RESUMO
DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequence.
Assuntos
Quadruplex G , Proteínas Proto-Oncogênicas c-myc , Humanos , DNA/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Transcription factor dysregulation is associated with many diseases, including cancer. Peptide-based molecules are increasingly recognised as important modulators of difficult intracellular protein-protein interaction targets, with peptide library screening consequently proven to be a viable strategy in developing inhibitors against a wide range of transcription factors (TFs). However, current strategies simply select the highest affinity of binding to a target TF rather than the ability to inhibit TF function. Here, we utilise our Transcription Block Survival (TBS) screening platform to enable high-throughput identification of peptides that inhibit TFs from binding to cognate DNA sites, hence inhibiting functionality. In this study, we explore whether the TBS can be expanded to derive a potent and functional peptide inhibitor of the BZLF1 transcription factor. The library-derived peptide, AcidicW, is shown to form a more stable dimer with BZLF1 than the BZLF1 homodimer, with a thermal denaturation temperature exceeding 80°C. AcidicW can also functionally inhibit the BZLF1:TRE DNA interaction with high potency and an IC50 of 612 nM.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , DNARESUMO
G-quadruplexes (G4s) are four-stranded DNA secondary structures that occur in the human genome and play key roles in transcription, replication, and genome stability. G4-specific molecular probes are of vital importance to elucidate the structure and function of G4s. The scFv antibody BG4 has been a widely used G4 probe but has various limitations, including relatively poor in vitro expression and the inability to be expressed intracellularly to interrogate G4s in live cells. To address these considerations, we describe herein the development of SG4, a camelid heavy-chain-only derived nanobody that was selected against the human Myc DNA G4 structure. SG4 exhibits low nanomolar affinity for a wide range of folded G4 structures in vitro. We employed AlphaFold combined with molecular dynamics simulations to construct a molecular model for the G4-nanobody interaction. The structural model accurately explains the role of key amino acids and Kd measurements of SG4 mutants, including arginine-to-alanine point mutations that dramatically diminish G4 binding affinity. Importantly, predicted amino acid-G4 interactions were subsequently confirmed experimentally by biophysical measurements. We demonstrate that the nanobody can be expressed intracellularly and used to image endogenous G4 structures in live cells. We also use the SG4 protein to positionally map G4s in situ and also on fixed chromatin. SG4 is a valuable, new tool for G4 detection and mapping in cells.
Assuntos
Quadruplex G , Humanos , DNA/química , CromatinaRESUMO
c-Myc is a transcription factor that is constitutively and aberrantly expressed in over 70% of human cancers. Its direct inhibition has been shown to trigger rapid tumor regression in mice with only mild and fully reversible side effects, suggesting this to be a viable therapeutic strategy. Here we reassess the challenges of directly targeting c-Myc, evaluate lessons learned from current inhibitors, and explore how future strategies such as miniaturisation of Omomyc and targeting E-box binding could facilitate translation of c-Myc inhibitors into the clinic.
Assuntos
Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Humanos , Terapia de Alvo Molecular , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
The Jumonji C lysine demethylases (KDMs) are 2-oxoglutarate- and Fe(II)-dependent oxygenases. KDM6A (UTX) and KDM6B (JMJD3) are KDM6 subfamily members that catalyze demethylation of N(ϵ)-methylated histone 3 lysine 27 (H3K27), a mark important for transcriptional repression. Despite reports stating that UTY(KDM6C) is inactive as a KDM, we demonstrate by biochemical studies, employing MS and NMR, that UTY(KDM6C) is an active KDM. Crystallographic analyses reveal that the UTY(KDM6C) active site is highly conserved with those of KDM6B and KDM6A. UTY(KDM6C) catalyzes demethylation of H3K27 peptides in vitro, analogously to KDM6B and KDM6A, but with reduced activity, due to point substitutions involved in substrate binding. The results expand the set of human KDMs and will be of use in developing selective KDM inhibitors.
Assuntos
Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Metilação , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade da EspécieRESUMO
The process of displaying functional peptides by 'grafting' them onto loops of a stable protein scaffold can be used to impart binding affinity for a target, but it can be difficult to predict the affinity of the grafted peptide and the effect of grafting on scaffold stability. In this study, we show that a series of peptides that bind to the E3 ubiquitin ligase Keap1 can be grafted into the inter-repeat loop of a consensus-designed tetratricopeptide repeat (CTPR) protein resulting in proteins with high stability. We found that these CTPR-grafted peptides had similar affinities to their free peptide counterparts and achieved a low nanomolar range. This result is likely due to a good structural match between the inter-repeat loop of the CTPR and the Keap1-binding peptide. The grafting process led to the discovery of a new Keap1-binding peptide, Ac-LDPETGELL-NH2, with low nanomolar affinity for Keap1, highlighting the potential of the repeat-protein class for application in peptide display.
Assuntos
Fator 2 Relacionado a NF-E2 , Ubiquitina-Proteína Ligases , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeos/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The proteins Keap1 and Nrf2 together act as a cytoprotective mechanism that enables cells to overcome electrophilic and oxidative stress. Research has shown that manipulating this system by modulating the Keap1-Nrf2 interaction either through inhibition at the binding interface or via the covalent modification of Keap1 could provide a powerful therapeutic strategy for a range of diseases. However, despite intensive investigation of the system and significant progress in the development of inhibitory small molecules, there is still much to learn about the pathways associated with the Keap1-Nrf2 system and the structural details underpinning its mechanism of action. In this review, we discuss how a deeper understanding could prove revolutionary in the development of new inhibitors and activators as well as guiding how to best harness Keap1 for targeted protein degradation.
Assuntos
Descoberta de Drogas , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/metabolismo , Sítios de Ligação , Proteínas Culina/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Peptide display approaches, in which peptide epitopes of known binding activities are grafted onto stable protein scaffolds, have been developed to constrain the peptide in its bioactive conformation and to enhance its stability. However, peptide grafting can be a lengthy process requiring extensive computational modeling and/or optimisation by directed evolution techniques. In this study, we show that ultra-stable consensus-designed tetratricopeptide repeat (CTPR) proteins are amenable to the grafting of peptides that bind the Kelch-like ECH-associated protein 1 (Keap1) onto the loop between adjacent repeats. We explore simple strategies to optimize the grafting process and show that modest improvements in Keap1-binding affinity can be obtained by changing the composition of the linker sequence flanking either side of the binding peptide.
Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/química , Fator 2 Relacionado a NF-E2/química , Peptídeos/química , Repetições de Tetratricopeptídeos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Modelos Moleculares , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and N(ϵ)-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known N(ϵ)-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the N(ϵ)-methyllysine of an H3K27me3 peptide binds similarly to N(ϵ)-methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the N(ϵ)-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes.