| 2018 |
Crystal structures of human GID4 alone and in complex with various Pro/N-degrons revealed the molecular mechanism of GID4-mediated Pro/N-degron recognition: GID4 binds substrates bearing N-terminal proline residues through a specific binding pocket, establishing GID4 as the N-recognin of the Pro/N-end rule pathway within the GID ubiquitin ligase complex. |
X-ray crystallography (crystal structures of apo and peptide-bound GID4) combined with biophysical analyses |
Nature chemical biology |
High |
29632410
|
| 2018 |
In yeast, Gid4 is tightly regulated at both the transcriptional and protein level: it is absent from the constitutive Gid complex and appears within minutes of glucose addition, acting as the inducible substrate recognition subunit that triggers ubiquitination of gluconeogenic enzymes. |
Transcriptional reporter assays, protein-level time-course experiments in Saccharomyces cerevisiae |
FEBS letters |
Medium |
30136317
|
| 2022 |
Small molecules that bind the substrate-recognition pocket of GID4 (subunit of the CTLH E3 complex) can redirect GID4-CTLH to induce proteasomal degradation of genetically recruited neo-substrates, establishing GID4 as a functional E3 ligase amenable to targeted protein degradation. X-ray co-structures revealed the molecular basis of GID4–small molecule interactions. |
NMR-based fragment screen, DNA-encoded library screen, X-ray co-crystallography, in vitro binding assays (Kd), cellular degradation assays |
Journal of medicinal chemistry |
High |
36117290
|
| 2024 |
The chemical probe PFI-7 antagonizes Pro/N-degron binding to human GID4. Proximity-dependent biotinylation and quantitative proteomics using PFI-7 identified GID4 interactors enriched for nucleolar proteins including Pro/N-degron-containing RNA helicases DDX21 and DDX50, and a subset of GID4-regulated proteins including HMGCS1, revealing both degradative and nondegradative functions of GID4. |
Structure-guided chemical probe development, BioID2 proximity labeling, quantitative proteomics, cellular protein-level assays |
Nature chemical biology |
High |
38773330
|
| 2024 |
The hGID complex (via GID4 substrate-binding pocket) binds and ubiquitinates ARHGAP11A, targeting this RhoGAP for proteasomal degradation. GID4 depletion or pocket inhibition with PFI-7 stabilizes ARHGAP11A at the cell periphery, where it inactivates RhoA and impairs cell motility and directed cell migration. |
BioID2 proximity labeling, biochemical ubiquitination assays, GID4 depletion (siRNA/knockout), PFI-7 inhibitor treatment, cell migration assays, RhoA activity assays |
Life science alliance |
High |
39389782
|
| 2025 |
GID4 can be leveraged for PROTAC-mediated targeted protein degradation using a noncovalent small molecule. The PROTAC NEP162 eliminates endogenous BRD4 in a GID4- and ubiquitin-proteasome system-dependent manner. Crystal structures of GID4-PROTAC-BRD4 ternary complexes in three distinct states revealed plastic interactions between GID4 and BRD4, deciphering the molecular basis of neo-substrate recruitment. |
PROTAC design and synthesis, crystal structures of ternary complexes, in vitro ubiquitination assays, GID4 knockout cellular assays, xenograft tumor model |
Nature structural & molecular biology |
High |
40295770
|
| 2016 |
In yeast, loss of Vid24p (Gid4 ortholog) function leads to accumulation of the gluconeogenic enzyme Mdh2p (malate dehydrogenase), and disruption of Mdh2p in a VID24 mutant background abolishes the high-malate-production phenotype, placing Vid24p/Gid4 upstream of Mdh2p degradation in the ubiquitin ligase pathway. |
Genetic disruption (VID24 mutation and MDH2 deletion), metabolite measurements, whole genome comparative analysis in Saccharomyces cerevisiae |
Journal of bioscience and bioengineering |
Medium |
26983942
|
| 2024 |
In yeast, the GID complex E3 ubiquitin ligase and its adaptor Gid4 mediate polyubiquitination of released Rps26 at Lys66 and Lys70, targeting it for degradation via the Pro/N-degron pathway (dependent on the N-terminal proline of Rps26). This degradation enables accumulation of Rps26-deficient specialized ribosomes during high-salt stress and is required for salt stress resistance. |
Ubiquitination site mapping (mass spectrometry), N-terminal proline-to-serine mutation, GID complex/Gid4 genetic deletion, ribosome population analysis in Saccharomyces cerevisiae |
bioRxiv (preprint)preprint |
Medium |
bio_10.1101_2024.08.15.608112
|
| 2026 |
The small molecule CLEO4-88 functions as a molecular glue that binds solely to GID4 and induces an allosteric conformational change that promotes GID4 interaction with the peroxisomal thiolase ACAA1 in vitro and in cells. Ternary complex formation inhibits ACAA1 thiolase activity, but ACAA1 cannot be recruited by GID4 to the full CTLH holoenzyme for ubiquitination. |
Atomic structure determination of ternary complex, in vitro binding and enzymatic assays, cellular interaction assays |
Nature chemical biology |
High |
41957281
|
| 2025 |
Molecular dynamics simulations revealed that GID4's binding pocket undergoes alternating open-closed conformational states mediated by hairpin loops L1–L4 in the apo state, and that substrate recognition follows a combined conformational selection and induced fit mechanism. Mutation of the N-terminal proline reduces its binding contribution but has minimal impact on interactions with other residues. |
Molecular dynamics simulations and binding energy calculations (computational study) |
Biophysical journal |
Low |
40382678
|
| 2024 |
Human GID4 was engineered via directed evolution and yeast-surface display to create N-terminal proline binders with increased binding affinity to Nt-Pro. A specific A252V mutation reduced the influence of residues at positions 2 and 3 following the N-terminal proline on binding, demonstrating that residues beyond the binding pocket rim tune substrate selectivity. |
Directed evolution, yeast-surface display, fluorescence-activated cell sorting (FACS), binding assays |
Biotechnology and bioengineering |
Medium |
39450770
|
| 2025 |
CTLH-MKLN1 complex assemblies (which include GID4) are required for B-cell somatic hypermutation and class switch recombination; Mkln1-/- mice lacking CTLH-MKLN1 showed increased UNG2 levels and reduced antibody diversification, placing CTLH-MKLN1/GID4-containing complexes upstream of UNG2 regulation in B cells. |
Mkln1 knockout mouse model, B-cell functional assays (somatic hypermutation frequency, class switch recombination), protein-level measurements |
Journal of immunology |
Medium |
40838616
|