Identification of a novel AU-rich-element-binding protein which is related to AUF1

The AU-rich element (ARE) is an important instability determinant for a large number of early-response-gene mRNAs. AREs also mediate the stabilization of certain pro-inflammatory mRNAs, such as tumour necrosis factor (TNF)-α and cyclo-oxygenase-2 (COX-2), in response to inflammatory stimuli. To understand how AREs control mRNA stability, it is necessary to identify trans-acting factors. We have purified a new ARE-binding protein and identified it as CArG box-binding factor-A (CBF-A). The amino acid sequence of CBF-A is highly similar to that of the ARE-binding protein AUF1. Recombinant CBF-A bound the COX-2 and TNF-α AREs, but not a non-specific control RNA. In contrast, in an electrophoretic-mobility-shift assay (EMSA) of crude RAW 264.7 macrophage-like cell extracts, an antiserum that recognizes both AUF1 and CBF-A failed to supershift complexes formed on the TNF-α ARE, but did supershift a complex specific for the COX-2 ARE. CBF-A exists as two isoforms, p37 and p42, that differ by a 47-amino-acid insertion close to the C-terminus. By expressing epitope-tagged isoforms of CBF-A it was shown that the p42 isoform binds the COX-2 ARE in EMSA of crude cell extracts. In a HeLa-cell tetracycline-regulated reporter system, overexpression of the p42 CBF-A isoform resulted in stabilization of a COX-2 ARE reporter mRNA. Epitope-tagged p42 CBF-A expressed in HeLa cells co-immunoprecipitated with endogenous COX-2 mRNA, but not glyceraldehyde-3-phosphate dehydrogenase mRNA, as shown by reverse-transcription PCR. The similarity between CBF-A and AUF1 suggests that CBF-A could be re-named AUF2.

Download Full-text

Amino acid sequences characterization and anti-inflammatory potency evaluation of Portulaca oleracea L. oligopeptides in macrophages

RSC Advances ◽

10.1039/c9ra10465h ◽

2020 ◽

Vol 10 (12) ◽

pp. 7321-7327

Author(s):

Shihui Chang ◽

Liping Wang ◽

Ting Zhang ◽

Yan Nie ◽

Ruijie Liu ◽

...

Keyword(s):

Amino Acid ◽

Signaling Pathways ◽

Inflammatory Cytokine ◽

Cytokine Expression ◽

Amino Acid Sequences ◽

Portulaca Oleracea ◽

Tnf Α ◽

Cox 2 ◽

Anti Inflammatory

POP-1 performed excellent anti-inflammatory potency by attenuating the pro-inflammatory cytokine expression (TNF-α, NO, IL-1β); inhibiting iNOS and COX-2 expressions and regulating the MAPK, PI3K/Akt and NF-κB signaling pathways.

Download Full-text

A conserved motif within the flexible C-terminus of the translational regulator 4E-BP is required for tight binding to the mRNA cap-binding protein eIF4E

Biochemical Journal ◽

10.1042/bj20101481 ◽

2011 ◽

Vol 441 (1) ◽

pp. 237-245 ◽

Cited By ~ 26

Author(s):

Keum Soon Paku ◽

Yu Umenaga ◽

Tsunego Usui ◽

Ai Fukuyo ◽

Atsuo Mizuno ◽

...

Keyword(s):

Amino Acid ◽

Binding Protein ◽

Tight Binding ◽

Terminal Region ◽

Dynamics Simulation ◽

Initiation Factor ◽

Binding Region ◽

The Core ◽

C Terminus ◽

Conserved Region

Although the central α-helical Y(X)4LΦ motif (X, variable amino acid; Φ, hydrophobic amino acid) of the translational regulator 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] is the core binding region for the mRNA cap-binding protein eIF4E, the functions of its N- and C-terminal flexible regions for interaction with eIF4E remain to be elucidated. To identify the role for the C-terminal region in such an interaction, the binding features of full-length and sequential C-terminal deletion mutants of 4E-BPn (n=1–3) subtypes were investigated by SPR (surface plasmon resonance) analysis and ITC (isothermal titration calorimetry). Consequently, the conserved PGVTS/T motif within the C-terminal region was shown to act as the second binding region and to play an important role in the tight binding to eIF4E. The 4E-BP subtypes increased the association constant with eIF4E by approximately 1000-fold in the presence of this conserved region compared with that in the absence of this region. The sequential deletion of this conserved region in 4E-BP1 showed that deletion of Val81 leads to a considerable decrease in the binding ability of 4E-BP. Molecular dynamics simulation suggested that the conserved PGVTS/T region functions as a kind of paste, adhering the root of both the eIF4E N-terminal and 4E-BP C-terminal flexible regions through a hydrophobic interaction, where valine is located at the crossing position of both flexible regions. It is concluded that the conserved PGVTS/T motif within the flexible C-terminus of 4E-BP plays an auxiliary, but indispensable, role in strengthening the binding of eIF4E to the core Y(X)4LΦ motif.

Download Full-text

Identification and Functional Outcome of mRNAs Associated with RNA-Binding Protein TIA-1

Molecular and Cellular Biology ◽

10.1128/mcb.25.21.9520-9531.2005 ◽

2005 ◽

Vol 25 (21) ◽

pp. 9520-9531 ◽

Cited By ~ 156

Author(s):

Isabel López de Silanes ◽

Stefanie Galbán ◽

Jennifer L. Martindale ◽

Xiaoling Yang ◽

Krystyna Mazan-Mamczarz ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Cellular Damage ◽

Necrosis Factor Alpha ◽

Target Mrnas ◽

Common Motif ◽

Tnf Α ◽

Cox 2 ◽

Rna Complexes

ABSTRACT The RNA-binding protein TIA-1 (T-cell intracellular antigen 1) functions as a posttranscriptional regulator of gene expression and aggregates to form stress granules following cellular damage. TIA-1 was previously shown to bind mRNAs encoding tumor necrosis factor alpha (TNF-α) and cyclooxygenase 2 (COX-2), but TIA-1 target mRNAs have not been systematically identified. Here, immunoprecipitation (IP) of TIA-1-RNA complexes, followed by microarray-based identification and computational analysis of bound transcripts, was used to elucidate a common motif present among TIA-1 target mRNAs. The predicted TIA-1 motif was a U-rich, 30- to 37-nucleotide (nt)-long bipartite element forming loops of variable size and a bent stem. The TIA-1 motif was found in the TNF-α and COX-2 mRNAs and in 3,019 additional UniGene transcripts (∼3% of the UniGene database), localizing preferentially to the 3′ untranslated region. The interactions between TIA-1 and target transcripts were validated by IP of endogenous mRNAs, followed by reverse transcription and PCR-mediated detection, and by pulldown of biotinylated RNAs, followed by Western blotting. Further studies using RNA interference revealed that TIA-1 repressed the translation of bound mRNAs. In summary, we report a signature motif present in mRNAs that associate with TIA-1 and provide support to the notion that TIA-1 represses the translation of target transcripts.

Download Full-text

The gene G13 in the class III region of the human MHC encodes a potential DNA-binding protein

Biochemical Journal ◽

10.1042/bj3190081 ◽

1996 ◽

Vol 319 (1) ◽

pp. 81-89 ◽

Cited By ~ 14

Author(s):

Anu KHANNA ◽

R. Duncan CAMPBELL

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Leucine Zipper ◽

Binding Protein ◽

Coiled Coil ◽

Single Copy ◽

Dna Binding Protein ◽

Lymphoid Tissues ◽

Class Iii ◽

Cell Extracts

G13 is a single-copy gene lying approx. 75 kb centromeric of the complement gene cluster in the class III region of the human MHC. The gene spans approx. 17 kb of DNA and has been shown to encode mRNA of approx. 2.7 kb that is present in cell lines representing lymphoid and non-lymphoid tissues, indicating that it is ubiquitously expressed. The complete nucleotide sequence of the 2.7 kb mRNA has been derived from cDNA and genomic clones. The longest open reading frame obtained for G13 codes for a 703 amino acid protein of approx. 77 kDa in molecular mass. Comparison of the putative G13 amino acid sequence with the protein databases revealed significant similarities with DNA-binding proteins of the leucine zipper class, including a human cAMP response element binding protein. G13 contains a bZIP motif, a region rich in basic amino acids adjacent to a coiled-coil leucine zipper domain, common to this class of proteins that is known to be involved in dimerization and DNA binding. Antibodies raised against a fragment encoding the C-terminal half of the putative G13 protein recognized a major polypeptide of approx. 86 kDa and a minor polypeptide of approx. 78 kDa on immunoblotting of U937 cell extracts; this has been confirmed by immunoprecipitation experiments. Even though it contained at least one potential bipartite nuclear localization signal, the G13 protein was present both in the cytoplasm and the nucleus of the fibroblast cells. Thus G13 might be a novel DNA-binding protein that is perhaps translocated to the nucleus in a regulated manner.

Download Full-text

C-C Chemokine Ligand 2 Gene Expression in Nasal Polyp Fibroblasts: Possible Implication in the Pathogenesis of Nasal Polyposis

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348940511401112 ◽

2005 ◽

Vol 114 (11) ◽

pp. 879-885 ◽

Cited By ~ 5

Author(s):

Chia-Tung Shun ◽

Sze-Kwan Lin ◽

Chi-Yuan Hong ◽

Sang-Heng Kok ◽

Yun-Hsiang Juan ◽

...

Keyword(s):

Gene Expression ◽

Northern Blot ◽

Nasal Polyps ◽

Northern Blot Analysis ◽

Mobility Shift ◽

Tnf Α ◽

Chemokine Ligand ◽

Cox 2 ◽

Mobility Shift Assay ◽

Chemokine Ligand 2

Objectives: Recruitment of macrophages is essential to the pathogenesis of nasal polyps (NP), since this disease is inflammation-related. In this study, the effects of tumor necrosis factor α (TNF-α) on the expression of C-C chemokine ligand 2 (CCL2) in fibroblasts derived from nasal polyps (NPFs) were investigated. The roles of cyclooxygenase (COX) 2 and prostaglandins in the mediation of TNF-α–stimulated CCL2 gene expression were also investigated. Methods: Northern blot analysis was used to study the expression of CCL2 and c-Fos in cultured NPFs. An electrophoretic mobility shift assay was used to explore the interactions between activator protein 1 (AP-1) and DNA. Immunohistochemistry was used to explore the in vivo expressions of COX-2, CCL2, and CD68 in NPs. Results: The Northern blot analysis showed that TNF-α stimulated the expression of CCL2 and COX-2 genes, and the synthesis of CCL2 messenger RNA was COX-2-dependent. A transient elevation of c-Fos and c-Jun messenger RNAs was induced by TNF-α, whereas COX-2 inhibitors NS-398 and meloxicam abolished the up-regulation of c-Fos. The electrophoretic mobility shift assay revealed that TNF-α triggered AP-1 and DNA binding and again, NS-398 and meloxicam inhibited this reaction via reducing c-Fos synthesis. Curcumin (AP-1 inhibitor) markedly suppressed the TNF-α–induced CCL2 expression. The immunohistochemical staining of NP surgical specimens also revealed an intimate alignment between CCL2-positive fibroblasts and CD-68-positive macrophages. Conclusions: These data suggest that NPFs may contribute to NP development by synthesizing CCL2 to promote macrophage recruitment. Furthermore, COX-2 facilitates CCL2 transcription in NPFs via a c-Fos and AP-1 signaling pathway.

Download Full-text

Differential Effects of Amrinone and Milrinone Upon Myocardial Inflammatory Signaling

Circulation ◽

10.1161/01.cir.0000032904.33237.8e ◽

2002 ◽

Vol 106 (12_suppl_1) ◽

Author(s):

Nikhil K. Chanani ◽

Douglas B. Cowan ◽

Koh Takeuchi ◽

Dimitrios N. Poutias ◽

Lina M. Garcia ◽

...

Keyword(s):

Cytokine Production ◽

Inflammatory Responses ◽

Vascular Endothelial Cells ◽

Myocardial Dysfunction ◽

Ischemia Reperfusion ◽

Phosphodiesterase Inhibitors ◽

Mobility Shift ◽

Inflammatory Signaling ◽

Tnf Α ◽

Cox 2

Background Mounting evidence links systemic and local inflammatory cytokine production to myocardial dysfunction and injury occurring during ischemia-reperfusion, cardiopulmonary bypass, and heart failure. Phosphodiesterase inhibitors (PDEIs), used frequently in these states, can modulate inflammatory signaling. The mechanisms for these effects are unclear. We therefore examined the effects of 2 commonly used PDEIs, amrinone and milrinone, on cardiac cell inflammatory responses. Methods and Results Primary rat cardiomyocyte cultures were treated with endotoxin (LPS) or tumor necrosis factor-α (TNF-α), alone or in the presence of clinically relevant concentrations of amrinone or milrinone. Regulation of nuclear factor-kappa B (NFκB), nitric oxide synthase and cyclooxygenase isoforms, and cytokine production were assessed by electrophoretic mobility shift assays, Western immunoblotting, and enzyme-linked immunoassays, respectively. Both LPS and TNF-α induced significant NFκB activation, cyclooxygenase-2 (COX-2) expression, and inducible NO synthase (iNOS) and cytokine production; with the exception of COX-2 expression, all were significantly reduced by amrinone, beginning at concentrations of 10 to 50 μmol/L. In contrast, milrinone increased nuclear NFκB translocation, iNOS and COX-2 expression, and cardiomyocyte production of interleukin-1β. Cell-permeable cAMP increased inflammatory gene expression, whereas cell-permeable cGMP had no effect, indicating that the effects of amrinone were not due to phosphodiesterase inhibition. Similar results were seen in macrophages and coronary vascular endothelial cells. Conclusions Both amrinone and milrinone have significant effects on cardiac inflammatory signaling. Overall, amrinone reduces activation of the key transcription factor NFκB and limits the production of pro-inflammatory cytokines, whereas milrinone does not.

Download Full-text

The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

Infection and Immunity ◽

10.1128/iai.00135-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5595-5601 ◽

Cited By ~ 10

Author(s):

Cynthia L. Sears ◽

Simy L. Buckwold ◽

Jai W. Shin ◽

Augusto A. Franco

Keyword(s):

Biological Activity ◽

Amino Acid ◽

Point Mutations ◽

Bacteroides Fragilis ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Reverse Transcription Pcr ◽

C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

Download Full-text

IN SILICO DOCKING STUDIES ON KAEMPFERITRIN WITH DIVERSE INFLAMMATORY AND APOPTOTIC PROTEINS FUNCTIONAL APPROACH TOWARDS THE COLON CANCER

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2017v9i9.20500 ◽

2017 ◽

Vol 9 (9) ◽

pp. 199

Author(s):

Mydhili Govindarasu ◽

Mariyappan Palani ◽

Manju Vaiyapuri

Keyword(s):

Colon Cancer ◽

Cytochrome P450 ◽

Amino Acid ◽

Protein Kinase ◽

Caspase 3 ◽

Tnf Α ◽

Different Types ◽

Inflammatory Proteins ◽

Cox 2 ◽

Apoptotic Proteins

Objective: The objective of this research was to formulate the binding energies and interaction of amino acid residues in kaempferitrin with different types of apoptotic and inflammatory proteins of colon cancer.Methods: AutoDock Vina and MGL tool were used for docking calculations. Both programs require the pdbqt input files and allow for flexibility of all the torsional bonds of small molecules. Discovery Studio Visualizer v3.5 was used for removal of water molecules and ligands and the pymol program was used to do analysis of the docking with various apoptotic proteins BAX, Bcl-2, COX-2, Protein kinase B.Results: In our study was developed binding energy scoring function of kaempferitrin docked with different types of inflammatory proteins and apoptotic proteins. Binding score values for-6.9 (BAX),-7.2 (Bcl-2),-7.3 (caspase-3),-8.8 (Cox-2),-7.4 (Cytochrome P450),-6.7 (Proteinase kinase B),-8.0 (TNF-α) and-7.2 (VEGF) kcal/mol, respectively. Amino acid interaction of kaempferitrin with proteins for ARG-25, LEU-52, ASN-54, PHE-55, GLU-17, LYS-14, TRP-22, THR-21 GLY-16 (Protein Kinase B), ASP-102, ASN-48, GLN-52, ASP-104 (BAX), GLU-176, TRP-173, GLU-132, PHE-135 (Bcl-2), SER-249, ASP-2, ASN-208, GLN-217, LEU-242 (Caspase 3), TYR-55, HIS-39, SER-49, GLU-322, GLY-326 (COX-2), SER-95, LEU-94, ARG-82, VAL-123, ALA-96 (TNF-α), ASP-414, LYS-322, GLU-326, GLU-416, GLU-438, ALA-439, GLU-437 (Cytochrome P450) and LEU-47, GLN-46, CYS-61, CYS-60, ASP-63, GLU-67, GLY-65, LEU-66 (VEGF) respectively.Conclusion: The results obtained in this research work clearly indicated the docking scores of apoptotic and Inflammatory proteins imply that kaempferitrin is an effective inhibitory compound for colon cancer.

Download Full-text

Amino acid sequence homology between rat and human C-reactive protein

Biochemical Journal ◽

10.1042/bj2210903 ◽

1984 ◽

Vol 221 (3) ◽

pp. 903-906 ◽

Cited By ~ 12

Author(s):

J A Taylor ◽

C J Bruton ◽

J K Anderson ◽

J E Mole ◽

F C De Beer ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Binding Protein ◽

C Reactive Protein ◽

Rat Serum ◽

Reactive Protein ◽

Phosphoryl Choline ◽

Serum Amyloid P Component ◽

C Terminus ◽

Amyloid P

The rat serum protein that undergoes Ca2+-dependent binding to pneumococcal C-polysaccharide and to phosphocholine residues, and that is evidently a member of the pentraxin family of proteins by virtue of its appearance under the electron microscope, has been variously designated as rat C-reactive protein (CRP) [de Beer, Baltz, Munn, Feinstein, Taylor, Bruton, Clamp & Pepys (1982) Immunology 45, 55-70], ‘phosphoryl choline-binding protein’ [Nagpurkar & Mookerjea (1981) J. Biol. Chem. 256, 7440-7448] and rat serum amyloid P component (SAP) [Pontet, D'Asnieres, Gache, Escaig & Engler (1981) Biochim. Biophys. Acta 671, 202-210]. The partial amino acid sequence (45 residues) towards the C-terminus of this protein was determined, and it showed 71.7% identity with the known sequence of human CRP but only 54.3% identity with human SAP. Since human CRP and SAP are themselves approximately 50% homologous, the level of identity between the rat protein and human SAP is evidence only of membership of the pentraxin family. In contrast, the much greater resemblance to human CRP confirms that the rat C-polysaccharide-binding/phosphocholine-binding protein is in fact rat CRP.

Download Full-text

Role of NF-κB in TNF-α-induced COX-2 Expression in Synovial Fibroblasts from Human TMJ

Journal of Dental Research ◽

10.1177/154405910708600412 ◽

2007 ◽

Vol 86 (4) ◽

pp. 363-367 ◽

Cited By ~ 36

Author(s):

J. Ke ◽

X. Long ◽

Y. Liu ◽

Y.F. Zhang ◽

J. Li ◽

...

Keyword(s):

Synovial Fibroblasts ◽

Nuclear Factor Κb ◽

Mobility Shift ◽

Tnf Α ◽

Cox 2 ◽

Mobility Shift Assay ◽

Pre Treatment ◽

Factor Α ◽

Necrosis Factor

In the temporomandibular joint (TMJ) synovium, cyclo-oxygenase-2 (COX-2) expression has been believed to be directly related to joint pain and synovitis. Here we investigated the role of Nuclear Factor κB (NF-κB) in the regulation of COX-2 expression in synovial fibroblasts from human TMJ induced by tumor necrosis factor-α (TNF-α). By reverse-transcriptase/polymerase chain-reaction (RT-PCR) and Western blotting analysis, TNF-α induced a dose- and time-dependent increase in COX-2 expression. Electrophoretic mobility shift assay (EMSA) revealed that transient NF-κB activation in the COX-2 promoter was triggered by TNF-α. In parallel with transient NF-κB activation, the rapid translocation of NF-κB, particularly the p65 subunit, from the cytoplasm into the nucleus was demonstrated. Pre-treatment with pyrolidine dithiocarbamate (PDTC), one of the NF-κB inhibitors, prevented binding to the COX-2 promoter and expression of COX-2 protein in response to TNF-α. These findings indicate that activation of NF-κB is responsible for TNF-α-induced COX-2 expression in synovial fibroblasts from the TMJ.

Download Full-text