ABSTRACT
The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.
Subject(s)
Capsid Proteins/ultrastructure , HIV Infections/genetics , HIV-1/genetics , Sp1 Transcription Factor/ultrastructure , gag Gene Products, Human Immunodeficiency Virus/ultrastructure , Capsid/chemistry , Capsid/ultrastructure , Capsid Proteins/genetics , Crystallography, X-Ray , HIV Infections/virology , HIV Seropositivity , HIV-1/pathogenicity , HIV-1/ultrastructure , Humans , Molecular Dynamics Simulation , Protein Multimerization/genetics , Proteolysis , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/genetics , Virion/genetics , Virion/pathogenicity , Virus Assembly/genetics , gag Gene Products, Human Immunodeficiency Virus/chemistry , gag Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
We report the biosynthesis of size confined palladium nanoparticles (Pd-NPs). The 2-3 nm size Pd-NPs were grown in 12-mer protein stable protein 1 (SP1), which serves as a template for the NP formation. We further show that by controlling the protein expression levels in the cells we can alter the cells' catalytic activity. The in vivo grown Pd-NPs were utilized in a hydrogenation reaction, converting acetylene feedstock into ethylene and ethane. The presented concept can be further used for a wide range of applications by exploiting the synergetic effect of the biotic elements with the abiotic ones.
Subject(s)
Metal Nanoparticles/chemistry , Palladium/metabolism , Sp1 Transcription Factor/metabolism , Hydrogenation , Palladium/chemistry , Particle Size , Populus/chemistry , Sp1 Transcription Factor/chemistry , Surface PropertiesABSTRACT
During the maturation of HIV-1 particle, the Gag polyprotein is cleaved into several proteins by the HIV-1 protease. These proteins rearrange to form infectious virus particles. In this study, the solution structure and dynamics of a monomeric mutated domain encompassing the C-terminal of capsid, the spacer peptide SP1 and the nucleocapsid from Gag was characterized by Nuclear Magnetic Resonance in the presence of maturation inhibitor EP39, a more hydro-soluble derivative of BVM. We show that the binding of EP39 decreases the dynamics of CA-SP1 junction, especially the QVT motif in SP1, and perturbs the natural coil-helix equilibrium on both sides of the SP1 domain by stabilizing the transient alpha helical structure. Our results provide new insight into the structure and dynamics of the SP1 domain and how HIV-1 maturation inhibitors interfere with this domain. They offer additional clues for the development of new second generation inhibitors targeting HIV-1 maturation.
Subject(s)
Anti-HIV Agents/pharmacology , Gene Products, gag/metabolism , HIV-1/drug effects , Amino Acid Sequence , Binding Sites , Dimerization , Gene Products, gag/chemistry , HIV-1/physiology , Humans , Nuclear Magnetic Resonance, Biomolecular , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/metabolismABSTRACT
NAMI-A is highly reactive to Sp1, a tumor metastasis related protein, resulting in the perturbation of the protein structure and disruption of the DNA recognition of Sp1. Interestingly, Sp1 is more susceptible than other zinc finger proteins to NAMI-A, suggesting that Sp1 could be the anti-metastasis target of NAMI-A.
Subject(s)
Antineoplastic Agents/chemistry , Dimethyl Sulfoxide/analogs & derivatives , Organometallic Compounds/chemistry , Ruthenium Compounds/chemistry , Sp1 Transcription Factor/chemistry , DNA/metabolism , Dimethyl Sulfoxide/chemistry , Glutathione/chemistry , Protein Binding , Protein Multimerization/drug effects , Protein Structure, Secondary/drug effects , Protein Unfolding/drug effects , Sp1 Transcription Factor/metabolismABSTRACT
The oxidation of guanine to 8-oxoguanine (8-oxoG) is the most common type of oxidative DNA lesion. There is a growing body of evidence indicating that 8-oxoG is not only pre-mutagenic, but also plays an essential role in modulating gene expression along with its cognate repair proteins. In this study, we investigated the relationship between 8-oxoG formed under intrinsic oxidative stress conditions and gene expression in adipose and lung tissues of juvenile mice. We observed that transcriptional activity and the number of active genes were significantly correlated with the distribution of 8-oxoG in gene promoter regions, as determined by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), and 8-oxoG and RNA sequencing. Gene regulation by 8-oxoG was not associated with the degree of 8-oxoG formation. Instead, genes with GC-rich transcription factor binding sites in their promoters became more active with increasing 8-oxoG abundance as also demonstrated by specificity protein 1 (Sp1)- and estrogen response element (ERE)-luciferase assays in human embryonic kidney (HEK293T) cells. These results indicate that the occurrence of 8-oxoG in GC-rich Sp1 binding sites is important for gene regulation during adipose tissue development.
Subject(s)
Adipose Tissue/metabolism , GC Rich Sequence , Guanine/analogs & derivatives , Sp1 Transcription Factor/metabolism , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Genomics , Guanine/metabolism , HEK293 Cells , Humans , Mice , Nucleotide Motifs , Sp1 Transcription Factor/chemistryABSTRACT
Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.
Subject(s)
Histone Deacetylases/metabolism , Protein Biosynthesis , Sirtuins/metabolism , Sp1 Transcription Factor/metabolism , TOR Serine-Threonine Kinases/metabolism , Transcription, Genetic , Animals , Cardiomegaly/genetics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Histone Deacetylases/genetics , Humans , Mice , Mice, Knockout , Promoter Regions, Genetic , Signal Transduction , Sirtuins/genetics , Sp1 Transcription Factor/chemistry , Zinc FingersABSTRACT
BACKGROUND/AIMS: Peperomin E (PepE), a natural secolignan isolated from the whole plant of Peperomia dindygulensis, has been reported by ourselves and others to display potent anti-cancer effects in many types cancer cells, especially gastric cancer. However, the effects of PepE on the metastasis of poorly-differentiated gastric cancer cells and the underlying molecular mechanisms have not been well elucidated. METHODS: We evaluated PepE effects on gastric cancer cell invasion and migration in vitro via wound healing and transwell assays and those on growth and metastasis in vivo using an orthotopic xenograft NOD-SCID mouse model. DNA methyltransferase (DNMT) activity was determined using a colorimetric DNMT activity/inhibition assay kit. PepE binding kinetics to DNMTs were determined using the bio-layer interferometry binding assay. Gene and protein levels of DNMTs, AMPKα-Sp1 signaling molecules, and metastatic-suppressor genes in PepE-treated gastric cancer cells were determined using quantitative reverse transcription-PCR arrays and western blotting. The effect of PepE on Sp1 binding to the DNMT promoter was determined by electrophoretic mobility-shift assay. Global DNA methylation levels were determined using liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The methylation status of silenced metastatic-suppressor genes (MSGs) in gastric cancer cells was investigated by methylation-specific PCR. RESULTS: PepE can dose-dependently suppress invasion and migration of poorly-differentiated gastric cancer cells in vitro and in vivo with low toxicity against normal cells. Mechanistically, PepE not only covalently binds to the catalytic domain of DNMT1 and inhibits its activity (IC50 value 3.61 µM) but also down-regulates DNMT1, 3a, and 3b mRNA and protein expression in in gastric cancer cells, by disruption of the physical interaction of Sp1 with the DNMT1, 3a, and 3b promoter and mediation of the AMPKα-Sp1 signaling pathway. The dual inhibition activity of PepE toward DNMTs renders a relative global DNA hypomethylation, which induces MSG promoter hypomethylation (e.g., E-cadherin and TIMP3) and enhances their expression in gastric cancer cells. CONCLUSION: Collectively, our data indicated that PepE may represent a promising therapeutic lead compound for intervention in gastric cancer metastasis and may also exhibit potential as a DNA methylation inhibitor for use in epigenetic cancer therapy.
Subject(s)
Benzodioxoles/pharmacology , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/drug effects , AMP-Activated Protein Kinases/chemistry , AMP-Activated Protein Kinases/metabolism , Animals , Benzodioxoles/chemistry , Benzodioxoles/therapeutic use , Binding Sites , Cell Movement/drug effects , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferases/genetics , Female , Humans , Liver Neoplasms/pathology , Liver Neoplasms/secondary , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Dynamics Simulation , Promoter Regions, Genetic , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/metabolism , Stomach Neoplasms/drug therapy , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/metabolismABSTRACT
The crowding effect is prevalent in cellular environments due to high concentrations of biomacromolecules. It can alter the structures and dynamics of proteins and thus impact protein functions. The crowding effect is important not only in 3-dimensional cytoplasm but also for a 2-dimensional (2D) cell surface due to the presence of membrane proteins and glycosylation of membrane proteins and phospholipids. These proteins and phospholipids - with limited translational degrees of freedom along the surface normal - are confined in 2D space. Although the crowding effect at interfaces has been studied by adding crowding agents to bulk solution, the 2D crowding effect remains largely unexplored. This is mostly due to challenges in controlling 2D crowding and synergistic use of physical methods for in situ protein characterization. To address these challenges, we applied chiral vibrational sum frequency generation (SFG) spectroscopy to probe the sp1 zinc finger (ZnF), a 31-amino acid protein, folding into a ß-hairpin/α-helix (ßßα) motif upon binding to Zn2+. We anchored ZnF at the air/water interface via covalent linkage of ZnF to palmitic acid and controlled 2D crowding by introducing neutral lipid as a spacer. We obtained chiral amide I SFG spectra upon addition of Zn2+ and/or spacer lipid. The chiral SFG spectra show that interfacial crowding in the absence of spacer lipid hinders ZnF from folding into the ßßα structure even in the presence of Zn2+. The results establish a paradigm for future quantitative, systematic studies of interfacial crowding effects.
Subject(s)
Membrane Proteins/chemistry , Phospholipids/chemistry , Cations, Divalent , Cell Membrane/chemistry , Glycosylation , Palmitic Acid/chemistry , Protein Binding , Protein Conformation , Protein Folding , Sp1 Transcription Factor/chemistry , Spectrum Analysis/methods , Vibration , Water , Zinc/chemistry , Zinc FingersABSTRACT
OBJECTIVE: Sp1 is a member of super zinc finger structure family that participates in cancer cells' apoptosis, proliferation, survival, and differentiation. This study detected the expressions of miR-375 and sp1 in colorectal cancer tissue and cells to analyze their impact on cell proliferation. PATIENTS AND METHODS: Colorectal cancer patients in our hospital were enrolled. HCT-116 cell was transfected with miR-375 mimics, mimics control, and miR-375 + sp1, respectively. RT-PCR and Western blot were applied to detect expressions of miR-375 and sp1 at mRNA and protein level in colorectal cancer tissue, para-carcinoma tissue, and normal colorectal tissue. RT-PCR and Western blot were used to test levels of miR-375 and sp1 in HCT-116 cells after transfection. MTT assay was performed to determine HCT-116 cell proliferation. RESULTS: Our data showed that miR-375 was downregulated, while sp1 was overexpressed in colorectal cancer tissue compared with that in para-carcinoma tissue and normal control (p < 0.05). MiR-375 level was elevated, while sp1 mRNA was declined after miR-375 mimic transfection (p < 0.05). Compared with miR-375 mimic group, the levels of miR-375 and sp1 showed no difference in miR-375 + sp1 group (p > 0.05). Of note, the increase of MiR-375 and reduction of sp1 were in a time-dependent manner (p < 0.05). The cell proliferation rate in miR-375 mimic group was significantly decreased compared with that in mimic control and blank group (p < 0.05). The cell proliferation rate in miR-375 + sp1 group was significantly higher than that miR-375 group, but still lower than the control (p < 0.05). The proliferation rate gradually declined in a time-dependent manner (p < 0.05). CONCLUSIONS: MiR-375 was decreased and sp1 level was enhanced in colorectal cancer. MiR-375 suppresses the proliferation of colorectal cancer cells via the inhibition of sp1 expression at posttranscriptional level.
Subject(s)
Cell Proliferation , Colorectal Neoplasms/pathology , MicroRNAs/metabolism , Sp1 Transcription Factor/metabolism , 3' Untranslated Regions , Antagomirs/metabolism , Apoptosis , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Down-Regulation , Female , HCT116 Cells , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , RNA, Messenger/metabolism , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/geneticsABSTRACT
ATM (ataxia-telangiectasia mutated) is a central molecule for DNA quality control. Its activation by DNA damage promotes cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, persistent DNA damage has been implicated in ATM-dependent cell death via apoptosis; however, the mechanisms underlying this process remain elusive. Here we find that, in response to persistent DNA strand breaks, ATM phosphorylates transcription factor Sp1 and initiates its degradation. We show that Sp1 controls expression of the key base excision repair gene XRCC1, essential for DNA strand break repair. Therefore, degradation of Sp1 leads to a vicious cycle that involves suppression of DNA repair and further aggravation of the load of DNA damage. This activates transcription of pro-apoptotic genes and renders cells susceptible to elimination via both apoptosis and natural killer cells. These findings constitute a previously unrecognized 'gatekeeper' function of ATM as a detector of cells with persistent DNA damage.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Repair , Sp1 Transcription Factor/metabolism , Apoptosis , Cells, Cultured , DNA Damage , Down-Regulation , Humans , Killer Cells, Natural/physiology , Male , Phosphorylation , Serine/metabolism , Sp1 Transcription Factor/chemistry , X-ray Repair Cross Complementing Protein 1/biosynthesis , X-ray Repair Cross Complementing Protein 1/geneticsABSTRACT
Human polyomavirus 9 (HPyV9) was originally detected in the serum of a renal transplant patient. Seroepidemiological studies showed that ~20-50% of the human population have antibodies against this virus. HPyV9 has not yet been associated with any disease and little is known about the route of infection, transmission, host cell tropism, and genomic variability in circulating strains. Recently, the HPyV9 variant UF-1 with an eight base-pair deletion, a thirteen base-pair insertion and with point mutations, creating three putative Sp1 binding sites in the late promoter was isolated from an AIDS patient. Transient transfection studies with a luciferase reporter plasmid driven by HPyV9 or UF1 promoter demonstrated that UF1 early and late promoters were stronger than HPyV9 promoters in most cell lines, and that the UF1 late promoter was more potently activated by HPyV9 large T-antigen (LTAg). Mutation of two Sp1 motifs strongly reduced trans-activation of the late UF1 promoter by HPyV9 LTAg in HeLa cells. In conclusion, the mutations in the UF1 late promoter seem to strengthen its activity and its response to stimulation by HPyV9 LTAg in certain cells. It remains to be investigated whether these promoter changes have an influence on virus replication and affect the possible pathogenic properties of the virus.
Subject(s)
Antigens, Polyomavirus Transforming/metabolism , Nucleotide Motifs/genetics , Polyomavirus/genetics , Promoter Regions, Genetic , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/metabolism , Base Sequence , Cell Line , Humans , Mutation/genetics , Protein BindingABSTRACT
The expression of eukaryotic genes is precisely controlled by interactions between general transcriptional factors and promoter-specific transcriptional activators. The fourth element of TATA-box binding protein-associated factor (TAF4), an essential subunit of the general transcription factor TFIID, serves as a coactivator for various promoter-specific transcriptional regulators. Interactions between TAF4 and site-specific transcriptional activators, such as Sp1, are important for regulating the expression levels of genes of interest. However, only limited information is available on the molecular mechanisms underlying the interactions between these transcriptional regulatory proteins. We herein analyzed the interaction between the transcriptional factors Sp1 and TAF4 using high-resolution solution nuclear magnetic resonance spectroscopy. We found that four glutamine-rich (Q-rich) regions in TAF4 were largely disordered under nearly physiological conditions. Among them, the first Q-rich region in TAF4 was essential for the interaction with another Q-rich region in the Sp1 molecule, most of which was largely disordered. The residues responsible for this interaction were specific and highly localized in a defined region within a range of 20-30 residues. Nevertheless, a detailed analysis of 13 C-chemical shift values suggested that no significant conformational change occurred upon binding. These results indicate a prominent and exceptional binding mode for intrinsically disordered proteins other than the well-accepted concept of "coupled folding and binding."
Subject(s)
Intrinsically Disordered Proteins/chemistry , Sp1 Transcription Factor/chemistry , TATA-Binding Protein Associated Factors/chemistry , Transcription Factor TFIID/chemistry , Amino Acid Motifs , Binding Sites , Carbon Isotopes , Humans , Intrinsically Disordered Proteins/metabolism , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Folding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sp1 Transcription Factor/metabolism , TATA-Binding Protein Associated Factors/metabolism , Transcription Factor TFIID/metabolism , Transcription, GeneticABSTRACT
INTRODUCTION: Cytosine methylation at CpG dinucleotides is a chief mechanism in epigenetic modification of gene expression patterns. Previous studies demonstrated that increased CpG methylation of Sp1 sites at -268 and -346 of protein kinase C ε promoter repressed the gene expression. MATERIALS & METHODS: The present study investigated the impact of CpG methylation on the Sp1 binding via molecular modeling and electrophoretic mobility shift assay. Each of the Sp1 sites contain two CpGs. Methylation of either CpG lowered the binding affinity of Sp1, whereas methylation of both CpGs produced a greater decrease in the binding affinity. Computation of van der Waals (VDW) energy of Sp1 in complex with the Sp1 sites demonstrated increased VDW values from one to two sites of CpG methylation. Molecular modeling indicated that single CpG methylation caused underwinding of the DNA fragment, with the phosphate groups at C1, C4 and C5 reoriented from their original positions. Methylation of both CpGs pinched the minor groove and increased the helical twist concomitant with a shallow, hydrophobic major groove. Additionally, double methylation eliminated hydrogen bonds on recognition helix residues located at positions -1 and 1, which were essential for interaction with O6/N7 of G-bases. Bonding from linker residues Arg565, Lys595 and Lys596 were also reduced. Methylation of single or both CpGs significantly affected hydrogen bonding from all three Sp1 DNA binding domains, demonstrating that the consequences of cytosine modification extend beyond the neighboring nucleotides. RESULTS: The results indicate that cytosine methylation causes subtle structural alterations in Sp1 binding sites consequently resulting in inhibition of side chain interactions critical for specific base recognition and reduction of the binding affinity of Sp1.
Subject(s)
Cytosine/metabolism , DNA Methylation , DNA/metabolism , Models, Molecular , Sp1 Transcription Factor/metabolism , Binding Sites , Cytosine/chemistry , DNA/chemistry , Electrophoretic Mobility Shift Assay , Hydrogen Bonding , Protein Binding , Sp1 Transcription Factor/chemistryABSTRACT
ING4, a potential tumor suppressor, is implicated in cell cycle arrest, apoptosis, cell migration and angiogenesis. Here, we investigated the clinical value of ING4 and its impact on angiogenesis in colorectal cancer (CRC). In this study, we found that ING4 expression was significantly reduced in CRC tissues versus paired normal colon tissues. Moreover, low ING4 expression was significantly associated with increased lymph node metastasis, advanced TNM stage and poor overall survival. Multivariate Cox regression analysis showed that ING4 expression was an independent favourable prognostic factor for CRC (hazard ratio = 0.45, P = 0.001). In addition, we found that ING4 strongly inhibited CRC angiogenesis by suppressing Sp1 expression and transcriptional activity through ubiquitin degradation and down-regulating the expressions of Sp1 downstream pro-angiogenic genes, MMP-2 and COX-2. Moreover, ING4 might inhibit phosphorylation activity of cyclin/CDK2 complexes to trigger Sp1 degradation by inducing p21 expression in despite of p53 status. Our findings imply that reduced ING4 expression in CRC resulted in increased angiogenesis and contributed to CRC metastasis and poor prognosis. Restoration of ING4 may be a novel strategy for the treatment of metastatic CRC.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Colorectal Neoplasms/pathology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Neovascularization, Pathologic/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclooxygenase 2/metabolism , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Male , Matrix Metalloproteinase 2/metabolism , Mice , Neoplasm Staging , Neoplasm Transplantation , Neovascularization, Pathologic/genetics , Prognosis , Proteolysis , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/genetics , Survival AnalysisABSTRACT
The present study was carried out to evaluate dietary Mn requirements of broilers from 22 to 42 d of age using molecular biomarkers. Chickens were fed a conventional basal maize-soyabean meal diet supplemented with Mn as Mn sulphate in graded concentrations of 20 mg Mn/kg from 0 to 140 mg Mn/kg of diet for 21 d (from 22 to 42 d of age). The Mn response curves were fitted for ten parameters including heart Mn-containing superoxide dismutase (MnSOD) mRNA and its protein expression levels and the DNA-binding activities of specificity protein 1 (Sp1) and activating protein-2 (AP-2). Heart MnSOD mRNA and protein expression levels showed significant quadratic responses (P<0·01), and heart MnSOD activity showed a broken-line response (P<0·01), whereas Mn content and DNA-binding activities of Sp1 and AP-2 in the heart displayed linear responses (P<0·01) to dietary Mn concentrations, respectively. The estimates of dietary Mn requirements were 101, 104 and 94 mg/kg for full expressions of MnSOD mRNA level, MnSOD protein level and MnSOD activity in the heart, respectively. Our findings indicate that heart MnSOD mRNA expression level is a more reliable indicator than heart MnSOD protein expression level and its activity for the evaluation of Mn requirement of broilers, and about 100 mg Mn/kg of diet is required for the full expression of heart MnSOD in broilers fed the conventional basal maize-soyabean meal diet from 22 to 42 d of age.
Subject(s)
Avian Proteins/metabolism , Chickens/physiology , Gene Expression Regulation, Enzymologic , Manganese/administration & dosage , Myocardium/metabolism , Nutritional Requirements , Superoxide Dismutase/metabolism , Animals , Avian Proteins/chemistry , Avian Proteins/genetics , Biomarkers/metabolism , Chickens/growth & development , China , Energy Intake , Heart/growth & development , Male , Manganese/analysis , Manganese/metabolism , Manganese Compounds/administration & dosage , Myocardium/enzymology , RNA, Messenger/metabolism , Random Allocation , Reproducibility of Results , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/metabolism , Sulfates/administration & dosage , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics , Transcription Factor AP-2/chemistry , Transcription Factor AP-2/metabolism , Weight GainABSTRACT
The expression of eukaryotic genes is precisely controlled by specific interactions between general transcription initiation factors and gene-specific transcriptional activators. The general transcription factor TFIID, which plays an essential role in mediating transcriptional activation, is a multisubunit complex comprising the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). On the other hand, biochemical and genetic approaches have shown that the promoter-specific transcriptional activator Sp1 has the ability to interact with one of the components of TFIID, the TBP-associated factor TAF4. We herein report the structural details of the glutamine-rich domains (Q-domains) of Sp1 and TAF4 using circular dichroism (CD) and heteronuclear magnetic resonance (NMR) spectroscopy. We found that the two Q-domains of Sp1 and four Q-domains of TAF4 were disordered under physiological conditions. We also quantitatively analyzed the interaction between the Q-domains of Sp1 and TAF4 by NMR and surface plasmon resonance, and detected a weak but specific association between them. Nevertheless, a detailed analysis of CD spectra suggested that any significant conformational change did not occur concomitantly with this association, at least at the level of the overall secondary structure. These results may represent a prominent and exceptional binding mode for the IDPs, which are not categorized in a well-accepted concept of "coupled folding and binding."
Subject(s)
Sp1 Transcription Factor/chemistry , TATA-Binding Protein Associated Factors/chemistry , Transcription Factor TFIID/chemistry , Circular Dichroism , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Domains , Protein Structure, Secondary , Sp1 Transcription Factor/metabolism , Surface Plasmon Resonance , TATA-Binding Protein Associated Factors/metabolism , Transcription Factor TFIID/metabolismABSTRACT
cis-DDP presents reactivity towards the transcription factor Sp1-F3, as opposed to previous observations for Sp1-F2. Replacing the ammine ligands with the chelating ethylenediamine increases the reactivity giving a unique dinuclear {Pt(en)}2-bis(cysteine)-bridged product, confirmed by study of the binding sequence ACPECP.
Subject(s)
Coordination Complexes/chemistry , Platinum/chemistry , Sp1 Transcription Factor/chemistry , Amino Acid Sequence , Coordination Complexes/metabolism , Humans , Isomerism , Magnetic Resonance Spectroscopy , Mass Spectrometry , Protein Binding , Sp1 Transcription Factor/metabolism , Zinc FingersABSTRACT
The human high-affinity copper transporter 1 (hCtr1) transports both Cu(I) and cisplatin (cDDP). Because Cu deficiency is lethal yet Cu overload is poisonous, hCtr1 expression is transcriptionally upregulated in response to Cu deficiency but is downregulated under Cu replete conditions in controlling Cu homeostasis. The up- and down-regulation of hCtr1 is regulated by Specific protein 1 (Sp1), which itself is also correspondingly regulated under these Cu conditions. hCtr1 expression is also upregulated by cDDP via upregulation of Sp1. The underlying mechanisms of these regulations are unknown. Using gel-electrophoretic mobility shift assays, we demonstrated here that Sp1-DNA binding affinity is reduced under Cu replete conditions but increased under reduced Cu conditions. Similarly, Sp1-DNA binding affinity is increased by cDDP treatment. This in vitro system demonstrated, for the first time, that regulation of Sp1/hCtr1 expression by these agents is modulated by the stability of Sp1-DNA binding, the first step in the Sp1-mediated transcriptional regulation process.
Subject(s)
Cisplatin/chemistry , Copper/chemistry , DNA/chemistry , Sp1 Transcription Factor/chemistry , Biological Transport, Active , Cation Transport Proteins/biosynthesis , Copper Transporter 1 , DNA/metabolism , Humans , Oxidation-Reduction , Sp1 Transcription Factor/metabolismABSTRACT
Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.
Subject(s)
Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , MicroRNAs/genetics , Multiple Myeloma/genetics , Proto-Oncogene Proteins c-myc/genetics , Sp1 Transcription Factor/genetics , Animals , Base Sequence , Binding Sites , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , DNA Methylation , Disease Models, Animal , Down-Regulation , Gene Expression , Gene Expression Profiling , Gene Silencing , Genes, Reporter , Humans , MicroRNAs/chemistry , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , RNA, Messenger/chemistry , RNA, Messenger/genetics , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/metabolismABSTRACT
Organic nanoparticle induced self-assembly of proteins with periodic nanostructures is a promising and burgeoning strategy to develop functional biomimetic nanomaterials. Cricoid proteins afford monodispersed and well-defined hollow centers, and can be used to multivalently interact with geometrically symmetric nanoparticles to form one-dimensional protein nanoarrays. Herein, we report that core-cross-linked micelles can direct cricoid stable protein one (SP1) to self-assembling nanowires through multiple electrostatic interactions. One micelle can act as an organic nanoparticle to interact with two central concaves of SP1 in an opposite orientation to form a sandwich structure, further controlling the assembly direction to supramolecular protein nanowires. The reported versatile supramolecular scaffolds can be optionally manipulated to develop multifunctional integrated or synergistic biomimetic nanomaterials. Artificial light-harvesting nanowires are further developed to mimic the energy transfer process of photosynthetic bacteria for their structural similarity, by means of labeling donor and acceptor chromophores to SP1 rings and spherical micelles, respectively. The absorbing energy can be transferred within the adjacent donors around the ring and shuttling the collected energy to the nearby acceptor chromophore. The artificial light-harvesting nanowires are designed by mimicking the structural characteristic of natural LH-2 complex, which are meaningful in exploring the photosynthesis process in vitro.