scholarly journals Turning Off Estrogen Receptor β-Mediated Transcription Requires Estrogen-Dependent Receptor Proteolysis

2006 ◽  
Vol 26 (21) ◽  
pp. 7966-7976 ◽  
Author(s):  
Yukiyo Tateishi ◽  
Raku Sonoo ◽  
Yu-ichi Sekiya ◽  
Nanae Sunahara ◽  
Miwako Kawano ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Active Form ◽  
Estrogen Receptor Β ◽  
Interacting Protein ◽  
Amino Acid Region ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Interfering Rna ◽  
Shed Light ◽  
Acid Region

ABSTRACT Recent studies have shed light on the ligand-dependent transactivation mechanisms of nuclear receptors (NRs). When the ligand dose is reduced, the transcriptional activity of NRs should be downregulated. Here we show that a ubiquitin-proteasome pathway plays a key role in turning off transcription mediated by estrogen receptor β (ERβ). ERβ shows estrogen-dependent proteolysis, and its degradation is regulated by two regions in the receptor. The N-terminal 37-amino acid-region is necessary for the recruitment of the ubiquitin ligase, i.e., the carboxyl terminus of HSC70-interacting protein (CHIP), to degrade ERβ. In contrast, the C-terminal F domain protects ligand-unbound ERβ from proteolysis to abrogate proteasome association. Suppression of CHIP by interfering RNA inhibited this switching off of receptor-mediated transcription when the ligand dose was reduced. Our results suggest that after ligand withdrawal, the active form of the NR is selectively eliminated via ligand-dependent proteolysis to downregulate receptor-mediated transcription.

scholarly journals A Unique Protection Signal in Cubitus interruptus Prevents Its Complete Proteasomal Degradation

2008 ◽  
Vol 28 (18) ◽  
pp. 5555-5568 ◽  
Author(s):  
Yifei Wang ◽  
Mary Ann Price
Keyword(s):  
Zinc Finger ◽  
Limited Proteolysis ◽  
Cubitus Interruptus ◽  
Ubiquitin Proteasome Pathway ◽  
Amino Acid Region ◽  
C Terminus ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Zinc Finger Region ◽  
Acid Region

ABSTRACT The limited proteolysis of Cubitus interruptus (Ci), the transcription factor for the developmentally and medically important Hedgehog (Hh) signaling pathway, triggers a critical switch between transcriptional repressor and activator forms. Ci repressor is formed when the C terminus of full-length Ci is degraded by the ubiquitin-proteasome pathway, an unusual reaction since the proteasome typically completely degrades its substrates. We show that several regions of Ci are required for generation of the repressor form: the zinc finger DNA binding domain, a single lysine residue (K750) near the degradation end point, and a 163-amino-acid region at the C terminus. Unlike other proteins that are partially degraded by the proteasome, dimerization is not a key feature of Ci processing. Using a pulse-chase assay in cultured Drosophila cells, we distinguish between regions required for initiation of degradation and those required for the protection of the Ci N terminus from degradation. We present a model whereby the zinc finger region and K750 together form a unique protection signal that prevents the complete degradation of Ci by the proteasome.

scholarly journals The yeast silent information regulator Sir4p anchors and partitions plasmids.

1997 ◽  
Vol 17 (12) ◽  
pp. 7061-7068 ◽  
Author(s):  
A Ansari ◽  
M R Gartenberg
Keyword(s):  
Coiled Coil ◽  
Axial Rotation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interacting Protein ◽  
Segregation Behavior ◽  
Amino Acid Region ◽  
Dividing Cells ◽  
Nuclear Component ◽  
Acid Region

Circular plasmids containing telomeric TG1-3 arrays or the HMR E silencer segregate efficiently between dividing cells of the yeast Saccharomyces cerevisiae. Subtelomeric X repeats augment the TG1-3 partitioning activity by a process that requires the SIR2, SIR3, and SIR4 genes, which are also required for silencer-based partitioning. Here we show that targeting Sir4p to DNA directly via fusion to the bacterial repressor LexA confers efficient mitotic segregation to otherwise unstable plasmids. The Sir4p partitioning activity resides within a 300-amino-acid region (residues 950 to 1262) which precedes the coiled-coil dimerization motif at the extreme carboxy end of the protein. Using a topology-based assay, we demonstrate that the partitioning domain also retards the axial rotation of LexA operators in vivo. The anchoring and partitioning properties of LexA-Sir4p chimeras persist despite the loss of the endogenous SIR genes, indicating that these functions are intrinsic to Sir4p and not to a complex of Sir factors. In contrast, inactivation of the Sir4p-interacting protein Rap1p reduces partitioning by a LexA-Sir4p fusion. The data are consistent with a model in which the partitioning and anchoring domain of Sir4p (PAD4 domain) attaches to a nuclear component that divides symmetrically between cells at mitosis; DNA linked to Sir4p by LexA serves as a reporter of protein movement in these experiments. We infer that the segregation behavior of telomere- and silencer-based plasmids is, in part, a consequence of these Sir4p-mediated interactions. The assays presented herein illustrate two novel approaches to monitor the intracellular dynamics of nuclear proteins.

scholarly journals MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

2008 ◽  
Vol 19 (3) ◽  
pp. 899-911 ◽  
Author(s):  
Shoshiro Hirayama ◽  
Yuji Yamazaki ◽  
Akira Kitamura ◽  
Yukako Oda ◽  
Daisuke Morito ◽  
...  
Keyword(s):  
Heat Shock ◽  
Ubiquitin Ligase ◽  
Wild Type ◽  
Hsp 70 ◽  
Human Genetic Disease ◽  
Interacting Protein ◽  
Bardet Biedl Syndrome ◽  
Heat Shock Cognate 70 ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

McKusick–Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet–Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin–proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.

scholarly journals Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

2008 ◽  
Vol 19 (4) ◽  
pp. 1328-1336 ◽  
Author(s):  
Daisuke Morito ◽  
Kazuyoshi Hirao ◽  
Yukako Oda ◽  
Nobuko Hosokawa ◽  
Fuminori Tokunaga ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Yeast Cells ◽  
Glutathione S Transferase ◽  
Assembly Factor ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Membrane Spanning ◽  
Interfering Rna

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.

scholarly journals Proteins containing ubiquitin-like (Ubl) domains not only bind to 26S proteasomes but also induce their activation

2020 ◽  
Vol 117 (9) ◽  
pp. 4664-4674 ◽  
Author(s):  
Galen A. Collins ◽  
Alfred L. Goldberg
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Phosphatase ◽  
Atp Hydrolysis ◽  
Active Form ◽  
Protein Homeostasis ◽  
Cellular Functions ◽  
Peptide Hydrolysis ◽  
Unfolded Protein ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

During protein degradation by the ubiquitin–proteasome pathway, latent 26S proteasomes in the cytosol must assume an active form. Proteasomes are activated when ubiquitylated substrates bind to them and interact with the proteasome-bound deubiquitylase Usp14/Ubp6. The resulting increase in the proteasome’s degradative activity was recently shown to be mediated by Usp14’s ubiquitin-like (Ubl) domain, which, by itself, can trigger proteasome activation. Many other proteins with diverse cellular functions also contain Ubl domains and can associate with 26S proteasomes. We therefore tested if various Ubl-containing proteins that have important roles in protein homeostasis or disease also activate 26S proteasomes. All seven Ubl-containing proteins tested—the shuttling factors Rad23A, Rad23B, and Ddi2; the deubiquitylase Usp7, the ubiquitin ligase Parkin, the cochaperone Bag6, and the protein phosphatase UBLCP1—stimulated peptide hydrolysis two- to fivefold. Rather than enhancing already active proteasomes, Rad23B and its Ubl domain activated previously latent 26S particles. Also, Ubl-containing proteins (if present with an unfolded protein) increased proteasomal adenosine 5′-triphosphate (ATP) hydrolysis, the step which commits substrates to degradation. Surprisingly, some of these proteins also could stimulate peptide hydrolysis even when their Ubl domains were deleted. However, their Ubl domains were required for the increased ATPase activity. Thus, upon binding to proteasomes, Ubl-containing proteins not only deliver substrates (e.g., the shuttling factors) or provide additional enzymatic activities (e.g., Parkin) to proteasomes, but also increase their capacity for proteolysis.

scholarly journals BAG2-Mediated Inhibition of CHIP Expression and Overexpression of MDM2 Contribute to the Initiation of Endometriosis by Modulating Estrogen Receptor Status

2021 ◽  
Vol 8 ◽  
Author(s):  
Li-Juan Chen ◽  
Bin Hu ◽  
Zhi-Qiang Han ◽  
Jian-Hua Zhu ◽  
Xu Fan ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Molecular Mechanisms ◽  
Estrogenic Activity ◽  
Estrogen Receptor Status ◽  
Therapeutic Effects ◽  
Childbearing Age ◽  
E3 Ligases ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Endometriosis is an estrogen-dependent gynecological disease primarily affecting women of childbearing age, which gives rise to pelvic pain calling for multiple operations, and sometimes leading to infertility. However, the etiology of endometriosis remains poorly understood. In this study we investigated the roles of two Ubiquitin E3 Ligases, namely hsc70-interacting protein (CHIP) and mouse double minute 2 (MDM2), in the abnormal estrogenic activity in endometriosis. We first collected endometrial tissues from 91 cases of endometriosis and 78 cases of uterine myomas. Next, we established a murine endometriosis model by ectopic endometrial tissue implantation. In other studies, we isolated human endometrial stromal cells (HESCs) were isolated from the endometrial tissues, and performed HA- or FLAG-immunoprecipitation assays and immunoblotting with an anti-ubiquitin antibody to test the interactions among BAG2, CHIP, MDM2, estrogen receptor α (ERα), and ERβ. The expression of ERα was downregulated while that of ERβ, BAG2, and MDM2 was upregulated in human endometriosis and in the mouse model. CHIP degraded ERβ instead of ERα via the ubiquitin-proteasome pathway, while BAG2 impaired the CHIP-mediated degradation of ERβ in cultured HESCs derived from human endometriosis. The degradation of ERα by MDM2 in cultured endometriosis-HESCs also occurred through the ubiquitin-proteasome pathway. Knockdown of both BAG2 and MDM2 alleviated the development of endometriosis in mice. Our findings suggest that the interference of BAG2 and MDM2 may have therapeutic effects in endometriosis. Understanding better the molecular mechanisms underlying the regulation of the abnormal estrogenic activity in endometriosis is crucial for the advancement of targeted therapeutic strategies.

scholarly journals MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters

2003 ◽  
Vol 23 (5) ◽  
pp. 1656-1665 ◽  
Author(s):  
Francesca Lembo ◽  
Raffaela Pero ◽  
Tiziana Angrisano ◽  
Carmen Vitiello ◽  
Rodolfo Iuliano ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Interacting Protein ◽  
Amino Acid Region ◽  
Gtp Binding ◽  
Acid Region

ABSTRACT We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.

scholarly journals Posttranslational regulation of thioredoxin-interacting protein

2012 ◽  
Vol 50 (1) ◽  
pp. 59-71 ◽  
Author(s):  
Katherine A Robinson ◽  
Jonathan W Brock ◽  
Maria G Buse
Keyword(s):  
Kinase Inhibitor ◽  
Pi3 Kinase ◽  
Interacting Protein ◽  
Hek 293 ◽  
Ubiquitin Proteasome Pathway ◽  
Thioredoxin Interacting Protein ◽  
Protein Levels ◽  
L6 Myotubes ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Thioredoxin-interacting protein (Txnip) is a metabolic regulator, which modulates insulin sensitivity and likely plays a role in type 2 diabetes. We studied the regulation of Txnip in 3T3-L1 adipocytes. Cells were incubated under different conditions and Txnip was measured by immunoblotting. We confirmed that high glucose markedly increases Txnip expression by promoting transcription. Insulin decreases Txnip protein levels. Rapamycin under most conditions decreased Txnip, suggesting that mTOR complex-1 is involved. The acute effects of insulin are mainly posttranscriptional; insulin (100 nM) accelerates Txnip degradation more than tenfold. This effect is cell type specific. It works in adipocytes, preadipocytes and in L6 myotubes but not in HepG2 or in HEK 293 cells or in a pancreatic β-cell line. The ubiquitin/proteasome pathway is involved. Degradation of Txnip occurred within 15 min in the presence of 3 nM insulin and overnight with 0.6 nM insulin. Proteasomal Txnip degradation is not mediated by a cysteine protease or an anti-calpain enzyme. Okadaic acid (OKA), an inhibitor of phosphoprotein phosphatases (pp), markedly reduced Txnip protein and stimulated its further decrease by insulin. The latter occurred after incubation with 1 or 1000 nM OKA, suggesting that insulin enhances the phosphorylation of a pp2A substrate. Incubation with 0.1 μM Wortmannin, a PI3 kinase inhibitor, increased Txnip protein twofold and significantly inhibited its insulin-induced decrease. Thus, while OKA mimics the effect of insulin, Wortmannin opposes it. In summary, insulin stimulates Txnip degradation by a PI3 kinase-dependent mechanism, which activates the ubiquitin/proteasome pathway and likely serves to mitigate insulin resistance.

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