scholarly journals Dual Regulation of c-Myc by p300 via Acetylation-Dependent Control of Myc Protein Turnover and Coactivation of Myc-Induced Transcription

2005 ◽  
Vol 25 (23) ◽  
pp. 10220-10234 ◽  
Author(s):  
Francesco Faiola ◽  
Xiaohui Liu ◽  
Szuying Lo ◽  
Songqin Pan ◽  
Kangling Zhang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Dependent Manner ◽  
Dual Roles ◽  
Human Telomerase Reverse Transcriptase ◽  
Lysine Residues ◽  
Human Telomerase

ABSTRACT The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. Here, we report a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. We show that p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independently of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene, and p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. Our results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Functional Domains of the TOL Plasmid Transcription Factor XylS

2000 ◽  
Vol 182 (4) ◽  
pp. 1118-1126 ◽  
Author(s):  
Niilo Kaldalu ◽  
Urve Toots ◽  
Victor de Lorenzo ◽  
Mart Ustav
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dependent Manner ◽  
Tol Plasmid ◽  
Terminal Fragment ◽  
Central Regions ◽  
Basic Features ◽  
Regulatory Functions

ABSTRACT The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 245-255 ◽  
Author(s):  
M. Van Doren ◽  
H.M. Ellis ◽  
J.W. Posakony
Keyword(s):  
Dna Binding ◽  
Cell Fate ◽  
Protein Complexes ◽  
Competitive Inhibitor ◽  
Binding Activity ◽  
Regulatory Function ◽  
Biochemical Basis ◽  
Dna Binding Activity

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three ‘E box’ consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

Bioflavonoids protect cells against halogenated boroxine-induced genotoxic damage by upregulation of hTERT expression

2019 ◽  
Vol 74 (5-6) ◽  
pp. 125-129 ◽  
Author(s):  
Maida Hadzic ◽  
Sanin Haveric ◽  
Anja Haveric ◽  
Naida Lojo-Kadric ◽  
Borivoj Galic ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Housekeeping Genes ◽  
Inhibitory Effects ◽  
Human Telomerase Reverse Transcriptase ◽  
Cycle Progression ◽  
Genotoxic Damage ◽  
Malignant Skin ◽  
Human Telomerase

Abstract Plant bioflavonoids are widely present in the human diet and have various protective properties. In this study, we have demonstrated the capacity of delphinidin and luteolin to increase human telomerase reverse transcriptase (hTERT) expression level and act as protective agents against halogenated boroxine-induced genotoxic damage. Halogenated boroxine K2(B3O3F4OH) (HB), is a novel compound with potential for the treatment of both benign and malignant skin changes. In vivo and in vitro studies have confirmed the inhibitory effects of HB on carcinoma cell proliferation and cell cycle progression as well as enzyme inhibition. However, minor genotoxic effects of HB are registered in higher applied concentrations, but those can be suppressed by in vitro addition of delphinidin and luteolin in appropriate concentrations. Fresh peripheral blood samples were cultivated for 72 h followed by independent and concomitant treatments of HB with luteolin or delphinidin. We analyzed the differences in relative hTERT expression between series of treatments compared with controls, which were based on normalized ratios with housekeeping genes. The obtained results have shown that selected bioflavonoids induce upregulation of hTERT that may contribute to the repair of genotoxic damage in vitro.

scholarly journals Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

1996 ◽  
Vol 16 (7) ◽  
pp. 3576-3586 ◽  
Author(s):  
C H Yang ◽  
J Tomkiel ◽  
H Saitoh ◽  
D H Johnson ◽  
W C Earnshaw
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
In Vitro Assays ◽  
Centromeric Heterochromatin ◽  
Structural Protein ◽  
Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

scholarly journals SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay in Caenorhabditis elegans

2004 ◽  
Vol 24 (17) ◽  
pp. 7483-7490 ◽  
Author(s):  
Andrew Grimson ◽  
Sean O'Connor ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Null Alleles ◽  
Dependent Manner ◽  
Messenger Rnas ◽  
Phosphatidylinositol Kinase ◽  
Nonsense Mediated Mrna Decay

ABSTRACT Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

scholarly journals Possible Role for Cellular Karyopherins in Regulating Polyomavirus and Papillomavirus Capsid Assembly

2008 ◽  
Vol 82 (20) ◽  
pp. 9848-9857 ◽  
Author(s):  
Gregory Bird ◽  
Malinda O'Donnell ◽  
Junona Moroianu ◽  
Robert L. Garcea
Keyword(s):  
Dna Binding ◽  
Capsid Assembly ◽  
Dependent Manner ◽  
Cellular Mechanisms ◽  
Virion Assembly ◽  
Localization Signal ◽  
Murine Polyomavirus ◽  
Cytoplasmic Assembly

ABSTRACT Polyomavirus and papillomavirus (papovavirus) capsids are composed of 72 capsomeres of their major capsid proteins, VP1 and L1, respectively. After translation in the cytoplasm, L1 and VP1 pentamerize into capsomeres and are then imported into the nucleus using the cellular α and β karyopherins. Virion assembly only occurs in the nucleus, and cellular mechanisms exist to prevent premature capsid assembly in the cytosol. We have identified the karyopherin family of nuclear import factors as possible “chaperones” in preventing the cytoplasmic assembly of papovavirus capsomeres. Recombinant murine polyomavirus (mPy) VP1 and human papillomavirus type 11 (HPV11) L1 capsomeres bound the karyopherin heterodimer α2β1 in vitro in a nuclear localization signal (NLS)-dependent manner. Because the amino acid sequence comprising the NLS of VP1 and L1 overlaps the previously identified DNA binding domain, we examined the relationship between karyopherin and DNA binding of both mPy VP1 and HPV11 L1. Capsomeres of L1, but not VP1, bound by karyopherin α2β1 or β1 alone were unable to bind DNA. VP1 and L1 capsomeres could bind both karyopherin α2 and DNA simultaneously. Both VP1 and L1 capsomeres bound by karyopherin α2β1 were unable to assemble into capsids, as shown by in vitro assembly reactions. These results support a role for karyopherins as chaperones in the in vivo regulation of viral capsid assembly.

scholarly journals Inhibiting lysine 353 oxidation of GRP78 by a hypochlorous probe targeting endoplasmic reticulum promotes autophagy in cancer cells

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Junya Ning ◽  
Zhaomin Lin ◽  
Xuan Zhao ◽  
Baoxiang Zhao ◽  
Junying Miao
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Growth ◽  
Cancer Cells ◽  
Cell Fate ◽  
Hypochlorous Acid ◽  
Dependent Manner ◽  
Autophagy Flux ◽  
Post Translational Modifications

Abstract The level of hypochlorous acid (HOCl) in cancer cells is higher than that in non-cancer cells. HOCl is an essential signal for the regulation of cell fate and works mainly through the protein post-translational modifications in cancer cells. However, the mechanism of HOCl regulating autophagy has not been clarified. Here we reported that a HOCl probe named ZBM-H targeted endoplasmic reticulum and induced an intact autophagy flux in lung cancer cells. Furthermore, ZBM-H promoted the binding of GRP78 to AMPK and increased the phosphorylation of AMPK in a dose- and time-dependent manner. GRP78 knockdown inhibited ZBM-H-induced AMPK phosphorylation and ZBM-H-stimulated autophagy. In addition, mass spectrometry combined with point mutation experiments revealed that ZBM-H increased GRP78 activity by inhibiting HOCl-induced lysine 353 oxidation of GRP78. Following ZBM-H treatment in vitro and in vivo, cell growth was significantly inhibited while apoptosis was induced. Nevertheless, exogenous HOCl partially reversed ZBM-H-inhibited cell growth and ZBM-H-induced GRP78 activation. In brief, we found that an endoplasmic reticulum-targeted HOCl probe named ZBM-H, acting through attenuating HOCl-induced GRP78 oxidation, inhibited tumor cell survival by promoting autophagy and apoptosis. Overall, these data demonstrated a novel mechanism of hypochlorous acid regulating autophagy by promoting the oxidation modification of GRP78.

Inhibition of Hemoglobin Degrading Protease Falcipain-2 as a Mechanism for Anti-Malarial Activity of Triazole-Amino Acid Hybrids

2020 ◽  
Vol 20 (5) ◽  
pp. 377-389 ◽  
Author(s):  
Vigyasa Singh ◽  
Rahul Singh Hada ◽  
Amad Uddin ◽  
Babita Aneja ◽  
Mohammad Abid ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Antimalarial Activity ◽  
Docking Studies ◽  
Dependent Manner ◽  
Trophozoite Stage ◽  
Hemoglobin Degradation

Background: Novel drug development against malaria parasite over old conventional antimalarial drugs is essential due to rapid and indiscriminate use of drugs, which led to the emergence of resistant strains. Methods: In this study, previously reported triazole-amino acid hybrids (13-18) are explored against Plasmodium falciparum as antimalarial agents. Among six compounds, 15 and 18 exhibited antimalarial activity against P. falciparum with insignificant hemolytic activity and cytotoxicity towards HepG2 mammalian cells. In molecular docking studies, both compounds bind into the active site of PfFP-2 and block its accessibility to the substrate that leads to the inhibition of target protein further supported by in vitro analysis. Results: Antimalarial half-maximal inhibitory concentration (IC50) of 15 and 18 compounds were found to be 9.26 μM and 20.62 μM, respectively. Blood stage specific studies showed that compounds, 15 and 18 are effective at late trophozoite stage and block egress pathway of parasites. Decreased level of free monomeric heme was found in a dose dependent manner after the treatment with compounds 15 and 18, which was further evidenced by the reduction in percent of hemoglobin hydrolysis. Compounds 15 and 18 hindered hemoglobin degradation via intra- and extracellular cysteine protease falcipain-2 (PfFP-2) inhibitory activity both in in vitro and in vivo in P. falciparum. Conclusion: We report antimalarial potential of triazole-amino acid hybrids and their role in the inhibition of cysteine protease PfFP-2 as its mechanistic aspect.

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