scholarly journals Amino Acid Residues Required for Physical and Cooperative Transcriptional Interaction of STAT3 and AP-1 Proteins c-Jun and c-Fos

2007 ◽  
Vol 27 (18) ◽  
pp. 6300-6308 ◽  
Author(s):  
Michael Ginsberg ◽  
Elmar Czeko ◽  
Patrick Müller ◽  
Zhiyong Ren ◽  
Xiaomin Chen ◽  
...  
Keyword(s):  
Target Genes ◽  
Amino Acid Residues ◽  
Chromosomal Dna ◽  
Protein Interfaces ◽  
Specific Contact ◽  
Dna Elements ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Chromosomal Loci

ABSTRACT Cooperation between STAT3 and c-Jun in driving transcription during transfection of reporter constructs is well established, and both proteins are present on some interleukin-6 (IL-6) STAT3-dependent promoters on chromosomal loci. We report that small interfering RNA knockdown of c-Jun or c-Fos diminishes IL-6 induction of some but not all STAT3-dependent mRNAs. Specific contact sites in STAT3 responsible for interaction of a domain of STAT3 with c-Jun were known. Here we show that the B-zip domain of c-Jun interacts with STAT3 and that c-Jun mutation R261A or R261D near but not in the DNA binding domain blocks in vitro STAT3-c-Jun interaction and decreases costimulation of transcription in transfection assays. Cooperative binding to DNA of tyrosine-phosphorylated STAT3 and both wild-type and R261A mutant c-Jun was observed. Even c-Jun mutant R261D, which on its own did not bind DNA, bound DNA weakly in the presence of STAT3. We conclude that a functional interaction between STAT3 and c-Jun while bound to chromosomal DNA elements exists and is necessary for driving transcription on at least some STAT3 target genes. Identifying such required interactive protein interfaces should be a stimulus to search for compounds that could ultimately inhibit the activity of STAT3 in tumors dependent on persistently active STAT3.

scholarly journals Phosphorylation by Cyclin C/Cyclin-Dependent Kinase 2 following Mitogenic Stimulation of Murine Fibroblasts Inhibits Transcriptional Activity of LSF during G1 Progression

2009 ◽  
Vol 29 (9) ◽  
pp. 2335-2345 ◽  
Author(s):  
Utsav H. Saxena ◽  
Christina M. H. Powell ◽  
Jill K. Fecko ◽  
Roxanne Cacioppo ◽  
Hubert S. Chou ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Cyclin Dependent Kinase ◽  
Mitogenic Stimulation ◽  
Mouse Fibroblasts ◽  
Cyclin C ◽  
Murine Fibroblasts ◽  
Interfering Rna ◽  
Stimulation Of

ABSTRACT Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G1/S boundary. Given the constant level of LSF protein from G0 through S, we investigated whether LSF is regulated by phosphorylation in G1. In vitro, LSF is phosphorylated by cyclin E/cyclin-dependent kinase 2 (CDK2), cyclin C/CDK2, and cyclin C/CDK3, predominantly on S309. Phosphorylation of LSF on S309 is maximal 1 to 2 h after mitogenic stimulation of quiescent mouse fibroblasts. This phosphorylation is mediated by cyclin C-dependent kinases, as shown by coimmunoprecipitation of LSF and cyclin C in early G1 and by abrogation of LSF S309 phosphorylation upon suppression of cyclin C with short interfering RNA. Although mouse fibroblasts lack functional CDK3 (the partner of cyclin C in early G1 in human cells), CDK2 compensates for this absence. By transient transfection assays, phosphorylation at S309, mediated by cyclin C overexpression, inhibits LSF transactivation. Moreover, overexpression of cyclin C and CDK3 inhibits induction of endogenous Tyms expression at the G1/S transition. These results identify LSF as only the second known target (in addition to pRb) of cyclin C/CDK activity during progression from quiescence to early G1. Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G1.

scholarly journals Targeted Deletion of the Murine apobec-1 Complementation Factor (acf) Gene Results in Embryonic Lethality

2005 ◽  
Vol 25 (16) ◽  
pp. 7260-7269 ◽  
Author(s):  
Valerie Blanc ◽  
Jeffrey O. Henderson ◽  
Elizabeth P. Newberry ◽  
Susan Kennedy ◽  
Jianyang Luo ◽  
...  
Keyword(s):  
Rna Binding ◽  
Cytidine Deaminase ◽  
Mrna Editing ◽  
Targeted Deletion ◽  
Apob Mrna ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Binding Subunit ◽  
Apparently Healthy

ABSTRACT apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf +/ − mice were apparently healthy and fertile, no viable acf − / − mice were identified. Mutant acf − / − embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf − / − blastocysts were detectable following implantation at E4.5, and isolated acf − / − blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

scholarly journals Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors

2007 ◽  
Vol 27 (11) ◽  
pp. 4018-4027 ◽  
Author(s):  
Rong Hu ◽  
Sudarshana M. Sharma ◽  
Agnieszka Bronisz ◽  
Ruchika Srinivasan ◽  
Uma Sankar ◽  
...  
Keyword(s):  
Target Genes ◽  
Zinc Finger Protein ◽  
Osteoclast Differentiation ◽  
Direct Interaction ◽  
Cathepsin K ◽  
Gene Promoters ◽  
Basal Expression ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Myeloid Progenitors

ABSTRACT Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.

scholarly journals BMP9 regulates endoglin-dependent chemokine responses in endothelial cells

Blood ◽  
2012 ◽  
Vol 120 (20) ◽  
pp. 4263-4273 ◽  
Author(s):  
Kira Young ◽  
Barbara Conley ◽  
Diana Romero ◽  
Eric Tweedie ◽  
Christine O'Neill ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Target Genes ◽  
Endothelial Cell Migration ◽  
Quantitative Mass Spectrometry ◽  
Chemokine Cxcl12 ◽  
Vessel Maturation ◽  
Rna Knockdown ◽  
Deficient Mice

Abstract BMP9 signaling has been implicated in hereditary hemorrhagic telangiectasia (HHT) and vascular remodeling, acting via the HHT target genes, endoglin and ALK1. This study sought to identify endothelial BMP9-regulated proteins that could affect the HHT phenotype. Gene ontology analysis of cDNA microarray data obtained after BMP9 treatment of primary human endothelial cells indicated regulation of chemokine, adhesion, and inflammation pathways. These responses included the up-regulation of the chemokine CXCL12/SDF1 and down-regulation of its receptor CXCR4. Quantitative mass spectrometry identified additional secreted proteins, including the chemokine CXCL10/IP10. RNA knockdown of endoglin and ALK1 impaired SDF1/CXCR4 regulation by BMP9. Because of the association of SDF1 with ischemia, we analyzed its expression under hypoxia in response to BMP9 in vitro, and during the response to hindlimb ischemia, in endoglin-deficient mice. BMP9 and hypoxia were additive inducers of SDF1 expression. Moreover, the data suggest that endoglin deficiency impaired SDF1 expression in endothelial cells in vivo. Our data implicate BMP9 in regulation of the SDF1/CXCR4 chemokine axis in endothelial cells and point to a role for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation.

scholarly journals The Effects of Dickkopf-4 on the Proliferation, Differentiation, and Apoptosis of Osteoblasts

Endocrinology ◽  
2013 ◽  
Vol 154 (12) ◽  
pp. 4618-4626 ◽  
Author(s):  
Shiro Hiramitsu ◽  
Masakazu Terauchi ◽  
Toshiro Kubota
Keyword(s):  
Alkaline Phosphatase ◽  
Bone Formation ◽  
Target Genes ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line ◽  
Alkaline Phosphatase Assay ◽  
Proliferation And Differentiation ◽  
Phosphatase Assay ◽  
Rna Knockdown ◽  
Interfering Rna

The Dickkopf family of proteins is comprised of four members (Dkk1, Dkk2, Dkk3, Dkk4) that are known to modulate Wnt/β-catenin signaling, which is activated during bone formation. Although the effects of Dkk1 on Wnt/β-catenin signaling have been well studied, little is known about the effects of Dkk4. Therefore, to evaluate the role of Dkk4 in osteoblastogenesis, we used the mouse osteoblastic cell line MC3T3-E1, in which Dkk4 expression was suppressed by small interfering RNA knockdown. Our results showed that the suppression of Dkk4 expression promoted osteoblast proliferation and differentiation and suppressed apoptosis. In colony-forming unit alkaline phosphatase assay, Dkk4 knockdown cells possessed markedly higher alkaline phosphatase activity compared with Dkk1 knockdown cells. Reduced Dkk4 expression also led to the up-regulation of β-catenin levels, β-catenin/T cell factor activity, and Wnt-target genes. In contrast, overexpression of Dkk4 in MC3T3-E1 cells led to inhibition of osteoblast differentiation. Our findings reveal that Dkk4 functions as an inhibitor of osteoblastogenesis through Wnt/β-catenin signaling, providing new insights into the relationship between Wnt/β-catenin signaling and Dkk4 in bone formation.

scholarly journals Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

2007 ◽  
Vol 81 (11) ◽  
pp. 5449-5459 ◽  
Author(s):  
Beau J. Fenner ◽  
Winnie Goh ◽  
Jimmy Kwang
Keyword(s):  
Rna Silencing ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Alanine Scanning Mutagenesis ◽  
Marine Aquaculture ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Fish Cells ◽  
Interfering Rna

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

scholarly journals Cloning and Characterization of Lipase Gene from a Local Isolate of Pseudoxanthomonas sp.

2017 ◽  
Vol 14 (2) ◽  
pp. 503-507 ◽  
Author(s):  
Yogi Yopa Kristia ◽  
Syifa F Syihab ◽  
Akhmaloka Akhmaloka
Keyword(s):  
Crystal Structure ◽  
3D Structure ◽  
Catalytic Triad ◽  
Catalytic Residue ◽  
Pseudomonas Sp ◽  
Amino Acid Residues ◽  
Chromosomal Dna ◽  
Lipase Gene

ABSTRACT: Lipase gene from Pseudoxanthomonas sp. was cloned through in vitro amplification from total chromosomal DNA. The gene was sequenced and characterized, coding for 312 amino acid residues. Homological analysis showed that the gene has 98% similarity to lipolytic gene from Uncultured Pseudomonas sp (GenBank No. AKA58891.1). Further analysis appeared that the sequences showed similar unique motifs of lipase sub-family I.1, such as pentapeptide (GHSHG) motif, tetrapeptide (GMLG) motif, and catalytic triad. In additional, 3D structure analysis based on crystal structure of Pseudomonas aeruginose (PDB ID 1ex9) showed that both structure of lipases are similar except on the conformation of catalytic residue of His277 showing to shift more far away compared to that the control.

scholarly journals Endoplasmic Reticulum (ER) Mannosidase I Is Compartmentalized and Required for N-Glycan Trimming to Man5–6GlcNAc2 in Glycoprotein ER-associated Degradation

2008 ◽  
Vol 19 (1) ◽  
pp. 216-225 ◽  
Author(s):  
Edward Avezov ◽  
Zehavit Frenkel ◽  
Marcelo Ehrlich ◽  
Annette Herscovics ◽  
Gerardo Z. Lederkremer
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Local Concentration ◽  
Mannose Residue ◽  
Low Concentrations ◽  
High Concentrations ◽  
Rna Knockdown ◽  
Interfering Rna

We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man9GlcNAc2. A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to four α1,2-linked mannose residues. Using small interfering RNA knockdown of ERManI, we show that this enzyme is required for trimming to Man5–6GlcNAc2 and for ERAD in cells in vivo, leading to the accumulation of Man9GlcNAc2 and Glc1Man9GlcNAc2 on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI is strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knockdown prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation.

scholarly journals AMSH is an endosome-associated ubiquitin isopeptidase

2004 ◽  
Vol 166 (4) ◽  
pp. 487-492 ◽  
Author(s):  
John McCullough ◽  
Michael J. Clague ◽  
Sylvie Urbé
Keyword(s):  
Egf Receptor ◽  
Sh3 Domain ◽  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Isopeptidase Activity ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Inactivating Mutation ◽  
Free Ubiquitin

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif has been proposed to provide the active site for isopeptidase activity associated with the Rpn11/POH1 subunit of the 19S-proteasome and the Csn5-subunit of the signalosome. We have looked for similar activity in associated molecule with the SH3 domain of STAM (AMSH), a JAMM domain–containing protein that associates with the SH3-domain of STAM, a protein, which regulates receptor sorting at the endosome. We demonstrate isopeptidase activity against K48-linked tetraubiquitin and K63-linked polyubiquitin chains to generate di-ubiquitin and free ubiquitin, respectively. An inactivating mutation (D348A) in AMSH leads to accumulation of ubiquitin on endosomes and the concomitant stabilization of a ubiquitinated form of STAM, which requires an intact ubiquitin interaction motif (UIM) within STAM. Short interfering RNA knockdown of AMSH enhances the degradation rate of EGF receptor (EGFR) following acute stimulation and ubiquitinated EGFR provides a substrate for AMSH in vitro. We propose that AMSH is a deubiquitinating enzyme with functions at the endosome, which oppose the ubiquitin-dependent sorting of receptors to lysosomes.

scholarly journals Hypoxia-inducible factor-1α mRNA: a new target for destabilization by tristetraprolin in endothelial cells

2011 ◽  
Vol 22 (18) ◽  
pp. 3366-3378 ◽  
Author(s):  
Sandrine Chamboredon ◽  
Delphine Ciais ◽  
Agnès Desroches-Castan ◽  
Pierre Savi ◽  
Françoise Bono ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Half Life ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Control Of Gene Expression ◽  
Hypoxia Inducible Factor 1Α ◽  
Protein Levels ◽  
Ca Ix ◽  
Rna Knockdown ◽  
Interfering Rna

Endothelial cells (ECs) are the primary sensors of variations in blood oxygen concentrations. They use the hypoxia-sensitive stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription factor to engage specific transcriptional programs in response to oxygen changes. The regulation of HIF-1α expression is well documented at the protein level, but much less is known about the control of its mRNA stability. Using small interfering RNA knockdown experiments, reporter gene analyses, ribonucleoprotein immunoprecipitations, and mRNA half-life determinations, we report a new regulatory mechanism of HIF-1α expression in ECs. We demonstrate that 1) sustained hypoxia progressively decreases HIF-1α mRNA while HIF-1α protein levels rapidly peak after 3 h and then slowly decay; 2) silencing the mRNA-destabilizing protein tristetraprolin (TTP) in ECs reverses hypoxia-induced down-regulation of HIF-1α mRNA; 3) the decrease in the half-life of Luciferase-HIF-1α-3′UTR reporter transcript that is observed after prolonged hypoxia is mediated by TTP; 4) TTP binds specifically to HIF-1α 3′UTR; and 5) the most distal AU-rich elements present in HIF-1α 3′UTR (composed of two hexamers) are sufficient for TTP-mediated repression. Finally, we bring evidence that silencing TTP expression enhances hypoxia-induced increase in HIF-1α protein levels with a concomitant increase in the levels of the carbonic anhydrase enzyme CA IX, thus suggesting that TTP physiologically controls the expression of a panel of HIF-1α target genes. Altogether, these data reveal a new role for TTP in the control of gene expression during the response of endothelial cell to hypoxia.

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