH4/JJJ3) is a type III J-protein cochaperone that catalyzes the first step of diphthamide biosynthesis on translation elongation factor 2 (eEF2) (PMID:15485916, PMID:18765564). It has a bipartite architecture, with a J-domain and a CSL zinc-finger domain joined by a flexible linker-helix, and binds iron in tetrahedral coordination through CSL-domain cysteines; the iron-bound protein oligomerizes and acts as a redox/electron carrier required for diphthamide synthesis (PMID:22367199). In this role its reduced, iron-loaded CSL domain serves as an electron donor to the Fe-S cluster of the Dph1-Dph2 enzyme complex, analogous to bacterial flavodoxins (PMID:24422557). DNAJC24 has a non-redundant function among cytosolic J proteins that cannot be substituted by any other (PMID:17438278), and it localizes to the cytoskeleton rather than partitioning into the DPH1/DPH2/DPH3 complex (PMID:18765564). Loss of DNAJC24 abolishes eEF2 diphthamide modification, conferring resistance to diphtheria toxin and Pseudomonas exotoxin A while sensitizing cells to TNF-mediated apoptosis via pre-activation of NF-κB and death receptor pathways (PMID:26261303); in cancer cells its expression is silenced by reversible promoter CpG hypermethylation (PMID:22509046). Beyond diphthamide biosynthesis, DNAJC24 regulates podocyte adhesion and cytoskeletal spreading (PMID:31566424), and in lung adenocarcinoma it interacts with PCNA and drives PI3K/AKT phosphorylation to promote proliferation and invasion (PMID:38713100).

1. 2004 Medium

   Established that DNAJC24/DPH4 is genetically required for diphthamide modification of eEF2, defining its core biological function and conservation from yeast to mammals.

   Evidence Genetic complementation, sequence homology, and functional knockout across yeast and mammals

   PMID:15485916

   Open questions at the time

   • Did not define the biochemical step DPH4 performs
   • No structural or mechanistic detail of how it supports diphthamide synthesis
2. 2007 Medium

   Showed that the cytosolic J protein Jjj3/DPH4 performs a specialized, non-redundant function distinct from all other cytosolic J proteins.

   Evidence Comprehensive cross-rescue screen of 13 yeast cytosolic J-protein deletion strains

   PMID:17438278

   Open questions at the time

   • Did not identify the molecular basis of its specialized role
   • Performed in yeast; human specificity inferred
3. 2008 High

   Confirmed in mammals that DPH4 loss abolishes eEF2 diphthamide and confers diphtheria toxin resistance, and localized the protein to the cytoskeleton and outside the DPH1/DPH2/DPH3 complex.

   Evidence Mouse genetic mutant analysis, eEF2 modification biochemistry, toxin resistance, and reporter localization imaging

   PMID:18765564

   Open questions at the time

   • Mechanism of how a non-complex protein contributes to the DPH1-DPH2 reaction unresolved
   • Functional meaning of cytoskeletal localization unclear
4. 2012 High

   Resolved the bipartite J-domain/CSL-domain structure and demonstrated iron binding through CSL cysteines with redox/electron-carrier activity, providing a biochemical mechanism for its role in diphthamide synthesis.

   Evidence NMR structure, UV-vis and EPR spectroscopy of iron-bound form, in vitro redox and oligomerization assays, cross-species comparison

   PMID:22367199

   Open questions at the time

   • Did not directly demonstrate electron transfer to the Dph1-Dph2 complex
   • Physiological relevance of iron-dependent Hsp70 functions not established
5. 2014 High

   Defined the electron-donor mechanism by which the reduced iron-bound CSL protein feeds electrons to the Fe-S cluster of the Dph1-Dph2 complex for the first diphthamide step.

   Evidence In vitro reconstitution of the first diphthamide step, EPR spectroscopy, iron-binding and electron-donor assays (studying the equivalent CSL protein Dph3/KTI11)

   PMID:24422557

   Open questions at the time

   • Direct biochemical demonstration was on Dph3/KTI11 with DPH4 inferred by analogy
   • Relative contributions of DPH3 vs DPH4 as electron donors not delineated
6. 2012 High

   Connected DPH4 expression to drug response by showing epigenetic silencing via promoter CpG hypermethylation prevents diphthamide synthesis and confers immunotoxin resistance.

   Evidence Immunotoxin-resistant cell lines, EF2 ADP-ribosylation assays, methylation analysis, and 5-azacytidine rescue

   PMID:22509046

   Open questions at the time

   • Trigger of the methylation event in resistant cells unknown
   • In vivo relevance to clinical toxin resistance not addressed
7. 2015 High

   Demonstrated that complete DPH4 knockout pre-activates NF-κB and death receptor pathways, sensitizing cells to TNF apoptosis while conferring toxin resistance, linking diphthamide loss to broader cellular signaling.

   Evidence CRISPR knockout in MCF7 cells with eEF2 modification verification, toxin and TNF apoptosis assays, and NF-κB activity measurements

   PMID:26261303

   Open questions at the time

   • Mechanistic link between loss of diphthamide and NF-κB pre-activation not defined
   • Whether the TNF phenotype is mediated solely through eEF2 unclear
8. 2019 Medium

   Extended DPH4 function to podocyte biology, showing its loss alters adhesion and cytoskeletal spreading via the diphthamide pathway.

   Evidence Genome-scale RNAi screen, shRNA and CRISPR validation in human podocytes, and Drosophila nephrocyte knockdown

   PMID:31566424

   Open questions at the time

   • Mechanistic connection from diphthamide synthesis to adhesion is inferential
   • Effector linking DPH4 to cytoskeletal organization unidentified
9. 2024 Medium

   Identified a diphthamide-independent oncogenic role in which DNAJC24 binds PCNA and activates PI3K/AKT signaling to promote tumor proliferation and invasion.

   Evidence Co-IP/mass spectrometry, AKT phosphorylation western blot, and knockdown/overexpression proliferation and invasion assays in LUAD cell lines

   PMID:38713100

   Open questions at the time

   • Single lab; PCNA interaction lacks reciprocal validation
   • Whether AKT activation is direct or downstream of diphthamide function unresolved

• How DNAJC24's iron/redox cochaperone activity is mechanistically connected to its downstream cellular phenotypes (NF-κB pre-activation, podocyte adhesion, PCNA/AKT-driven proliferation) remains unresolved.
• No structural model of DPH4 within the diphthamide enzyme machinery in vivo
• Whether the cancer and podocyte roles depend on diphthamide synthesis or represent moonlighting functions is unknown