Region Of DNA-binding Domain Research Articles

Targeting Human Papillomavirus 33 E2 DNA Binding Domain With Polyphenols: Unveiling Interactions Through Biophysical and In Silico Methods.

The human papillomavirus (HPV) 33 is a high-risk strain that causes lesions with potential cancerous outcomes. Its E2 protein regulates the viral protein transcription and life cycle maintenance. The DNA binding domain (DBD) of the E2 protein plays a crucial role in the viral life cycle. The DBD region of the E2 protein is particularly interesting for targeting and finding potential inhibitors to inhibit its function or dimerization. Given the limited research on HPV 33 and its proteins, the present work delved into the interaction of two natural polyphenolic compounds, resveratrol, and baicalein, with the E2 DBD of HPV 33 using biophysical and in silico studies. Fluorescence studies of the E2 DBD-polyphenol complexes showed fluorescence quenching with a binding constant of the order of 106 M-1. Circular dichroism data reveal conformational changes upon binding with the polyphenols, possibly due to distinct binding sites of the E2 DBD. Differential scanning calorimetry exhibited higher melting temperatures for the two complexes than alone DBD, suggesting the complexes' stability. ITC experiment suggested favorable binding reactions with kd values in the micromolar range. Molecular docking and dynamic simulation studies revealed that the resveratrol binds to the helical region and baicalein near the central dimeric interface of E2 DBD with a good binding affinity, forming a stable protein-ligand complex during the run of 100 ns simulation. Therefore, the current study identifies both polyphenolic compounds as promising candidates for potential antiviral drug development.

LBi-DBP, an accurate DNA-binding protein prediction method based lightweight interpretable BiLSTM network

Accurate prediction of the DNA-binding protein (DBP) helps to uncover the underlying mechanism of DNA-protein interaction, thus contributing to the understanding of many biochemical processes. Although the computational methods have made great process, there is still much room for improving the DBP prediction. In this study, a lightweight and interpretable sequence-based method, named LBi-DBP, is well-designed to improve the performance of DBP prediction. In LBi-DBP, five sequence feature sources are first employed to extract important sequence context information using a slight bidirectional long short-term memory (BiLSTM)-based neural network module; then, a multi-layer perceptron (MLP) module is utilized to learn the optimal/sub-optimal parameters with sequence context information extracted by BiLSTM module and pseudo sequence order feature as inputs. Experimental results on two independent test sets, i.e., UniSwiss-Tst and PDB2272, demonstrate that LBi-DBP can achieve Matthew’s correlation coefficient values of 0.762 and 0.574 respectively, which are both higher than those of the second-best existing DBP prediction method (0.741 and 0.424). Meanwhile, good prediction result on a disease-related test set also shows the value of LBi-DBP. Through interpretability analysis, we find that the BiLSTM module can focus on the residues in real native DNA-binding domain region without prior knowledge, thus improving DNA-binding protein prediction performance.

42P Efficacy of maintenance with PARPi in advanced ovarian cancer according to the location of BRCA mutation

Currently little is known about correlations between the type and the location of the BReast CAncer genes (BRCA) 1/2 mutations and response to Poly (ADP-ribose) polymerase inhibitors (PARPi) in ovarian cancer (OC). Clinical data from PAOLA1 study suggest that patients with mutations in DNA binding domain (DNA-BD) of BRCA1 are more sensitive to the combination olaparib/bevacizumab and those in the DNA-BD of BRCA2 confer excellent outcomes. We investigated if the type and the location of the BRCA 1/2 mutations are related with PARPi sensitivity. This multicenter and retrospective study evaluated the efficacy of PARPi according to the mutation type and location in the functional domain (FD) of BRCA1 (RING, DNA-BD or BRCA1 C terminus [BRCT]) and BRCA2 (PALB2, RAD51-BD; DNA-BD). BRCA1/2 germline mutated OC patients receiving PARPi as maintenance, after a response to platinum-based first line chemotherapy were enrolled. Baseline characteristics and survival data were collected from 3 academic centers. Of 202 patients treated between December 2008 and October 2021, 122 (60.4%) received PARPi in the first-line setting and 80 (39.6%) at recurrence. Among the first group, 73 (60%) and 49 (40%) harbored BRCA1 and BRCA2 mutations, respectively. BRCA1 mutations in FDs of RING, DNA-BD and BRCT were found in 3 (2.5%), 8 (6.6%) and 21 (17.2%) patients, respectively. BRCA2 mutation were detected in FDs of RAD51-BD and DNA-BD in 25 (20.5%) and 5 (4.1%) patients, respectively. Median follow-up was of 20 (4-100) months. In patients with DNA-BD mutation of BRCA1/2, 2-yr Progression Free Survival PFS was 100%, compared with 80% in mutation in the other domains (log rank p= 0.079). With regard to the mutation type, patients harboring a missense BRCA1 mutation had a longer PFS (2-yr PFS 100%), compared with 50% of those with a splicing mutation (log rank p= 0.021) and 75% of those with a nonsense mutation (log rank p= 0.049). Mutation types in BRCA2 were not analyzed due to the small numbers. The results confirmed that mutations in the DNA-BD region in the BRCA 1/2 genes confer longer PFS respect to other location of mutation. Besides, missense mutation has the best prognosis, compared with splicing and nonsense mutation.

Comprehensive Analysis of TP53 Mutation Characteristics and Identification of Patients with Inferior Prognosis and Enhanced Immune Escape in Diffuse Large B Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphatic malignancy from both the clinical and molecular standpoints. TP53 mutations have been observed in DLBCL with high frequency mutations, which play a crucial role in lymphomagenesis. Considering the complex pathophysiological mechanisms involved in DLBCL, we herein assume that the interaction of TP53 with other genetic variants together promote the development of DLBCL. We also explore that these genetic interactions shape the discrepant immune landscape in DLBCL.Methods: We performed a comprehensive analysis of TP53 mutations in the DNA-binding domain (DBD) region. Genome alterations that were mutually exclusive or co-occurring with TP53 mutations were identified, and their potential value in prognosis and possible relationships with tumor microenvironment (TME) were further examined using targeted next-generation sequencing (n=176), transcriptome sequencing (n=152) and circulating tumor DNA sequencing (n=38).Results: TP53 was frequently mutated in DLBCL with a rate of 30% (53 of 176). The occurrence of TP53 mutations was skewed toward the early stages of DLBCL pathogenesis. Among these variants of TP53, 74% were missense mutations, and the remaining were inactivating frameshift indels, nonsense mutations, coding sequencing indels and splicing mutations. Importantly, most mutations (87.5%) occurred in exons 5-8, which encoded the DNA-binding domain (DBD) region of TP53. Codons 175, 273, and 248 of the p53 protein had the highest mutation frequency, which are also the hot spots of TP53 mutation found in most human cancers. However, TP53 alone is insufficient to effectively differentiate the risk of DLBCL, even when only considering mutations in the DBD region. CD58 mutations, which are mutually exclusive from TP53 mutations, in combination with TP53 mutations, could significantly differentiate the prognosis of DLBCL. The survival of patients with either one of the mutually exclusive mutation patterns, namely, TP53MUT&CD58WT or TP53WT&CD58MUT, was inferior to those harboring both wild-type TP53 and CD58. Notably, patients with TP53WT&CD58MUT mutation pattern had the worst outcome and were characterized by a higher overall tumor mutation burden (TMB) and immune score. An enhanced immune escape feature, including the abundant infiltration of inflammatory cells and upregulation of inhibitory immunomodulatory molecules such as PD-1, TIM3, LAG3, KLRC1, KLRC3, KLRD1, IDO1, IDO2 and TDO2, was observed in these patients with TP53WT&CD58MUT mutation pattern. A larger number of terms related to cytokine and chemokine and inflammatory pathways were enriched in the TP53WT&CD58MUT group, including cytokine and chemokine production, binding and activity, and interferon-γ pathway. Additionally, GSEA demonstrated that interferon-α and -γ responses and IL-6/JAK/STAT3 signaling enriched in the TP53WT&CD58MUT group. These patients with TP53WT&CD58MUT mutation pattern represent the candidate populations for immune therapy.Conclusions: Our findings indicated that the mutation patterns of TP53 and CD58 accurately stratified patients with DLBCL to permit the optional immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Unraveling the Allosteric Mechanism of Four Cancer-related Mutations in the Disruption of p53-DNA Interaction.

The p53 protein plays active roles in the physiological regulation of cell cycle as well as in cancer developments. In more than half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain (DBD), and some mutations located in the β-sandwich region of DBD are reported to decrease p53-DNA binding affinities. To understand the long-range correlation between p53 β-sandwich and DNA, and the allosteric mechanism of β-sandwich mutations in the disruption of p53-DNA interactions, we first identify three regions with a strong correlation with DNA based on microsecond molecular dynamics (MD) simulations of wild-type p53-DNA complex and then perform multiple MD simulations on four cancer-related mutants L145Q, P151S, Y220C, and G266R, which are located in these three regions. Our simulations show that these mutations allosterically destabilize the structural stability of the DNA-binding groove in p53 and disrupt the p53-DNA interactions. Network analyses reveal optimal correlation paths through which the mutation-induced allosteric signal passes to DNA, and the disturbance effect of these mutations on the global connectivity and dynamical correlation of the p53-DNA complex. This work paves the way for the in-depth understanding of the mutation-induced loss in p53's DNA-recognition ability and the pathological mechanism of cancer development.

Characterizing the Binding Interactions between DNA-Binding Proteins, XPA and XPE: A Molecular Dynamics Approach.

The scaffold nature of Xeroderma pigmentosum complementation group A (XPA) protein makes it an important member of nucleotide excision repair (NER) that removes bulky DNA lesions with the help of various protein–protein interactions (PPI) and DNA–protein interactions. However, many structural insights of XPA’s interaction and the binding patterns with other NER proteins are yet to be understood. Here, we have studied one such crucial PPI of XPA with another NER protein, Xeroderma pigmentosum complementation group A (XPE), by using the previously identified binding site of XPA (residues 185–226) in the Assisted Model Building With Energy Refinement force-field-mediated dynamic system. We studied the relationship between XPA185–226–XPE complex using three different docked models. The major residues observed in all of the models that were responsible for the PPI of this complex were Arg20, Arg47, Asp51, and Leu57 from XPE and the residues Leu191, Gln192, Val193, Trp194, Glu198, Glu202, Glu205, Arg207, Glu209, Gln216, and Phe219 from XPE185–226. During the simulation study, the orientation of XPA was also noted to be changed by almost 180° in models 1 and 3, which remain unchanged in model 2, indicating that XPA interacts with XPE with its N-terminal end facing downward and C-terminal end facing upward. The same was concurrent with the binding of DNA-binding domain region of XPA (aa98–239) with XPE. The N-terminal of XPE was stretched for accommodating XPA. Using the per-residue energy decomposition analysis for the interface residues of all models, the binding affinity between these proteins were found to be dependent on R20, R47, and L57 of XPE and the residues L191, V193, W194, E198, E202, E205, R207, and F219 of XPA. The net binding free energy of the XPA185–226–XPE protein complex was found to be −48.3718 kcal mol–1 for model 1, −49.09 kcal mol–1 for model 2, and −56.51 kcal mol–1 for model 3.

Fli1 Expression Level Affects Megakaryopoiesis and Thrombopoiesis: More Is Not Always a Bad Thing

Friend leukemia integration 1 (FLI1) is a critical transcription factor responsible for terminal megakaryocyte differentiation. This transcription factor is amongst the genes missing in the inherited disorder Jacobsen syndrome, resulting from a hemizygous deletion on chromosome 11q. The deletion causes dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Also, FLI1 mutations in its DNA-binding domain region results in thrombocytopenia in affected patients. We examined induced pluripotent stem cell- (iPSC) derived megakaryocytes (iMegs) to determine if the platelet disorder observed in PTSx could be replicated and if varied levels of Fli1 expression affected megakaryopoiesis and thrombopoiesis. Beginning with a normal control (WT) iPSC line, genome editing was performed to generate three lines with: 1) one copy of FLI1 disrupted (FLI1+/-), 2) hemizygous transgene expression of Fli1 in the adeno-associated virus site 1 (AAVS1) safe harbor locus using a megakaryocyte-specific GP1balpha promoter (WT-overexpressing line, WT-OE), and 3) homozygous transgene expression in AAVS1 (WT-OE2). Additionally, we established an iPSC line from a PTSx patient and edited this line for a similar hemizygous transgene expression of FLI1 (PTSx-OE). Data described here are summarized in the tables below. We confirm our genome editing strategies by examining mRNA and protein levels of iMegs and found WT-OE and WT-OE2 iMegs have ~3X higher mRNA and ~7X higher protein levels than WT iMegs. FLI1+/- and PTSx iMegs both have lower mRNA and ~0.5X the protein levels of WT iMegs, while PTSx-OE iMegs had comparable levels of mRNA and protein as WT-OE iMegs. Megacult colony assays showed WT-OE and WT-OE2 yielded more CFU-Megs than WT HPCs (p²0.01, p²0.05, respectively). PTSx and FLI1+/- had much less CFU-Megs compared to PTSx-OE and WT (p²0.0001). After growth in liquid culture, WT-OE and -OE2 lines had an increase in iMeg numbers (p=0.29, p²0.01) while FLI1+/- and PTSx iMeg numbers were 43% and 10% that of the WT control (p²0.01, p²0.001). PTSx-OE iMeg numbers were comparable to WT. Surface marker CD41 and CD42b levels were increased compared to WT iMegs in the WT-OE and -OE2 iMegs (p=0.25, p²0.05) and less on FLI1+/- and PTSx iMegs (p²0.01, p²0.05). PTSx-OE iMegs were normal compared to WT, but p²0.05 vs. PTSx. Infused CD41+CD42b+ iMegs into NSG mice showed a trend toward same yield of platelets in the OE lines and lower yield in FLI1+/- and PTSx lines compared to WT. However, when calculations were made from HPCs rather than iMegs infused, the FLI1+/- and PTSx iMegs generated significantly less number of released platelets (p²0.05, p²0.01). The half-life of WT-OE and -OE2 released platelets were increased at 4 and 10 hours compared to WT, whereas FLI1+/- and PTSx released platelets have lower half-lives of 2 hours and 0.5 hours, respectively. These decreased platelet half-lives are due to the majority of platelets being defective and cleared at a higher rate. This was corrected to WT in the PTSx-OE-released platelets. Both in vitro iMegs and in vivo released platelets within the murine blood were assessed for function. Analysis of activation by thrombin was performed via FACS for PAC-1 binding and cell surface P-selectin levels, which are both indicators of platelet activation. Initial studies show no difference between WT and the WT-OE and WT-OE2 lines, but FLI1+/- platelet activation was slightly impaired. In summary, we show that studies of iMegs with decreased Fli1 levels replicate many of the clinical features previously described: less iMegs, lower CD41 and CD42b density with less platelets released while having shorter half-lives. On the other hand, increased Fli1 levels resulted in higher number of iMegs with more surface antigen density and released platelets with increased half-lives. Based on overexpression studies of other transcription factors during megakaryopoiesis, such as GATA1, the expectation would have been that the excess of Fli1 in the OE lines would have also resulted in defects in megakaryopoiesis and thrombopoiesis, but instead improvements were seen. The basis for this difference when overexpressing different transcriptional factors needs further analysis. That high Fli1 levels enhance iMeg yield with maintained numbers and function of released platelets may be of value for generating platelets for clinical use from in vitro grown Megs. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

STAT3 selectively interacts with Smad3 to antagonize TGF-β signalling.

Smad and STAT proteins are critical signal transducers and transcription factors in controlling cell growth and tumorigenesis. Here we report that the STAT3 signaling pathway attenuates TGF-β-induced responses through a direct Smad3-STAT3 interplay. Activated STAT3 blunts TGF-β-mediated signaling. Depletion of STAT3 promotes TGF-β-mediated transcriptional and physiological responses, including cell cycle arrest, apoptosis and epithelial-to-mesenchymal transition. STAT3 directly interacts with Smad3 in vivo and in vitro, resulting in attenuation of the Smad3-Smad4 complex formation and suppression of DNA-binding ability of Smad3. The N-terminal region of DNA-binding domain of STAT3 is responsible for the STAT3-Smad3 interaction and also indispensable for STAT3-mediated inhibition of TGF-β signaling. Thus, our finding illustrates a direct crosstalk between the STAT3 and Smad3 signaling pathways that may contribute to tumor development and inflammation.

Investigating DNA Binding and Conformational Variation in Temperature Sensitive p53 Cancer Mutants Using QM-MM Simulations.

The tp53 gene is found to be mutated in 50% of all the cancers. The p53 protein, a product of tp53 gene, is a multi-domain protein. It consists of a core DNA binding domain (DBD) which is responsible for its binding and transcription of downstream target genes. The mutations in p53 protein are responsible for creating cancerous conditions and are found to be occurring at a high frequency in the DBD region of p53. Some of these mutations are also known to be temperature sensitive (ts) in nature. They are known to exhibit partial or strong binding with DNA in the temperature range (298–306 K). Whereas, at 310 K and above they show complete loss in binding. We have analyzed the changes in binding and conformational behavior at 300 K and 310 K for three of the ts-mutants viz., V143A, R249S and R175H. QM-MM simulations have been performed on the wild type and the above mentioned ts-mutants for 30 ns each. The optimal estimate of free energy of binding for a particular number of interface hydrogen bonds was calculated using the maximum likelihood method as described by Chodera et. al (2007). This parameter has been observed to be able to mimic the binding affinity of the p53 ts-mutants at 300 K and 310 K. Thus the correlation between MM-GBSA free energy of binding and hydrogen bonds formed by the interface residues between p53 and DNA has revealed the temperature dependent nature of these mutants. The role of main chain dihedrals was obtained by performing dihedral principal component analysis (PCA). This analysis, suggests that the conformational variations in the main chain dihedrals (ϕ and ψ) of the p53 ts-mutants may have caused reduction in the overall stability of the protein. The solvent exposure of the side chains of the interface residues were found to hamper the binding of the p53 to the DNA. Solvent Accessible Surface Area (SASA) also proved to be a crucial property in distinguishing the conformers obtained at 300 K and 310 K for the three ts-mutants from the wild type at 300 K.

Next Generation Sequencing (NGS) of TP53 Gene Allows to Identify a Very High Risk Group of Philadelphia Negative Acute Lymphoblastic Leukemia (ALL) Patients

Background and Aim of the StudyFor both childhood and adult Acute Lymphoblastic Leukemia (ALL) patients, clinical risk factors such as age, white cell count, response to steroids, time to complete remission, as well as biologic characteristics such as immunophenotype and cytogenetic at diagnosis are important but not sufficient in predicting clinical outcome. Aberrations of TP53 play a crucial role in the molecular pathogenesis of leukemias and lymphomas in which their presence is associated to disease progression and represents a strong predictor of poor clinical outcome. In childhood ALL, hereditary and acquired TP53 mutations are involved both in the pathogenesis and progression of the disease. In adult ALL, TP53 mutations are frequent in patients negative for recurrent fusion genes and correlate with poor response to induction therapy (Chiaretti S. et al, Haematologica 2013). The aim of this study was to evaluate the impact of TP53 alterations, analyzed by Next Generation Sequencing (NGS), on the outcome of a cohort of T (n= 57) and B (n= 114) precursor, Philadelphia (Ph) negative, adult ALL patients enrolled into the NILG-ALL 09/2000 clinical trial (ClinicalTrials.gov identifier: NCT00358072, Bassan R. et al, Blood 2009) in which molecular minimal residual disease was used to guide post-remissional therapy.Patients and Study designAmong the 171 patients who were investigated for TP53 mutations, 16 proved also positive for t(4;11) and 3 for t(1;19). We analyzed DNA isolated from mononuclear cells obtained from bone marrow or peripheral blood samples containing at least 30% of blasts at diagnosis. The TP53 gene was sequenced using 454 ultra-deep sequencing (Roche Diagnostics) for alterations in exons 4 to 11, following the protocol developed in the IRON-II consortium. The sequencing data were analyzed by the Roche Diagnostics GS Run Browser and GS Amplicon Variant Analyzer software. The probabilities of survival were estimated using the Kaplan Meier method. The log-rank test was used to compare survival probabilities between subgroups of patients.Results and DiscussionThe data obtained by NGS allowed to identify 15 coding mutations detected in the DNA binding domain region (exons 5 to 8). These alterations were observed at diagnosis in 14 patients (8%), (11 B-precursor ALL and 3 T-ALL). In 12 cases these aberrations were single nucleotide changes, in 2 cases we found a duplication (one of 4 and the other of 8 nucleotides) and in one case there was an 11 base pair DNA insertion. Remarkably, all of these DNA alterations led to missense or frame-shift mutations that introduced a premature stop codon. Moreover, they were detected with a wide range of allele burden (from 5% to 97%) pointing out that TP53 mutations can be present at diagnosis in different proportions within the leukemic clones. All patients carrying a TP53 alteration reached complete remission after induction therapy but 13 out of 14 suffered an early relapse. Frequency of relapses was significantly higher in mutated than in wild-type cases (p=0.019). Relapse DNA samples were available in 3 patients and in all of them we detected the same TP53 mutation found at diagnosis, indicating the presence of a stable mutated clone. The univariate analysis enlightens a clear relationship between TP53 mutation with an increasing age (p= 0.0003) but no correlation with other clinical features such as gender, hemoglobin, white blood count, platelets, percentage of blasts and cytogenetics at diagnosis. Moreover, patients with mutated TP53 showed a Disease Free Survival (DFS) and Overall Survival (OS) dramatically shorter than wild-type patients. The 2 years DFS was 43% in the TP53 non-mutated subjects compared to 7% in the mutated (p=0.0007). Similarly, the 2 years OS was of 50% in wild-type patients and of 7% in mutated patients (p=0.0011) (Figure 1).ConclusionsIn adult ALL, response to induction chemotherapy is not different in patients with a wild-type or a TP53 mutated gene, but in these latter cases the leukemia relapse rate is dramatically higher. The frequency of these mutations observed at diagnosis and the poor clinical outcome indicate the need of their identification during the diagnostic work up of adult ALL to guide treatment strategies. The use of a highly sensitive deep sequencing approach is crucial to identify also minor leukemic clones carrying TP53 mutations that may lead to the rapid emergence of a treatment resistant disease. [Display omitted] DisclosuresKohlmann:AstraZeneca: Employment.

The Basis for Evolution of DNA-Binding Specificity of the Aft1 Transcription Factor in Yeasts

The Saccharomyces cerevisiae Aft1 and Kluyveromyces lactis KlAft are orthologous yeast transcription activators that regulate the expression of the same group of iron-uptake genes but bind to the different DNA sites: TGCACCC for Aft1 and PuCACCC for KlAft. To establish whether the DNA-binding mechanisms of Aft1 and KlAft have diverged during the evolution of the Aft-type transcription factor, we examined the function of a nonconserved region in their DNA-binding domains. A large part of this region is composed of a sequence predicted to be disordered in structure and potentially phosphorylated. We show with deletion mutant analyses that this sequence is essential for the binding of Aft1 to its DNA site and for the iron uptake and growth of S. cerevisiae under iron-limited conditions. We constructed hybrid proteins by exchanging the nonconserved regions of Aft1 and KlAft. We show that the Aft1 region is necessary and sufficient for KlAft to bind efficiently to the Aft1 DNA site in S. cerevisiae and to complement the iron-dependent phenotype of the aft1Δaft2Δ mutant. This demonstrates that the changes in the nonconserved region of the Aft-type DNA-binding domain have led to changes in the DNA-binding specificity and have major consequences for the regulation of iron homeostasis. The combination of bioinformatic and experimental analyses indicates that the sequence TGCACCC is the most probable ancestral Aft-type element. Our findings suggest that the changes in the nonconserved region of the DNA-binding domain are responsible for the evolution of the TGCACCC sequence toward PuCACCC in the K. lactis species.

Highly sequence-specific binding is retained within the DNA-binding domain of  theSaccharomyces castelliiCdc13 telomere-binding protein

The essential protein Cdc13p binds the single-stranded telomeric 3' overhangs in Saccharomyces cerevisiae and takes part in the regulation of telomere length. The DNA-binding domain (DBD) of Cdc13p is structurally established by an oligonucleotide/oligosaccharide-binding (OB)-fold domain. The sequence homolog in Saccharomyces castellii (scasCDC13) was characterized previously, and the full-length protein was found to bind telomeric DNA specifically. Here, the DBD of scasCdc13p was defined to the central part (402-658) of the protein. The region necessary for forming the scasCdc13p-DBD is larger than the minimal DBD of S. cerevisiae Cdc13p. Deletion of this extended DBD region from the full-length protein completely abolished the DNA binding, indicating the importance of the extended region for the correct formation of a binding-competent DBD. The scasCdc13p-DBD bound the same 8-mer minimal binding site as the full-length protein, but an extension of the target site in the 3' end increased the stability of the DNA-protein complex. Significantly, scasCdc13p-DBD showed a retained high sequence specific binding, where the four nucleotides of most importance for the sequence specificity are highly conserved in eukaryotic telomeric repeats. Thus, the unique single-stranded DNA-binding properties of the full-length protein are entirely retained within the isolated scasCdc13p-DBD.

7,7′-Dihydroxy bursehernin inhibits the expression of inducible nitric oxide synthase through NF-κB DNA binding suppression

This study isolated a lignan, 7,7′-dihydroxy bursehernin, from Geranium thunbergii and investigated whether or not the lignan affects the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). The gel shift analysis and luciferase reporter gene assays using the iNOS promoter and nuclear factor-κB (NF-κB) minimal promoter showed that a treatment with 7,7′-dihydroxy bursehernin reduced the reporter activities and binding of NF-κB to the NF-κB consensus sequence, while it had no effect on the nuclear translocation of p65 and the phosphorylation/degradation of I-κBα. It was reported that a few natural compounds directly suppressed the binding activity of the NF-κB components to DNA. The NF-κB binding activity was not reversed by the in vitro exposure of the nuclear extracts to 7,7′-dihydroxy bursehernin, which suggest that a metabolite(s) of 7,7′-dihydroxy bursehernin might target the binding of the NF-κB complex to the DNA binding domain region in the promoter region of the iNOS gene. After incubation of RAW264.7 cells with 7,7-dihydroxy bursehernin for 18 h, the levels of parent compound were negligible; while a main metabolite, 4-[4-( n-hydroxy-phenyl)-2,3-dimethyl-buta-1,3-dienyl]-benzene-1,2-diol was detected in cell lysates and culture medium.

GCNF-dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility.

To determine the function of germ cell nuclear factor (GCNF) in female reproduction, we generated an oocyte-specific GCNF knockout mouse model (GCNF(fl/fl)Zp3Cre(+)). These mice displayed hypofertility due to prolonged diestrus phase of the estrous cycle and aberrant steroidogenesis. These reproductive defects were secondary to a primary defect in the oocytes, in which expression of the paracrine transforming growth factor-beta signaling molecules, bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9), were up-regulated in GCNF(fl/fl)Zp3Cre(+) females at diestrus. This was a direct effect of GCNF, as molecular studies showed that GCNF bound to DR0 elements within the BMP-15 and GDF-9 gene promoters and repressed their reporter activities. Consistent with these findings, abnormal double-oocyte follicles, indicative of aberrant BMP-15/GDF-9 expression, were observed in GCNF(fl/fl)Zp3Cre(+) females. The Cre/loxP knockout of GCNF in the oocyte has uncovered a new regulatory pathway in ovarian function. Our results show that GCNF directly regulates paracrine communication between the oocyte and somatic cells by regulating the expression of BMP-15 and GDF-9, to affect female fertility.

Peptide insertion in the DNA-binding domain of fish glucocorticoid receptor is encoded by an additional exon and confers particular functional properties

The trout glucocorticoid receptor (rtGR) contains an additional sequence of nine amino acids located between the two zinc fingers of the DNA-binding domain (DBD) (Endocrinology 136 (1995) 3774). Polymerase chain reaction on trout genomic DNA and sequencing were performed in the DBD region, demonstrating that this peptide is encoded by an additional exon of 27 nucleotides between the two exons encoding the two zinc fingers of other nuclear receptors. This additional sequence in the rtGR confers a better binding affinity of the receptor to a single GRE, as shown by gel shift experiments with GST-DBDrtGR fusion proteins, deleted or not of the nine amino acids (Δ9). This higher affinity is correlated with a higher constitutive transcriptional activity of the receptor on a reporter gene driven by a single GRE, but not with the ligand-induced transcriptional activity. Nevertheless, on a double GRE, the wild type and rtGR-Δ9 are equally active on both constitutive or dexamethasone-induced transcriptional activity. This original DBD structure could have emerged during evolution such as to allow better regulation of glucocorticoid dependent genes in relation to the large spectrum of cortisol physiological functions in fish.

Conformation-dependent phosphorylation of p53.

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.

Characterization and expression of a cDNA encoding the human androgen receptor.

We report the isolation of a cDNA that encodes the complete human androgen receptor. The cDNA predicts a protein of 917 amino acids with a molecular weight of 98,918. Introduction of the cDNA into heterologous mammalian cells caused expression of high levels of a protein that binds dihydrotestosterone with the affinity, specificity, and sedimentation properties characteristic of the native human androgen receptor. Comparisons with the amino acid sequence of previously cloned steroid hormone receptors revealed a high degree of sequence conservation with the progesterone, glucocorticoid, and mineralocorticoid receptors in the putative hormone and DNA-binding domain regions.