88 research outputs found
The CDK9 C-helix Exhibits Conformational Plasticity That May Explain the Selectivity of CAN508
Correct regulation of transcription is essential for maintaining a healthy cellular state. During transcription RNA polymerase II (Pol II) proceeds in a regulated manner through several transitions to ensure appropriate control of synthesis and enable correct processing of the pre-RNA. Shortly after initiation Pol II is caused to pause by the binding of factors, DSIF and NELF. To enable transition of Pol II into the elongation phase CDK9/cyclin T phosphorylates the C-terminal domain (CTD) of Pol II, DSIF and NELF. This phosphorylation releases the paused state and provides an alternative set of post-transcriptional modifications on the CTD to generate a binding platform for elongation, histone modifying and termination factors. CDK9/cyclin T is itself regulated within multicomponent complexes. A small activated complex, containing Brd4, recruits CDK9/cyclin T to active sites of transcription, thereby promoting the elongation of transcription. The role of CDK9/cyclin T in the regulation of transcription has resulted in its validation as a drug target against several disease states including cancer, HIV and cardiac hypertrophy.In this thesis, I present the crystallographic structures of a series of 2-amino-4-heteroaryl-pyrimidine compounds and the roscovitine derivative, (S)-CR8, bound to CDK9/cyclin T and CDK2/cyclin A. In combination with thermal denaturation data and kinetic analysis, these structures have suggested chemical modifications that might be made to increase the CDK9 specificity of these compounds. I have also validated the use of a mutated form of cyclin T for use in the development of CDK9/cyclin T inhibitors.In addition, I present both structural and kinetic analysis of the Brd4-CDK9/cyclin T interaction. I show that C-terminal fragments of Brd4 enhance the in vitro kinase activity of CDK9/cyclin T against the Pol II CTD. Furthermore, I demonstrate that this enhancement may be inhibited by Plk1-mediated phosphorylation of Brd4. Finally, I show that Brd4 binds to a site that spans CDK9 and cyclin T and I propose detailed molecular models of the Brd4-cyclin T interaction.This thesis is not currently available via ORA
Experimental Investigation of the Surface Roughness for Aluminum Alloy AA6061 in Milling Operation by Taguchi Method with the ANOVA Technique
The surface roughness of the machined parts is the most important parameter to predict the performance of mechanical components. Moreover, predicting the optimal machining parameters conditions is the preferable method for cost reduction and achieving the desired surface quality of the product. This study investigates three cutting parameters, such as depth of cut, spindle speed, and feed for the milling aluminium alloy AA6061, to predict the surface roughness quality. The experimental work utilized a manual milling machine with a coated carbide cutter. Furthermore, the experiments were arranged using the Taguchi L9 orthogonal array (OA) method. The average surface roughness (Ra) was measured and converted to signal-to-noise (S/N) ratio and then analyzed in the statistical method of analysis of variance (ANOVA). Finally, the optimal combination set speed, feed, and depth of cut was 2400 rpm, 30 mm/min, and 0.5 mm, respectively. Also, according to the ANOVA test, the most influential parameter was the spindle speed among the selected parameters, with the highest P value of (66.42%). In comparison, the lowest P value is a depth of cut (5.34%). Furthermore, spindle speed was the only significant factor statistically. By selecting a high spindle speed (2400 rpm), surface quality was enhanced, but the preferable level was low for depth of cut and feed.
Removal of Surface Oxygen Vacancies Increases Conductance Through TiO2 Thin Films for Perovskite Solar Cells
Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1
The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology
Caffeine Prevents Transcription Inhibition and P-TEFb/7SK Dissociation Following UV-Induced DNA Damage
Background: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. Methodology/Principal Findings: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. Conclusion/Significance: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibitio
Protein Phosphatase-1 Activates CDK9 by Dephosphorylating Ser175
The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription
Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon
Supplementary Data are available at NAR OnlineThe human immunodeficiency virus type 1 (HIV-1)
regulator Tat is essential for viral replication
because it achieves complete elongation of viral
transcripts. Tat can be released to the extracellular
space and taken up by adjacent cells, exerting
profound cytoskeleton rearrangements that lead to
apoptosis. In contrast, intracellular Tat has been
described as protector from apoptosis. Tat gene is
composed by two coding exons that yield a protein
of 101 amino acids (aa). First exon (1–72aa) is sufficient
for viral transcript elongation and second
exon (73–101 aa) appears to contribute to
non-transcriptional functions. We observed that
Jurkat cells stably expressing intracellular Tat101
showed gene expression deregulation 4-fold
higher than cells expressing Tat72. Functional
experiments were performed to evaluate the effect
of this deregulation. First, NF-iB-, NF-AT- and
Sp1-dependent transcriptional activities were
greatly enhanced in Jurkat-Tat101, whereas Tat72
induced milder but efficient activation. Second,
cytoskeleton-related functions as cell morphology,
proliferation, chemotaxis, polarization and actin
polymerization were deeply altered in Jurkat-
Tat101, but not in Jurkat-Tat72. Finally, expression
of several cell surface receptors was dramatically
impaired by intracellular Tat101 but not by Tat72.
Consequently, these modifications were greatly
dependent on Tat second exon and they could be
related to the anergy observed in HIV-1-infected
T cells.Plan Nacional del SIDA (MVI 1434/05–5), FIPSE 36584/
06 and 36633/07, VIRHORST Network from Comunidad
de Madrid (Spain), FIS PI040614 and PI0808752,
ISCIII-RETIC RD06/0006, EUROPRISE Network of
Excellence of the EU (Grant no. LSHP CT-2006-
037611), and BIO2008-04384 from the Ministerio de
Ciencia e Innovacio´ n, Espan˜ a. Funding for open access
charge: Instituto de Salud Carlos III, Ministry of Science
and Technology, Spain.Peer reviewe
Malleable Machines in Transcription Regulation: The Mediator Complex
The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein–protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function
A conserved mediator hinge revealed in the structure of the MED7·MED21 (Med7·Srb7) heterodimer.
The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. We present the 3.0-Å crystal structure of a highly conserved part of the Mediator, the MED7·MED21 (Med7·Srb7) heterodimer. The structure is very extended, spanning one-third of the Mediator length and almost the diameter of Pol II. It shows a four-helix bundle domain and a coiled-coil protrusion connected by a flexible hinge. Four putative protein binding sites on the surface allow for assembly of the Mediator middle module and for binding of the conserved subunit MED6, which is shown to bridge to the Mediator head module. A flexible MED6 bridge and the MED7·MED21 hinge could account for changes in overall Mediator structure upon binding to Pol II or activators. Our results support the idea that transcription regulation involves conformational changes within the general machinery
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