scholarly journals CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

2008 ◽  
Vol 181 (6) ◽  
pp. 959-972 ◽  
Author(s):  
Xueni Li ◽  
Mei Huang ◽  
Huiling Zheng ◽  
Yinyin Wang ◽  
Fangli Ren ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Osteoblast Differentiation ◽  
Degradation Mechanism ◽  
Interacting Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
C Terminus ◽  
Osteoblast Lineage

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

scholarly journals Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity in RPTEC/TERT1 cells

2020 ◽  
Vol 9 (2) ◽  
pp. 91-100 ◽  
Author(s):  
Xuan Qiu ◽  
Yufa Miao ◽  
Xingchao Geng ◽  
Xiaobing Zhou ◽  
Bo Li
Keyword(s):  
Transcriptional Activation ◽  
Messenger Rna ◽  
Aristolochic Acid ◽  
Kidney Injury ◽  
Drug Induced ◽  
Vitro Assay ◽  
Protein Levels ◽  
Aristolochic Acid I

Abstract There have been intensive efforts to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage before significant impairment occurs. Kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β2-microglobulin and cystatin C (CysC) have been validated as clinical or preclinical biomarkers in urinary and plasma predictive of acute and chronic kidney injuries and diseases. A high-throughput in vitro assay predictive of nephrotoxicity could potentially be implemented in early drug discovery stage to reduce attrition at later stages of drug development. To assess the potential of these known in vivo biomarkers for in vitro evaluation of drug-induced nephrotoxicity, we selected four nephrotoxic agents (cisplatin, cyclosporin, aristolochic acid I and gentamicin) and detected their effects on the protein levels of nephrotoxic biomarkers in RPTEC/TERT1 cells. The protein levels of clusterin, CysC, GSTπ and TIMP-1 significantly increased in the conditioned media of RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin. The messenger RNA levels of clusterin, CysC, GSTπ and TIMP-1 also increased in RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin, indicating that drug-induced upregulation involves transcriptional activation. Taken together, the results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, clusterin, CysC, GSTπ and TIMP-1 can be effectively used as in vitro biomarkers for drug-induced nephrotoxicity in RPTEC/TERT1 cells.

scholarly journals WBP-2, a WW domain binding protein, interacts with the thyroid-specific transcription factor Pax8

2004 ◽  
Vol 377 (3) ◽  
pp. 553-560 ◽  
Author(s):  
Roberto NITSCH ◽  
Tina DI PALMA ◽  
Anna MASCIA ◽  
Mariastella ZANNINI
Keyword(s):  
Protein Interaction ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Interaction Domain ◽  
Ww Domain ◽  
Transcriptional Activation Domain ◽  
Culture Cells ◽  
C Terminus

The Pax gene family encodes transcription factors that are essential in organogenesis and in the differentiation of various organs in higher eukaryotes. Pax proteins have a DNA binding domain at the N-terminus, and a transcriptional activation domain at the C-terminus. How these domains interact with the transcriptional machinery of the cell is still unclear. In the present paper, we describe the identification by means of immunological screening of the WW domain binding protein WBP-2 as a biochemical interactor of Pax8 (a WW domain is a protein-interaction domain containing two conserved tryptophan residues). Pax8 is required for the morphogenesis of the thyroid gland and for the maintenance of the thyroid differentiated cellular phenotype. WBP-2 was identified originally as a WW domain binding protein, and its function is still unknown. WBP-2 binds to Pax8 in vitro in pulldown assays, and in vivo in tissue culture cells in co-immunoprecipitation assays. Interestingly, Pax8 does not contain a WW domain. Our results point to the identification of a new protein-interacting domain that is present in the C-terminal portion of Pax8 and that is required for protein–protein interaction with WBP-2. Our results demonstrate that WBP-2 is not a transcriptional co-activator of Pax8, but rather behaves as an adaptor molecule, as suggested in other studies.

scholarly journals Ketamine induces endoplasmic reticulum stress in rats and SV-HUC-1 human uroepithelial cells by activating NLRP3/TXNIP aix

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Lingjuan Cui ◽  
Xiaoyan Jiang ◽  
Chengjun Zhang ◽  
Danxia Li ◽  
Shengqiang Yu ◽  
...  
Keyword(s):  
Caspase 3 ◽  
B Cell Lymphoma ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interacting Protein ◽  
Protein Levels ◽  
Tnf Α ◽  
Uroepithelial Cells

Abstract Many clinical studies have been conducted on ketamine-associated cystitis. However, the underlying mechanisms of ketamine-associated cystitis still remain unclear. Bladder tissues of rats were stained by Hematoxylin and Eosin (HE). The viability of human uroepithelial cells (SV-HUC-1 cells) was determined by cell counting kit-8 (CCK-8). Apoptosis and reactive oxygen species (ROS) were examined by flow cytometry. Additionally, the expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and IL-18 were respectively determined by reverse transcription quantitative (RTq)-PCR and enzyme-linked immunosorbent assay (ELISA). The mRNA and protein levels of B-cell lymphoma/leukemia-2 (Bcl2), Bcl-2-associated X protein (Bax), cleaved caspase 3, glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), NOD-like receptor 3 (NLRP3), thioredoxin-interacting protein (TXNIP), Catalase and MnSOD were examined by RT-qPCR and Western blot. Small interfering RNA target TXNIP transfection was performed using Lipofectamine™ 2000. We found that ketamine effectively damaged bladder tissues of rats and promoted apoptosis through regulating the expression levels of GRP78, CHOP, Bcl-2, Bax and cleaved Caspase-3 proteins in vivo and in vitro. NLRP3 inflammatory body and TXNIP were activated by ketamine, which was supported by the changes in TNF-α, IL-6, IL-1 and IL-18 in vivo and in vitro. Furthermore, knocking down TXNIP reversed the effects of ketamine on apoptosis and NLRP3 inflammatory body in SV-HUC-1 cells. Meanwhile, the changes of Catalase and MnSOD showed that ROS was enhanced by ketamine, however, such an effect was ameliorated by down-regulation of TXNIP in SV-HUC-1 cells. Ketamine promoted cell apoptosis and induced inflammation in vivo and in vitro by regulating NLRP3/TXNIP aix.

scholarly journals Function and regulation of an aldehyde dehydrogenase essential for ethanol and methanol metabolism of the yeast, Komagataella phaffii

2020 ◽  
Author(s):  
Kamisetty Krishna Rao ◽  
Umakant Sahu ◽  
Pundi N Rangarajan
Keyword(s):  
Transcriptional Activation ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Ethanol Metabolism ◽  
Mrna Levels ◽  
Methanol Metabolism ◽  
Protein Levels ◽  
Komagataella Phaffii

AbstractThe genome of the methylotrophic yeast, Komagataella phaffii harbours multiple genes encoding putative alcohol dehydrogenases and aldehyde dehydrogenases (ALDs). Here, we demonstrate that one of the ALDs denoted as ALD-A is essential for ethanol metabolism. A zinc finger transcription factor known as Mxr1p regulates ALD-A transcription by binding to Mxr1p response elements (MXREs) in the ALD-A promoter. Mutations which abrogate Mxr1p binding to ALD-A MXREs in vitro abolish transcriptional activation from ALD-A promoter in vivo. Mxr1p regulates ALD-A expression during ethanol as well as methanol metabolism. ALD-A is essential for the utilization of methanol and Δald-a is deficient in alcohol oxidase (AOX), a key enzyme of methanol metabolism. AOX protein but not mRNA levels are down regulated in Δald-a. ALD-A and AOX localize to cytosol and peroxisomes respectively during methanol metabolism suggesting that they are unlikely interact with each other in vivo. This study has led to the identification of Mxr1p as a key regulator of ALD-A transcription during ethanol and methanol metabolism of K. phaffii. Post-transcriptional regulation of AOX protein levels by ALD-A during methanol metabolism is another unique feature of this study.

scholarly journals Spatial Interplay between PIASy and FIP200 in the Regulation of Signal Transduction and Transcriptional Activity

2008 ◽  
Vol 28 (8) ◽  
pp. 2771-2781 ◽  
Author(s):  
Nadine Martin ◽  
Klaus Schwamborn ◽  
Henning Urlaub ◽  
Boyi Gan ◽  
Jun-Lin Guan ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Ring Finger ◽  
Subcellular Fractionation ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Bona Fide ◽  
Independent Effects ◽  
P21 Promoter

ABSTRACT The members of the protein inhibitor of activated STAT (PIAS) family of proteins are implicated in fundamental cellular processes, including transcriptional regulation, either through action as E3 SUMO ligases or through SUMO-independent effects. We report here the identification of FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) as a new PIASy-interacting protein. We show that the interaction depends on the integrity of the RING finger of PIASy and the carboxy terminus of FIP200. Both in vitro and in vivo sumoylation assays failed to reveal any sumoylation of FIP200, suggesting that FIP200 is not a bona fide SUMO substrate. Immunofluorescence microscopy and subcellular fractionation, either upon forced PIASy expression or in the absence of PIASy, revealed that interaction with PIASy redistributes FIP200 from the cytoplasm to the nucleus, correlating with abrogation of FIP200 regulation of TSC/S6K signaling. Conversely, FIP200 enhances the transcriptional activation of the p21 promoter by PIASy whereas PIASy transcription activity is severely reduced upon FIP200 depletion by RNA interference. Chromatin immunoprecipitation analysis demonstrates that endogenous PIASy and FIP200 are corecruited to the p21 promoter. Altogether, these results provide the first evidence for the existence of a close—spatially controlled—mode of regulation of FIP200 and PIASy nucleocytoplasmic functions.

scholarly journals Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tatenda Mahlokozera ◽  
Bhuvic Patel ◽  
Hao Chen ◽  
Patrick Desouza ◽  
Xuan Qu ◽  
...  
Keyword(s):  
E3 Ligase ◽  
Treatment Resistance ◽  
Interacting Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
Proteomic Approach ◽  
Sox2 Gene ◽  
Functional Consequences ◽  
Bona Fide

AbstractThe pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.

scholarly journals Role for SUMO Modification in Facilitating Transcriptional Repression by BKLF

2005 ◽  
Vol 25 (4) ◽  
pp. 1549-1559 ◽  
Author(s):  
José Perdomo ◽  
Alexis Verger ◽  
Jeremy Turner ◽  
Merlin Crossley
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Site Directed Mutagenesis ◽  
E3 Ligases ◽  
Zinc Finger Transcription Factor ◽  
Sumo Modification ◽  
Lysine Residues ◽  
Protein Moiety

ABSTRACT Small ubiquitin-like modifier (SUMO) is a protein moiety that is ligated to lysine residues on a variety of target proteins. Many known SUMO substrates are transcription factors or coregulators of transcription, and in most cases, modification with SUMO leads to the attenuation of transcriptional activation. We have examined basic Krüppel-like factor/Krüppel-like factor 3 (BKLF), a zinc finger transcription factor that is known to function as a potent transcriptional repressor. We show that BKLF recruits the E2 SUMO-conjugating enzyme Ubc9 and can be modified by the addition of SUMO-1 in vitro and in vivo. The SUMO E3 ligases PIAS1, PIASγ, PIASxα, and PIASxβ but not Pc2 enhance the sumoylation of BKLF. Site-directed mutagenesis identified two lysines (K10 and K197) of BKLF as the sumoylation sites. Sumoylation does not detectably affect DNA binding by BKLF, but mutation of the sumoylation sites reduces transcriptional repression activity. Most interestingly, when mutations preventing sumoylation are combined with an additional mutation that eliminates contact with the C-terminal binding protein (CtBP) corepressor, BKLF becomes an activator of transcription. These results link SUMO modification to transcriptional repression and demonstrate that both recruitment of CtBP and sumoylation are required for full repression by BKLF.

scholarly journals The E1B 19K/Bcl-2–binding Protein Nip3 is a Dimeric Mitochondrial Protein that Activates Apoptosis

1997 ◽  
Vol 186 (12) ◽  
pp. 1975-1983 ◽  
Author(s):  
Gao Chen ◽  
Reena Ray ◽  
Don Dubik ◽  
Lianfa Shi ◽  
Jeannick Cizeau ◽  
...  
Keyword(s):  
Cell Death ◽  
Topoisomerase I ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Mitochondrial Protein ◽  
Interacting Protein ◽  
Protein Levels ◽  
Yeast Two Hybrid System

Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3163) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3163 mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3163 protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

scholarly journals The Papillomavirus E2 Protein Binds to and Synergizes with C/EBP Factors Involved in Keratinocyte Differentiation

2003 ◽  
Vol 77 (9) ◽  
pp. 5253-5265 ◽  
Author(s):  
Dirk Hadaschik ◽  
Korinna Hinterkeuser ◽  
Monika Oldak ◽  
Herbert J. Pfister ◽  
Sigrun Smola-Hess
Keyword(s):  
Transcriptional Activation ◽  
Proximal Promoter ◽  
Keratinocyte Differentiation ◽  
Bovine Papillomavirus ◽  
Late Gene Expression ◽  
C Terminus ◽  
Viral Maturation ◽  
E6 And E7

ABSTRACT The papillomavirus life cycle is closely linked to the differentiation program of the host keratinocyte. Thus, late gene expression and viral maturation are restricted to terminally differentiated keratinocytes. A variety of cellular transcription factors including those of the C/EBP family are involved in the regulation of keratinocyte differentiation. In this study we show that the papillomavirus transcription factor E2 cooperates with C/EBPα and -β in transcriptional activation. This synergism was independent of an E2 binding site. E2 and C/EBP factors synergistically transactivated a synthetic promoter construct containing classical C/EBPβ sites and the C/EBPα-responsive proximal promoter of the involucrin gene, which is naturally expressed in differentiating keratinocytes. C/EBPα or -β coprecipitated with E2 proteins derived from human papillomavirus type 8 (HPV8), HPV16, HPV18, and bovine papillomavirus type 1 in vitro and in vivo, indicating complex formation by the cellular and viral factors. The interaction domains could be mapped to the C terminus of E2 and amino acids 261 to 302 located within the bZIP motif of C/EBPβ. Our data suggest that E2, via its interaction with C/EBP factors, may contribute to enhancing keratinocyte differentiation, which is suppressed by the viral oncoproteins E6 and E7 in HPV-induced lesions.

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