RESUMO
Deubiquitinases have emerged as promising drug targets for cancer therapy. The two DUBs USP25 and USP28 share high similarity but vary in their cellular functions. USP28 is known for its tumor-promoting role, whereas USP25 is a regulator of the innate immune system and, recently, a role in tumorigenesis was proposed. We solved the structures of the catalytic domains of both proteins and established substantial differences in their activities. While USP28 is a constitutively active dimer, USP25 presents an auto-inhibited tetramer. Our data indicate that the activation of USP25 is not achieved through substrate or ubiquitin binding. USP25 cancer-associated mutations lead to activation in vitro and in vivo, thereby providing a functional link between auto-inhibition and the cancer-promoting role of the enzyme. Our work led to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.
Assuntos
Carcinogênese/genética , Neoplasias/genética , Ubiquitina Tiolesterase/genética , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Humanos , Mutação/genética , Neoplasias/tratamento farmacológico , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , Ubiquitina/genética , Ubiquitina Tiolesterase/químicaRESUMO
The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets.
Assuntos
Ubiquitina Tiolesterase , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Humanos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Sítios de Ligação , Piridinas/química , Piridinas/farmacologia , Ligação Proteica , Modelos MolecularesRESUMO
G-quadruplex (G4s) DNA structures have been implicated in inducing genomic instability and contributing to cancer development. However, the relationship between G4s and cancer-related single nucleotide variants (cSNVs) in clinical settings remains unclear. In this large-scale study, we integrated experimentally validated G4s with genomic cSNVs from 13480 cancer patients to investigate the spatial association of G4s with the cellular cSNV landscape. Our findings demonstrate an increase in local genomic instability with increasing local G4 content in cancer patients, suggesting a potential role for G4s in driving cSNVs. Notably, we observed distinct spatial patterns of cSNVs and common single nucleotide variants (dbSNVs) in relation to G4s, implying different mechanisms for their generation and accumulation. We further demonstrate large, cancer-specific differences in the relationship of G4s and cSNVs, which could have important implications for a new class of G4-stabilizing cancer therapeutics. Moreover, we show that high G4-content can serve as a prognostic marker for local cSNV density and patient survival rates. Our findings underscore the importance of considering G4s in cancer research and highlight the need for further investigation into the underlying molecular mechanisms of G4-mediated genomic instability, especially in the context of cancer.
Assuntos
Quadruplex G , Instabilidade Genômica , Neoplasias , Polimorfismo Genético , Humanos , DNA/química , Instabilidade Genômica/genética , Neoplasias/genéticaRESUMO
Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical, and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2'-deoxyuridine (PhedU). The crosslinking ability of PhedU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2 + 2] cycloaddition within the DNA duplex, resulting in efficient formation of a PhedU dimer after short irradiation times of a few seconds. A PhedU-dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology.
Assuntos
DNA , Nucleosídeos , Conformação de Ácido Nucleico , Pareamento de Bases , DNA/química , Metais , Reagentes de Ligações Cruzadas/químicaRESUMO
Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.
Assuntos
Ciclina H , Quinases Ciclina-Dependentes , Ciclo Celular , Chaetomium/química , Chaetomium/enzimologia , Ciclina H/química , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fosforilação , Transcrição Gênica , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Poli A/metabolismo , Domínios Proteicos/fisiologia , RNA Bacteriano/metabolismo , Proteínas Repressoras/metabolismo , Salmonella enterica/metabolismoRESUMO
Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five 'structural' subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD's DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD's affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62's high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a RNA/ultraestrutura , Fator de Transcrição TFIIH/ultraestrutura , Sítios de Ligação/efeitos da radiação , Microscopia Crioeletrônica , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/efeitos da radiação , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas de Ligação a RNA/genética , Imagem Individual de Molécula , Fator de Transcrição TFIIH/genética , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/ultraestruturaRESUMO
The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB's ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB's ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.
Assuntos
Adenosina Trifosfatases/química , Chaetomium/química , DNA/genética , Proteínas Fúngicas/química , Subunidades Proteicas/química , Fator de Transcrição TFIIH/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Chaetomium/genética , Chaetomium/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição GênicaRESUMO
Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.
Assuntos
Proteínas de Drosophila/química , Canais Iônicos/química , Proteínas Centrais de snRNP/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Drosophila melanogaster , Humanos , Ligação de Hidrogênio , Camundongos , Microscopia Eletrônica , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Xenopus/química , Xenopus laevis , Proteínas Centrais de snRNP/ultraestruturaRESUMO
Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.
Assuntos
Sequência Conservada , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Animais , Evolução Molecular , Humanos , Lisina/genética , Lisina/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Ligação Proteica , Serina/genética , Serina/metabolismo , Troponina C/química , Troponina C/genética , Troponina C/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.
Assuntos
Fator de Transcrição TFIIH/química , Chaetomium/enzimologia , Proteínas Fúngicas/química , Humanos , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Fator de Transcrição TFIIH/genéticaRESUMO
p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.
Assuntos
Apoptose , Ciclo Celular , Proliferação de Células , DNA/metabolismo , Regiões Promotoras Genéticas , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Ciclo Celular/genética , Dano ao DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
FANCM is a highly conserved DNA remodeling enzyme that promotes the activation of the Fanconi anemia DNA repair pathway and facilitates replication traverse of DNA interstrand crosslinks. However, how FANCM interacts with the replication machinery to promote traverse remains unclear. Here, we show that FANCM and its archaeal homolog Hef from Thermoplasma acidophilum interact with proliferating cell nuclear antigen (PCNA), an essential co-factor for DNA polymerases in both replication and repair. The interaction is mediated through a conserved PIP-box; and in human FANCM, it is strongly stimulated by replication stress. A FANCM variant carrying a mutation in the PIP-box is defective in promoting replication traverse of interstrand crosslinks and is also inefficient in promoting FANCD2 monoubiquitination, a key step of the Fanconi anemia pathway. Our data reveal a conserved interaction mode between FANCM and PCNA during replication stress, and suggest that this interaction is essential for FANCM to aid replication machines to traverse DNA interstrand crosslinks prior to post-replication repair.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , DNA Helicases/genética , Humanos , Modelos Moleculares , Mutação , Estresse Fisiológico , Thermoplasma/enzimologiaRESUMO
Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a ß-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.
Assuntos
Dano ao DNA , Enzimas Reparadoras do DNA/química , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/química , 2-Acetilaminofluoreno/química , 2-Acetilaminofluoreno/metabolismo , Sequência de Aminoácidos , Cisplatino/química , Cisplatino/metabolismo , Cristalografia por Raios X , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Termodinâmica , Temperatura de TransiçãoRESUMO
There is growing awareness of the link between drug-target residence time and in vivo drug activity, and there are increasing efforts to determine the molecular factors that control the lifetime of a drug-target complex. Rational alterations in the drug-target residence time require knowledge of both the ground and transition states on the inhibition reaction coordinate, and we have determined the structure-kinetic relationship for 22 ethyl- or hexyl-substituted diphenyl ethers that are slow-binding inhibitors of bpFabI1, the enoyl-ACP reductase FabI1 from Burkholderia pseudomallei. Analysis of enzyme inhibition using a two-dimensional kinetic map demonstrates that the ethyl and hexyl diphenyl ethers fall into two distinct clusters. Modifications to the ethyl diphenyl ether B ring result in changes to both on and off rates, where residence times of up to â¼700 min (â¼11 h) are achieved by either ground state stabilization (PT444) or transition state destabilization (slower on rate) (PT404). By contrast, modifications to the hexyl diphenyl ether B ring result in residence times of 300 min (â¼5 h) through changes in only ground state stabilization (PT119). Structural analysis of nine enzyme:inhibitor complexes reveals that the variation in structure-kinetic relationships can be rationalized by structural rearrangements of bpFabI1 and subtle changes to the orientation of the inhibitor in the binding pocket. Finally, we demonstrate that three compounds with residence times on bpFabI1 from 118 min (â¼2 h) to 670 min (â¼11 h) have in vivo efficacy in an acute B. pseudomallei murine infection model using the virulent B. pseudomallei strain Bp400.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Burkholderia pseudomallei/efeitos dos fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Inibidores Enzimáticos/química , Melioidose/dietoterapia , Éteres Fenílicos/química , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica , Cinética , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Éteres Fenílicos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Baço/efeitos dos fármacos , Baço/microbiologia , Relação Estrutura-AtividadeRESUMO
Nucleotide excision repair is an important and highly conserved DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. Lesion verification is believed to be achieved by the helicases UvrB and XPD in the prokaryotic and eukaryotic processes, respectively. Using single molecule atomic force microscopy analyses, we demonstrate that UvrB and XPD are able to load onto DNA and pursue lesion verification in the absence of the initial lesion detection proteins. Interestingly, our studies show different lesion recognition strategies for the two functionally homologous helicases, as apparent from their distinct DNA strand preferences, which can be rationalized from the different structural features and interactions with other nucleotide excision repair protein factors of the two enzymes.
Assuntos
Proteínas de Bactérias/química , DNA Helicases/química , Reparo do DNA , DNA Bacteriano/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismoRESUMO
A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition-state destabilization rather than ground-state stabilization. In the present work, we evaluate the inhibition of InhA by 14 triazole-based diphenyl ethers and use a combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to 3 orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min, which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time.
Assuntos
Inibinas/antagonistas & inibidores , Termodinâmica , Triazóis/farmacologia , Cristalografia por Raios X , Humanos , Inibinas/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , Fatores de Tempo , Triazóis/químicaRESUMO
BACKGROUND: Corticotropin-independent Cushing's syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood. METHODS: We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro. RESULTS: Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617AâC in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing's syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased. CONCLUSIONS: Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).
Assuntos
Adenoma/genética , Neoplasias das Glândulas Suprarrenais/genética , Hiperplasia Suprarrenal Congênita/genética , Síndrome de Cushing/etiologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Mutação em Linhagem Germinativa , Adenoma/complicações , Adenoma/enzimologia , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/enzimologia , Adulto , Domínio Catalítico , Síndrome de Cushing/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Exoma , Humanos , Hidrocortisona/biossíntese , Pessoa de Meia-Idade , Mutação , Conformação Proteica , Análise de Sequência de DNARESUMO
The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.
Assuntos
Reparo do DNA/genética , Proteínas Fúngicas/genética , Fator de Transcrição TFIIH/genética , Transcrição Gênica , Proteína Grupo D do Xeroderma Pigmentoso/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Modelos Moleculares , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/metabolismoRESUMO
The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold â 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156.