| 1995 |
GMEB1 (88 kDa) and GMEB2 (67 kDa) were partially purified and characterized as two novel proteins that bind the glucocorticoid modulatory element (GME/CRE) as a heteromeric macromolecular complex of ~550 kDa (dissociable by deoxycholate), distinct from CREB/CREM/ATF family members, and capable of modulating glucocorticoid receptor transactivation properties (EC50 shift and partial agonist activity of antagonists). |
Partial protein purification, gel shift assays, size exclusion chromatography, molecular weight determination, partial peptide sequencing |
The Journal of biological chemistry |
High |
7665613
|
| 1998 |
GMEB2 (67 kDa, cloned by PCR/RACE) binds GME DNA in gel shift assays; its binding to GME DNA increases markedly when mixed with authentic GMEB-1, forming a heteromeric complex similar to that from HTC cell cytosol. GMEB2 shares the KDWKR domain with Drosophila DEAF-1, rat Suppressin, and C. elegans ORFs, defining a novel protein family. |
cDNA cloning, in vitro transcription/translation, gel shift assay, co-incubation with GMEB-1 |
The Journal of biological chemistry |
High |
9651376
|
| 1999 |
Human GMEB1 (hGMEB1, 573 aa, 85 kDa) was cloned using HSP27 as bait in a yeast two-hybrid screen. In vitro translated hGMEB1 bound specifically to GME oligonucleotides, forming a complex of similar size to that from rat liver nuclear extracts (confirmed by supershift with anti-hGMEB1 antibody). Co-immunoprecipitation confirmed the in vivo interaction of HSP27 with hGMEB1. |
Yeast two-hybrid screening, cDNA cloning, in vitro translation, EMSA/gel shift with supershift, co-immunoprecipitation |
FEBS letters |
High |
10386584
|
| 2000 |
GMEB-1 interacts with glucocorticoid receptor (GR) as shown by mammalian two-hybrid and pull-down assays. GMEB-1 possesses intrinsic transactivation activity in mammalian one-hybrid assays. Overexpression of GMEB-1 (alone or with GMEB-2) causes a reversible right shift in the GR dose-response curve and decreased partial agonist activity of antisteroids. Both GMEBs interact with CREB-binding protein (CBP) in two-hybrid assays; neither possesses histone acetyltransferase (HAT) activity. GMEB-1 and -2 share a 90-aa region (~80% identical) containing the KDWK core. |
Mammalian two-hybrid assay, GST pull-down, mammalian one-hybrid transactivation assay, reporter gene assay, HAT activity assay |
Molecular endocrinology (Baltimore, Md.) |
High |
10894151
|
| 2000 |
GME activity requires positioning within ~250 bp upstream of a tandem GRE driving a complex promoter; phasing between GME and downstream GREs is unimportant (unlike GREs); the GME does not affect GR binding to a single GRE. Changes in GME activity did not correlate with fold induction from the GRE, indicating that GME and GRE activities utilize parallel rather than common pathways. |
Transient transfection reporter gene assays with positional/distance mutants, gel shift assays |
Molecular and cellular endocrinology |
Medium |
10854715
|
| 2000 |
Mouse GMEB-1 was identified by yeast two-hybrid screening using the activation domain 2 of nuclear receptor coactivator TIF2 as bait. In vitro translated mGMEB-1 bound specifically to GME oligonucleotides alone and as a heterodimer with rGMEB-2. Transient transfection with TAT promoter reporter genes indicated a role as a transcriptional regulator of the TAT promoter. |
Yeast two-hybrid screening, in vitro translation, EMSA/gel shift, transient transfection reporter assay |
FEBS letters |
Medium |
10692587
|
| 2002 |
GMEB-1 domains were mapped for distinct activities: homooligomerization, heterooligomerization, DNA binding, binding to GR, binding to CBP, and GR modulation each require defined regions. The domain for GR modulation and the domain for intrinsic transactivation activity do not overlap, providing a structural basis for the independence of dose-response curve modulation from total levels of GR-induced gene expression. |
Deletion/truncation mutagenesis, mammalian two-hybrid assay, DNA binding assays, reporter gene assays |
The Journal of biological chemistry |
High |
11934901
|
| 2002 |
MURF-1 binds GMEB-1 (a transcriptional regulator) in vitro. Endogenous MURF-1 was detected in nuclei of some myocytes, suggesting that MURF-1's interaction with GMEB-1 may link myofibril signaling to muscle gene expression. |
In vitro binding assay, immunofluorescence/subcellular fractionation |
The Journal of cell biology |
Medium |
11927605
|
| 2002 |
GMEBs contact Ubc9 (mammalian E2 SUMO-conjugating enzyme), which also binds GR. Ubc9 modifies GR-induced gene expression (amount, fold induction, EC50, partial agonist activity) in a manner independent of its SUMO-1 transfer activity. The GME is proposed to act by increasing the local concentration of Ubc9 near the transcription machinery. |
Co-immunoprecipitation, reporter gene assay, transfection with Ubc9 mutants (SUMO-transfer deficient) |
The Journal of biological chemistry |
Medium |
11812797
|
| 2003 |
The 1.55 Å crystal structure of the GMEB1 SAND domain was determined. NMR and binding studies mapped DNA recognition to an alpha-helical region exposing the conserved KDWK motif. Site-directed mutagenesis identified key residues for DNA binding. The GMEB1 SAND domain also contains a zinc-binding motif (absent in Sp100b SAND domain); zinc is not required for DNA binding but determines C-terminal conformation of the domain. |
X-ray crystallography (1.55 Å), NMR spectroscopy, site-directed mutagenesis, DNA binding assays |
Molecular endocrinology (Baltimore, Md.) |
High |
12702733
|
| 2004 |
GMEB1 binds to the prodomain (CARD) of procaspase-2 and inhibits its autoproteolytic activation by oligomerization in a chemical compound-dependent system, identifying GMEB1 as an endogenous inhibitor of procaspase-2 autoactivation. |
Co-immunoprecipitation, in vitro binding to procaspase-2 prodomain, caspase activation assay |
Biochemical and biophysical research communications |
Medium |
15555560
|
| 2004 |
GMEB-2 structure/activity relationships mirror those of GMEB-1: homo- and heterooligomerization, GR binding, CBP binding, DNA binding, and GR transactivation modulation each require large regions of the protein; only intrinsic transactivation activity could be localized to a small region. Quantitative differences between GMEB-1 and -2 activities arise from amino acid sequence variation rather than global structural differences. |
Deletion/truncation mutagenesis, mammalian two-hybrid assay, DNA binding assays, reporter gene assays |
Biochemistry |
Medium |
14705952
|
| 2006 |
GMEB1 binds to procaspase-8 and procaspase-9 (in addition to procaspase-2) via their prodomains. GMEB1 attenuates Fas-mediated caspase-8 and -9 activation and subsequent apoptosis. siRNA knockdown of endogenous GMEB1 renders cells more sensitive to stress-induced apoptosis. Transgenic mice with neurospecific GMEB1 overexpression exhibit smaller cerebral infarcts and less brain swelling after transient focal ischemia. |
Co-immunoprecipitation, siRNA knockdown, caspase activation assays, transgenic mouse model with focal ischemia |
The Journal of biological chemistry |
High |
16497673
|
| 2008 |
GMEB1 prevents caspase activation and apoptosis in human neuroblastoma SK-N-MC cells subjected to hypoxia or oxidative stress, confirming its role as an endogenous inhibitor of initiator caspase activation in response to diverse stress stimuli. |
Caspase activation assays, cell viability/apoptosis assays under hypoxia and oxidative stress |
Neuroscience letters |
Medium |
18455874
|
| 2009 |
GMEB1/PIF p96 interacts with the N-terminal domain of RAG1 as identified by yeast two-hybrid assay. A WW-like motif within RAG1's N-terminal domain mediates this interaction; point mutations at conserved WW residues abolished binding. A luciferase reporter assay demonstrated that a protein complex containing RAG proteins and GMEB1 can assemble in cells. |
Yeast two-hybrid assay, point mutagenesis, luciferase reporter assay |
Nucleic acids research |
Medium |
19324890
|
| 2011 |
IL-12 induces GMEB1 expression in human T cells. siRNA knockdown of GMEB1 reverses the protective effect of IL-12 on dexamethasone-induced T cell apoptosis, placing GMEB1 downstream of IL-12/PI3K/Akt signaling as a mediator of anti-apoptotic protection against glucocorticoid-induced apoptosis. |
siRNA knockdown, apoptosis assays (flow cytometry), qPCR, PI3K inhibitor treatment |
Immunobiology |
Medium |
21840619
|
| 2012 |
GMEB1 was identified as a novel binding partner of FOXL2 transcription factor by yeast two-hybrid screening and confirmed by co-immunoprecipitation. GMEB1 is sequestered in aggregates formed by BPES-causing FOXL2 mutants. On most promoters GMEB1 acts as a transcriptional repressor; it increases wild-type FOXL2 activity on the Per2 promoter and to a greater extent increases the activity of the oncogenic p.C134W FOXL2 variant. |
Yeast two-hybrid screening, co-immunoprecipitation, immunofluorescence (aggregate sequestration), luciferase reporter assays |
Human molecular genetics |
Medium |
22544055
|
| 2019 |
GMEB1 interacts with CFLARL (c-FLIPL) in the cytosol and promotes its stability. The deubiquitinase USP40 catalyzes K48-linked deubiquitination of CFLARL; GMEB1 acts as a bridge protein promoting the binding of USP40 to CFLARL. USP40 knockdown abolishes GMEB1-mediated CFLARL stabilization. GMEB1 inhibits pro-caspase-8 activation and DISC formation upon TRAIL stimulation; CFLARL enhances the binding of GMEB1 to CASP8. GMEB1 knockdown inhibits A549 xenograft tumor growth in vivo. |
Co-immunoprecipitation, GST pull-down, Western blot (ubiquitination), immunofluorescence, flow cytometry (apoptosis), shRNA knockdown, xenograft mouse model |
Journal of experimental & clinical cancer research : CR |
High |
31046799
|
| 2019 |
In midbrain dopamine (mDA) neurons, Gmeb1 was identified as a transcription factor regulating expression of Th (tyrosine hydroxylase) and Dat (dopamine transporter). Gmeb1 knockdown in mDA neurons caused downregulation of Th and Dat and severe motor deficits, establishing Gmeb1 as a master regulator of mDA gene expression and function. |
Virus-based nuclear capture, RNA-seq, DNase-seq (chromatin accessibility), predictive modeling, in vivo AAV-mediated Gmeb1 knockdown with behavioral readout |
Nature communications |
High |
31175277
|
| 2020 |
TRAF3 interacts with GMEB1 as identified by yeast two-hybrid screening of a human B cell cDNA library and confirmed by co-immunoprecipitation in mammalian cells. TRAF3 overexpression enhances GMEB1's anti-apoptotic function; TRAF3 siRNA knockdown significantly reduces GMEB1-mediated inhibition of apoptosis. The RING and TRAF-C domains of TRAF3 are not required for this interaction. |
Yeast two-hybrid screening, co-immunoprecipitation, siRNA knockdown, cell viability/apoptosis assays |
Journal of biological research (Thessalonike, Greece) |
Medium |
32514408
|
| 2021 |
CircGlis3 promotes the degradation of GMEB1 by facilitating the interaction between GMEB1 and the E3 ubiquitin ligase MIB2, thereby suppressing GMEB1-dependent phosphorylation of HSP27. |
RNA pull-down, co-immunoprecipitation, Western blot (protein stability/ubiquitination), gain/loss-of-function assays |
Diabetologia |
Medium |
34751796
|
| 2023 |
GMEB1 binds to the YAP1 promoter region and positively regulates YAP1 expression in hepatocellular carcinoma cells. GMEB1 binding to the YAP1 promoter was confirmed by dual-luciferase reporter assay and chromatin immunoprecipitation-qPCR. |
Chromatin immunoprecipitation-qPCR, dual-luciferase reporter assay, Western blot, qRT-PCR, gain/loss-of-function (overexpression/knockdown) |
World journal of gastrointestinal oncology |
Medium |
37389116
|