RESUMO
For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag-Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag-Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.
Assuntos
Fármacos Anti-HIV/farmacologia , Benzamidas/farmacologia , HIV/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/efeitos dos fármacos , Acetilação , Sequência de Aminoácidos , Linhagem Celular , Cisteína/química , Proteínas de Fusão gag-pol/química , Proteínas de Fusão gag-pol/efeitos dos fármacos , Humanos , Lisina/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/químicaRESUMO
Replication foci are generated by many viruses to concentrate and localize viral DNA synthesis to specific regions of the cell. Expression of the HPV16 E1 and E2 replication proteins in keratinocytes results in nuclear foci that recruit proteins associated with the host DNA damage response. We show that the Brd4 protein localizes to these foci and is essential for their formation. However, when E1 and E2 begin amplifying viral DNA, Brd4 is displaced from the foci and cellular factors associated with DNA synthesis and homologous recombination are recruited. Differentiated HPV-infected keratinocytes form similar nuclear foci that contain amplifying viral DNA. We compare the different foci and show that, while they have many characteristics in common, there is a switch between early Brd4-dependent foci and mature Brd4-independent replication foci. However, HPV genomes encoding mutated E2 proteins that are unable to bind Brd4 can replicate and amplify the viral genome. We propose that, while E1, E2 and Brd4 might bind host chromatin at early stages of infection, there is a temporal and functional switch at later stages and increased E1 and E2 levels promote viral DNA amplification, displacement of Brd4 and growth of a replication factory. The concomitant DNA damage response recruits proteins required for DNA synthesis and repair, which could then be utilized for viral DNA replication. Hence, while Brd4 can enhance replication by concentrating viral processes in specific regions of the host nucleus, this interaction is not absolutely essential for HPV replication.
Assuntos
Replicação do DNA/fisiologia , DNA Viral/biossíntese , Genoma Viral/fisiologia , Papillomavirus Humano 16/fisiologia , Proteínas Nucleares/metabolismo , Infecções por Papillomavirus/metabolismo , Fatores de Transcrição/metabolismo , Replicação Viral/fisiologia , Proteínas de Ciclo Celular , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromatina/virologia , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Fatores de Transcrição/genéticaRESUMO
Ligand binding to the glucocorticoid receptor (GR) results in receptor binding to glucocorticoid response elements (GREs) and the formation of transcriptional regulatory complexes. Equally important, these complexes are continuously disassembled, with active processes driving GR off GREs. We found that co-chaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD). Next, we examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Overall, these studies showed that activities impinging on the LBD regulate GR exchange with GREs but that the dissociation of GR from GREs is independent from ligand dissociation.
Assuntos
Chaperonas Moleculares/metabolismo , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Clonagem Molecular , Recuperação de Fluorescência Após Fotodegradação , Humanos , Ligantes , Camundongos , Chaperonas Moleculares/genética , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de Glucocorticoides/genética , Elementos de RespostaRESUMO
Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Óxido Nítrico/química , Oxidiazóis/química , Oxidiazóis/farmacologia , Esquistossomose/tratamento farmacológico , Esquistossomicidas/química , Esquistossomicidas/farmacologia , Animais , Disponibilidade Biológica , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/química , Oxidiazóis/farmacocinética , Oxidiazóis/uso terapêutico , Conformação Proteica , Ratos , Schistosoma/efeitos dos fármacos , Esquistossomose/enzimologia , Esquistossomicidas/farmacocinética , Esquistossomicidas/uso terapêutico , Solubilidade , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
To investigate the determinants of promoter-specific gene regulation by the glucocorticoid receptor (GR), we compared the composition and function of regulatory complexes at two NFkappaB-responsive genes that are differentially regulated by GR. Transcription of the IL-8 and IkappaBalpha genes is stimulated by TNFalpha in A549 cells, but GR selectively represses IL-8 mRNA synthesis by inhibiting Ser2 phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). The proximal kappaB elements at these genes differ in sequence by a single base pair, and both recruited RelA and p50. Surprisingly, GR was recruited to both of these elements, despite the fact that GR failed to repress the IkappaBalpha promoter. Rather, the regulatory complexes formed at IL-8 and IkappaBalpha were distinguished by differential recruitment of the Ser2 CTD kinase, P-TEFb. Disruption of P-TEFb function by the Cdk-inhibitor, DRB, or by small interfering RNA selectively blocked TNFalpha stimulation of IL-8 mRNA production. GR competed with P-TEFb recruitment to the IL-8 promoter. Strikingly, IL-8 mRNA synthesis was repressed by GR at a post-initiation step, demonstrating that promoter proximal regulatory sequences assemble complexes that impact early and late stages of mRNA synthesis. Thus, GR accomplishes selective repression by targeting promoter-specific components of NFkappaB regulatory complexes.
Assuntos
Regulação da Expressão Gênica , Interleucina-8/genética , NF-kappa B/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Glucocorticoides/metabolismo , Imunoprecipitação da Cromatina , Diclororribofuranosilbenzimidazol/farmacologia , Glutationa Transferase/metabolismo , Humanos , Proteínas I-kappa B/genética , Modelos Biológicos , Inibidor de NF-kappaB alfa , Oligonucleotídeos , Fosforilação , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Members of the mammalian p160 family, such as GRIP1, are known as glucocorticoid receptor (GR) coactivators; at certain glucocorticoid response elements (GREs), however, GRIP1 acts as a GR corepressor. We characterized functional interactions of GR and GRIP1 in a repression complex where GR tethers to DNA-bound activator protein-1 (AP-1), as at the human collagenase-3 gene, and tested whether the identified interactions were similar or different at other response elements. At the AP-1 tethering GRE, we mapped the GRIP1 corepressor activity to a domain distinct from the two known GRIP1 activation domains; it exhibited intrinsic GR-independent repression potential when recruited to DNA via Gal4 DNA-binding domain. Interestingly, neither the domain nor the activity was detected in the other two p160 family members, SRC1 and RAC3. The same GRIP1 corepression domain was required for GR-mediated repression at the nuclear factor-kappaB (NF-kappaB) tethering GRE of the human IL-8 gene. In contrast, at the osteocalcin gene GRE, where GR represses transcription by binding to a DNA site overlapping the TATA box, both GRIP1 and SRC1 corepressed, and the GRIP1-specific repression domain was dispensable. Thus, in a single cell type, GR and GRIP1 conferred one mode of activation and two modes of repression by selectively engaging distinct surfaces of GRIP1 in a response element-specific manner.