RESUMO
HIV-1 transcription is specifically augmented by a transcriptional activator complex composed of Tat, an HIV-1-encoded activator, and the host transcription elongation factor P-TEFb, which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1. Several observations suggest that P-TEFb is an attractive anti-HIV-1 drug target. However, the long-term cytotoxicity of CDK9 inhibitors hinders their widespread use in HIV-1 therapy. Thus, novel and safe inhibitors are sorely needed. By performing molecular dynamics simulations of the 3D structure of Tat/P-TEFb, we previously identified a unique cavity structure of CDK9, the CDK9 hidden cavity, that is specifically induced by Tat binding. Here, we attempted to identify compounds that fit this cavity and inhibit CDK9 activity by in silico screening. We identified compounds that could inhibit CDK9 activity. One of such compound, 127, showed the strongest inhibitory activity against CDK9. Interestingly, it also inhibited CDK6 to a similar extent. We inspected the amino acid sequence and structural properties of the CDK9 hidden cavity to determine whether it is conserved in other CDKs, such as CDK6. The Ile61, comprising the center of the CDK9 hidden cavity, appears to be crucial for its kinase activity, thus indicating that the identification of the CDK9 hidden cavity may provide vital information for the development of novel CDK9 inhibitors.
Assuntos
Quinase 9 Dependente de Ciclina , HIV-1 , Quinase 9 Dependente de Ciclina/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Ciclina T/química , HIV-1/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina , Transcrição GênicaRESUMO
DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA polymerase II (Pol II) is not fully understood. Central to the DNA damage response, poly(ADP-ribose) polymerase 1 (PARP1) initiates DNA repair by translocating to the lesions where it catalyses protein poly(ADP-ribosylation). Here we report that PARP1 inhibits Pol II elongation by inactivating the transcription elongation factor P-TEFb, a CDK9-cyclin T1 (CycT1) heterodimer. After sensing damage, the activated PARP1 binds to transcriptionally engaged P-TEFb and modifies CycT1 at multiple positions, including histidine residues that are rarely used as an acceptor site. This prevents CycT1 from undergoing liquid-liquid phase separation that is required for CDK9 to hyperphosphorylate Pol II and to stimulate elongation. Functionally, poly(ADP-ribosylation) of CycT1 promotes DNA repair and cell survival. Thus, the P-TEFb-PARP1 signalling plays a protective role in transcription quality control and genomic stability maintenance after DNA damage.
Assuntos
Dano ao DNA , Fator B de Elongação Transcricional Positiva , ADP-Ribosilação , Ciclina T/química , Ciclina T/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
P-TEFb modulates RNA polymerase II elongation through alternative interaction with negative and positive regulation factors. While inactive P-TEFbs are mainly sequestered in the 7SK snRNP complex in a chromatin-free state, most of its active forms are in complex with its recruitment factors, Brd4 and SEC, in a chromatin-associated state. Thus, switching from inactive 7SK snRNP to active P-TEFb (Brd4/P-TEFb or SEC/P-TEFb) is essential for global gene expression. Although it has been shown that cellular signaling stimulates the disruption of 7SK snRNP, releasing dephosphorylated and catalytically inactive P-TEFb, little is known about how the inactive released P-TEFb is reactivated. Here, we show that the Cdk9/CycT1 heterodimer released from 7SK snRNP is completely dissociated into monomers in response to stress. Brd4 or SEC then recruits monomerized Cdk9 and CycT1 to reassemble the core P-TEFb. Meanwhile, the binding of monomeric dephosphorylated Cdk9 to either Brd4 or SEC induces the autophosphorylation of T186 of Cdk9. Finally, the same mechanism is employed during nocodazole released entry into early G1 phase of cell cycle. Therefore, our studies demonstrate a novel mechanism by which Cdk9 and CycT1 monomers are reassembled on chromatin to form active P-TEFb by its interaction with Brd4 or SEC to regulate transcription.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Ciclo Celular , Linhagem Celular , Ciclina T/química , Quinase 9 Dependente de Ciclina/química , Ativação Enzimática , Humanos , Modelos Biológicos , Fosforilação , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes , Ribonucleoproteínas Nucleares Pequenas/química , Estresse FisiológicoRESUMO
Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing1-3. The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation1,4,5. The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase-DYRK1A 6 -also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH1-3. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.
Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Ciclina T/química , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fosforilação , Fator B de Elongação Transcricional Positiva/metabolismo , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/metabolismo , Ativação Transcricional , Quinases DyrkRESUMO
BACKGROUND: HIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. Here we analyzed Tat phosphorylation in cultured cells and its functionality. RESULTS: Mass spectrometry analysis showed primarily Tat Ser-16 phosphorylation in cultured cells. In vitro, CDK2/cyclin E predominantly phosphorylated Tat Ser-16 and PKR-Tat Ser-46. Alanine mutations of either Ser-16 or Ser-46 decreased overall Tat phosphorylation. Phosphorylation of Tat Ser-16 was reduced in cultured cells treated by a small molecule inhibitor of CDK2 and, to a lesser extent, an inhibitor of DNA-PK. Conditional knock-downs of CDK2 and PKR inhibited and induced one round HIV-1 replication respectively. HIV-1 proviral transcription was inhibited by Tat alanine mutants and partially restored by S16E mutation. Pseudotyped HIV-1 with Tat S16E mutation replicated well, and HIV-1 Tat S46E-poorly, but no live viruses were obtained with Tat S16A or Tat S46A mutations. TAR RNA binding was affected by Tat Ser-16 alanine mutation. Binding to cyclin T1 showed decreased binding of all Ser-16 and Ser-46 Tat mutants with S16D and Tat S46D mutationts showing the strongest effect. Molecular modelling and molecular dynamic analysis revealed significant structural changes in Tat/CDK9/cyclin T1 complex with phosphorylated Ser-16 residue, but not with phosphorylated Ser-46 residue. CONCLUSION: Phosphorylation of Tat Ser-16 induces HIV-1 transcription, facilitates binding to TAR RNA and rearranges CDK9/cyclin T1/Tat complex. Thus, phosphorylation of Tat Ser-16 regulates HIV-1 transcription and may serve as target for HIV-1 therapeutics.
Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Serina/metabolismo , Transcrição Gênica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Ciclina T/química , Ciclina T/genética , Ciclina T/metabolismo , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/metabolismo , Técnicas de Silenciamento de Genes , Infecções por HIV/genética , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , RNA Viral , Ubiquitinação , Replicação Viral , eIF-2 Quinase/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.
Assuntos
Ciclina T/química , Quinase 9 Dependente de Ciclina/química , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Sítios de Ligação , Catálise , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM.
Assuntos
Ciclina T/química , Quinase 9 Dependente de Ciclina/química , DNA Viral/química , Repetição Terminal Longa de HIV , Proteínas Repressoras/química , Fatores de Elongação da Transcrição/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , DNA Viral/metabolismo , Medição da Troca de Deutério , HIV-1/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Provírus/genética , Proteínas Repressoras/metabolismo , Espalhamento a Baixo Ângulo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg2+ ion at site 1 (Mg12+). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg12+ ion, coupled with the Mg22+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg12+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg12+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg12+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.
Assuntos
Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , HIV-1/metabolismo , Magnésio/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Ciclina T/química , Quinase 9 Dependente de Ciclina/química , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2' helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1' and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound.
Assuntos
Ciclina T/química , Simulação de Dinâmica Molecular , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Ciclina T/metabolismo , HIV-1/química , Dados de Sequência Molecular , Ligação Proteica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. RESULTS: Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. CONCLUSIONS: Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription.
Assuntos
Ciclina T/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Ciclina T/química , Humanos , Ligação de Hidrogênio , Mutação Puntual , Dobramento de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. RESULTS: To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. CONCLUSIONS: This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription.
Assuntos
Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , HIV/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Células 3T3 , Animais , Ciclina T/química , Células HEK293 , Humanos , Camundongos , Mutação Puntual , Relação Estrutura-Atividade , Linfócitos T/virologia , Fatores de Transcrição , Fatores de Elongação da Transcrição , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
Developing anti-viral therapies targeting HIV-1 transcription has been hampered by the limited structural knowledge of the proteins involved. HIV-1 hijacks the cellular machinery that controls RNA polymerase II elongation through an interaction of HIV-1 Tat with the positive transcription elongation factor P-TEFb, which interacts with an AF4 family member (AFF1/2/3/4) in the super elongation complex (SEC). Because inclusion of Tatâ¢P-TEFb into the SEC is critical for HIV transcription, we have determined the crystal structure of the Tatâ¢AFF4â¢P-TEFb complex containing HIV-1 Tat (residues 1-48), human Cyclin T1 (1-266), human Cdk9 (7-332), and human AFF4 (27-69). Tat binding to AFF4â¢P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5' of Cyclin T1 and the α-3 10 helix of AFF4. The interaction between Tat and AFF4 provides structural constraints that explain tolerated Tat mutations. Analysis of the Tat-binding surface of AFF4 coupled with modeling of all other AF4 family members suggests that AFF1 and AFF4 would be preferred over AFF2 or AFF3 for interaction with Tatâ¢P-TEFb. The structure establishes that the Tat-TAR recognition motif (TRM) in Cyclin T1 interacts with both Tat and AFF4, leading to the exposure of arginine side chains for binding to TAR RNA. Furthermore, modeling of Tat Lys28 acetylation suggests that the acetyl group would be in a favorable position for H-bond formation with Asn257 of TRM, thereby stabilizing the TRM in Cyclin T1, and provides a structural basis for the modulation of TAR RNA binding by acetylation of Tat Lys28.
Assuntos
HIV-1/química , Modelos Moleculares , Complexos Multiproteicos/química , Fator B de Elongação Transcricional Positiva/química , Proteínas Repressoras/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Acetilação , Cristalização , Ciclina T/química , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Conformação Proteica , Proteínas Repressoras/metabolismo , Fatores de Elongação da Transcrição , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The positive transcription elongation factor b (P-TEFb) stimulates RNA polymerase elongation by inducing the transition of promoter proximally paused polymerase II into a productively elongating state. P-TEFb itself is regulated by reversible association with various transcription factors/cofactors to form several multisubunit complexes [e.g., the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)-P-TEFb complex] that constitute a P-TEFb network controlling cellular and HIV transcription. These complexes have been thought to share no components other than the core P-TEFb subunits cyclin-dependent kinase 9 (CDK9) and cyclin T (CycT, T1, T2a, and T2b). Here we show that the AF4/FMR2 family member 1 (AFF1) is bound to CDK9-CycT and is present in all major P-TEFb complexes and that the tripartite CDK9-CycT-AFF1 complex is transferred as a single unit within the P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 facilitates Tat's extraction of P-TEFb from 7SK snRNP and the formation of Tat-SECs for HIV transcription. Our data identify AFF1 as a ubiquitous P-TEFb partner and demonstrate that full Tat transactivation requires the complete SEC.
Assuntos
Ciclina T/química , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Fator B de Elongação Transcricional Positiva/química , Ribonucleoproteínas Nucleares Pequenas/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Alanina/genética , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Quinase 9 Dependente de Ciclina/química , Células HeLa , Humanos , Proteínas Nucleares/química , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Ativação Transcricional , Fatores de Elongação da TranscriçãoRESUMO
P-TEFb complex, a heterodimer of the kinase CDK9 and Cyclin T, is a critical factor that stimulates the process of transcription elongation. Here, we explored a fast and large-scale screening method to induce a temperature-dependent conditional disruption of the CDK9/Cyclin T interaction and developed an assay to validate their mutant phenotypes in a biological context. First, we used the yeast two-hybrid system to screen Drosophila melanogaster Cyclin T mutants at a large scale for temperature or cold sensitive (TS or CS) CDK9 interaction phenotypes. The isolated P-TEFb TS mutants were then expressed in Drosophila cells and were investigated for their effects on Drosophila hsp70 transcriptional activity. Our results showed that these P-TEFb TS mutants had a reduced level of hsp70 transcription at restrictive temperatures. A model structure of the Cyclin T and CDK9 complex suggested that the key TS mutations were found within the α2- and α3-helices at the interface of the complex, which may disrupt the binding of Cyclin T to CDK9 directly or indirectly by affecting the conformation of Cyclin T. The yeast two-hybrid-based screening strategy described here for isolating TS or CS interaction phenotypes can be directly applicable to other complexes in higher organisms. The use of TS or CS mutants will enable a 'real-time and reversible perturbation' restricted to specific protein-protein interactions, providing a mechanistic insight into the biological process mediated by a target complex.
Assuntos
Proteínas de Drosophila/genética , Mutação , Fator B de Elongação Transcricional Positiva/genética , Técnicas do Sistema de Duplo-Híbrido , Sequência de Aminoácidos , Animais , Ciclina T/química , Ciclina T/genética , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Modelos Moleculares , Dados de Sequência Molecular , Fator B de Elongação Transcricional Positiva/metabolismo , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Análise de Sequência de DNA , Temperatura , Transcrição GênicaRESUMO
Cyclin dependent kinase 9 (CDK9) is a protein that belongs to the cyclin-dependent kinases family, and its main role is in the regulation of the cell transcription processes. Since the increased activity of CDK9 is connected with the development of pathological processes such as tumor growth and survival and HIV-1 replication, inhibition of the CDK9 could be of particular interest for treating such diseases. The activation of CDK9 is initiated by the formation of CDK9/cyclin T1 complex, therefore disruption of its formation could be a promising strategy for the design of CDK9 inhibitors. In order to assist in the design of potential inhibitors of CDK9/cyclin T1 complex formation, a computational study of the CDK9/cyclin T1 interface was conducted. Ten peptides were designed using the information from the analysis of the complex, hot spot residues and fragment based design. The designed peptides were docked to CDK9 structures obtained by molecular dynamics simulations of CDK9/cyclin T1 complex and the CDK9 alone and their binding affinities were evaluated using molecular mechanics Poisson Boltzman surface area (MM-PBSA) method and steered molecular dynamics (SMD). Designed peptide sequences LQTLGF and ESIILQ, both derived from the surface of cyclin T1, as well as the peptide sequence PRWPE, derived from fragment based design, showed the most favorable binding properties and were selected for our further studies.
Assuntos
Ciclina T/química , Quinase 9 Dependente de Ciclina/química , Simulação de Acoplamento Molecular , Oligopeptídeos/química , Inibidores de Proteínas Quinases/química , Sítios de Ligação , Ciclina T/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Desenho de Fármacos , Humanos , Cinética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , TermodinâmicaRESUMO
Human immunodeficiency virus type 1 (HIV-1) transcription is essential for viral replication and the only step for viral genome amplification. Cyclin T1 (CycT1) interacts with HIV-1 Tat and transactivation-responsive (TAR) RNA, leading to the activation of viral transcription through the hyperphosphorylation of RNA polymerase II (RNAPII). Thus, the CycT1/Tat/TAR RNA interaction represents a novel target for inhibition of HIV-1 replication. In this study, we conducted in silico screening of compounds targeting the CycT1/Tat/TAR RNA complex and found that two structurally related compounds (C1 and C2) had high docking scores for a model of the complex. These compounds proved inhibitory to HIV-1 replication in tumor necrosis factor alpha-stimulated chronically infected cells. In addition, C3, a derivative of C1 and C2, was found to be a more potent inhibitor of HIV-1 replication in chronically infected cells. C3 also inhibited HIV-1 replication in acutely infected cells. The compound could suppress Tat-mediated HIV-1 long terminal repeat-driven gene expression and phosphorylation of RNAPII through inhibition of Tat binding to CycT1. Furthermore, the docking pose of C3 was defined by analyses for its in silico docking energy and in vitro antiviral activity, which indicates that C3 interacts with Tat-binding amino acids of CycT1. Thus, a series of compounds described herein are novel inhibitors of HIV-1 transcription through inhibition of CycT1/Tat interaction.
Assuntos
Fármacos Anti-HIV/farmacologia , Ciclina T/antagonistas & inibidores , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/efeitos dos fármacos , RNA Polimerase II/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/química , Sítios de Ligação , Linhagem Celular Tumoral , Ciclina T/química , Ciclina T/genética , Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Simulação de Acoplamento Molecular , Fosforilação , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Viral/metabolismo , Bibliotecas de Moléculas Pequenas/química , Termodinâmica , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Replicação Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
CDK9, the kinase of positive transcription elongation factor b (P-TEFb), stimulates transcription elongation by phosphorylating RNA polymerase II and transcription elongation factors. Using kinetic analysis of a human P-TEFb complex consisting of CDK9 and cyclin T, we show that the CDK9 C-terminal tail sequence is important for the catalytic mechanism and imposes an ordered binding of substrates and release of products. Crystallographic analysis of a CDK9/cyclin T complex in which the C-terminal tail partially blocks the ATP binding site reveals a possible reaction intermediate. Biochemical characterization of CDK9 mutants supports a model in which the CDK9 tail cycles through different conformational states. We propose that this mechanism is critical for the pattern of CTD Ser2 phosphorylation on actively transcribed genes.
Assuntos
Ciclina T/química , Quinase 9 Dependente de Ciclina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Diclororribofuranosilbenzimidazol/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fator B de Elongação Transcricional Positiva/química , Ligação Proteica , Inibidores de Proteínas Quinases/químicaRESUMO
HIV transcription is regulated at the step of elongation by the viral Tat protein and the cellular positive transcription elongation factor b (P-TEFb; Cdk9/cyclin T1). Herein, a human cyclin T1 mutant, cyclin T1-U7, which contains four substitutions and one deletion in the N-terminal cyclin box, was stably expressed in HeLa cells. HIV transcription was efficiently inhibited in HeLa-HA-CycT1-U7 stable cells. Cyclin T1-U7 bound Tat but did not modulate its expression levels, which remained high. Importantly cyclin T1-U7 failed to interact with Cdk9 or HEXIM1 and did not interfere with endogenous P-TEFb activity to stimulate MEF2C or NFkB mediated transcription. In a T cell line and primary CD4+ cells, cyclin T1-U7 also inhibited HIV transcription. We conclude that cyclin T1-U7 sequesters Tat from P-TEFb and inhibits HIV transcription. Importantly, N-terminal residues in cyclin T1 are specifically involved in the binding of cyclin T1 to HEXIM1 but not to Tat.
Assuntos
Ciclina T/genética , Ciclina T/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Ciclina T/química , Regulação Viral da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Células HeLa , Humanos , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/genética , Fatores de Transcrição , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
CDK9 is the kinase of positive transcription elongation factor b and facilitates the transition of paused RNA polymerase II to processive transcription elongation. CDK9 is a validated target for the treatment of cancer, cardiac hypertrophy, and human immunodeficiency virus. Here we analyze different CDK9/cyclin T variants to identify a form of the complex amenable to use in inhibitor design. To demonstrate the utility of this system, we have determined the crystal structures of CDK9/cyclin T and CDK2/cyclin A bound to the CDK9-specific inhibitor CAN508. Comparison of the structures reveals CDK9-specific conformational changes and identifies a CDK9-specific hydrophobic pocket, adjacent to the αC-helix. By comparison with a previously published structure of CDK9/cyclin T/human immunodeficiency virus TAT we find that the CDK9 αC-helix has a degree of conformational variability that has the potential to be exploited for inhibitor design.
Assuntos
Compostos Azo/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/química , Pirazóis/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Ciclina A/química , Ciclina A/metabolismo , Ciclina T/química , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Humanos , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
Transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, positive transcription elongation factor b, with the viral Tat protein at the transactivation response (TAR) element of nascent viral transcripts. The involvement of the interaction between Tat and CycT1 is known to be through the Tat-TAR recognition motif (TRM) on CycT1. Here, we have further characterized this molecular interaction and clarified the role of the CycT1 N-terminal region in Tat action. We found crucial and distinctive roles of Q46, Q50 and F176 of human CycT1 protein in Tat-mediated transcription by creating various Ala substitution mutants of CycT1 based on its three-dimensional structure. We confirmed the involvement of these amino acid residues in binding to Tat with Q46 and Q50, and to a lesser extent with F176, by in vitro pull-down assay. Relative transactivation activities of wild-type CycT1 chimeras and mutant derivatives on the HIV-1 long terminal repeat were determined by luciferase reporter assays. Whereas CycT1 Q46A alone had impaired transcriptional activity, the CycT1(Q46A)-Tat chimeric protein retained almost full activity of the wild-type CycT1. However, CycT1 mutants (C261Y, Q50A or F176A) or their chimeric counterparts had lost the transactivation capacity. Moreover, a triple-mutant chimera containing Q46A, Q50A and F176A mutations completely abolished the transcriptional activity, indicating that these amino acid residues are involved through distinct mechanisms. These findings provide new insights for the development of anti-HIV drugs.