RESUMO
Ribonuclease (RNase) P is a ubiquitous ribozyme that cleaves the 5' leader from precursor tRNAs. Here, we report cryo-electron microscopy structures of the human nuclear RNase P alone and in complex with tRNAVal. Human RNase P is a large ribonucleoprotein complex that contains 10 protein components and one catalytic RNA. The protein components form an interlocked clamp that stabilizes the RNA in a conformation optimal for substrate binding. Human RNase P recognizes the tRNA using a double-anchor mechanism through both protein-RNA and RNA-RNA interactions. Structural comparison of the apo and tRNA-bound human RNase P reveals that binding of tRNA induces a local conformational change in the catalytic center, transforming the ribozyme into an active state. Our results also provide an evolutionary model depicting how auxiliary RNA elements in bacterial RNase P, essential for substrate binding, and catalysis, were replaced by the much more complex and multifunctional protein components in higher organisms.
Assuntos
Microscopia Crioeletrônica , RNA de Transferência/química , Ribonuclease P/química , Sítios de Ligação , Evolução Molecular , Células HEK293 , Holoenzimas/química , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Domínios Proteicos , Estrutura Terciária de Proteína , RNA de Transferência/metabolismo , Ribonuclease P/isolamento & purificação , Ribonuclease P/metabolismoRESUMO
Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.
Assuntos
Proteínas de Ligação a DNA/química , Simulação de Acoplamento Molecular , Proteínas de Saccharomyces cerevisiae/química , Telomerase/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Pyruvate carboxylase (PC) catalyzes the two-step carboxylation of pyruvate to produce oxaloacetate, playing a key role in the maintenance of metabolic homeostasis in cells. Given its involvement in multiple diseases, PC has been regarded as a potential therapeutic target for obesity, diabetes, and cancer. Albeit acetyl-CoA has been recognized as the allosteric regulator of PC for over 60 years, the underlying mechanism of how acetyl-CoA induces PC activation remains enigmatic. Herein, by using time-resolved cryo-electron microscopy, we have captured the snapshots of PC transitional states during its catalytic cycle. These structures and the biochemical studies reveal that acetyl-CoA stabilizes PC in a catalytically competent conformation, which triggers a cascade of events, including ATP hydrolysis and the long-distance communication between the two reactive centers. These findings provide an integrated picture for PC catalysis and unveil the unique allosteric mechanism of acetyl-CoA in an essential biochemical reaction in all kingdoms of life.
Assuntos
Acetil-CoA Carboxilase , Piruvato Carboxilase , Humanos , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , Acetilcoenzima A/metabolismo , Regulação Alostérica , Microscopia Crioeletrônica , Conformação Molecular , Acetil-CoA Carboxilase/metabolismoRESUMO
Human mixed-lineage leukemia (MLL) family methyltransferases methylate histone H3 lysine 4 to different methylation states (me1/me2/me3) with distinct functional outputs, but the mechanism underlying the different product specificities of MLL proteins remains unclear. Here, we develop methodologies to quantitatively measure the methylation rate difference between mono-, di-, and tri-methylation steps and demonstrate that MLL proteins possess distinct product specificities in the context of the minimum MLL-RBBP5-ASH2L complex. Comparative structural analyses of MLL complexes by X-ray crystal structures, fluorine-19 nuclear magnetic resonance, and molecular dynamics simulations reveal that the dynamics of two conserved tyrosine residues at the "F/Y (phenylalanine/tyrosine) switch" positions fine-tune the product specificity. The variation in the intramolecular interaction between SET-N and SET-C affects the F/Y switch dynamics, thus determining the product specificities of MLL proteins. These results indicate a modified F/Y switch rule applicable for most SET domain methyltransferases and implicate the functional divergence of MLL proteins.
Assuntos
Histona-Lisina N-Metiltransferase , Leucemia , Humanos , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Lisina/metabolismo , Flúor/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Tirosina , FenilalaninaRESUMO
Standard first-line chemotherapy results in disease progression and death within one year in most patients with human epidermal growth factor receptor 2 (HER2)-negative gastro-oesophageal adenocarcinoma1-4. Nivolumab plus chemotherapy demonstrated superior overall survival versus chemotherapy at 12-month follow-up in gastric, gastro-oesophageal junction or oesophageal adenocarcinoma in the randomized, global CheckMate 649 phase 3 trial5 (programmed death ligand-1 (PD-L1) combined positive score ≥5 and all randomized patients). On the basis of these results, nivolumab plus chemotherapy is now approved as a first-line treatment for these patients in many countries6. Nivolumab and the cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor ipilimumab have distinct but complementary mechanisms of action that contribute to the restoration of anti-tumour T-cell function and induction of de novo anti-tumour T-cell responses, respectively7-11. Treatment combining 1 mg kg-1 nivolumab with 3 mg kg-1 ipilimumab demonstrated clinically meaningful anti-tumour activity with a manageable safety profile in heavily pre-treated patients with advanced gastro-oesophageal cancer12. Here we report both long-term follow-up results comparing nivolumab plus chemotherapy versus chemotherapy alone and the first results comparing nivolumab plus ipilimumab versus chemotherapy alone from CheckMate 649. After the 24.0-month minimum follow-up, nivolumab plus chemotherapy continued to demonstrate improvement in overall survival versus chemotherapy alone in patients with PD-L1 combined positive score ≥5 (hazard ratio 0.70; 95% confidence interval 0.61, 0.81) and all randomized patients (hazard ratio 0.79; 95% confidence interval 0.71, 0.88). Overall survival in patients with PD-L1 combined positive score ≥ 5 for nivolumab plus ipilimumab versus chemotherapy alone did not meet the prespecified boundary for significance. No new safety signals were identified. Our results support the continued use of nivolumab plus chemotherapy as standard first-line treatment for advanced gastro-oesophageal adenocarcinoma.
Assuntos
Adenocarcinoma , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Esofágicas , Neoplasias Gástricas , Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1 , Neoplasias Esofágicas/tratamento farmacológico , Junção Esofagogástrica , Seguimentos , Humanos , Ipilimumab/efeitos adversos , Ipilimumab/uso terapêutico , Nivolumabe/efeitos adversos , Nivolumabe/uso terapêutico , Neoplasias Gástricas/tratamento farmacológicoRESUMO
BRCT domains support myriad protein-protein interactions involved in genome maintenance. Although di-BRCT recognition of phospho-proteins is well known to support the genotoxic response, whether multi-BRCT domains can acquire distinct structures and functions is unclear. Here we present the tetra-BRCT structures from the conserved yeast protein Rtt107 in free and ligand-bound forms. The four BRCT repeats fold into a tetrahedral structure that recognizes unmodified ligands using a bi-partite mechanism, suggesting repeat origami enabling function acquisition. Functional studies show that Rtt107 binding of partner proteins of diverse activities promotes genome replication and stability in both distinct and concerted manners. A unified theme is that tetra- and di-BRCT domains of Rtt107 collaborate to recruit partner proteins to chromatin. Our work thus illustrates how a master regulator uses two types of BRCT domains to recognize distinct genome factors and direct them to chromatin for constitutive genome protection.
Assuntos
Instabilidade Genômica/genética , Proteínas Nucleares/ultraestrutura , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Cromatina/genética , Dano ao DNA/genética , Ligantes , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Domínios Proteicos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Methionine is important for intestinal development and homeostasis in various organisms. However, the underlying mechanisms are poorly understood. Here, we demonstrate that the methionine adenosyltransferase gene Mat2a is essential for intestinal development and that the metabolite S-adenosyl-L-methionine (SAM) plays an important role in intestinal homeostasis. Intestinal epithelial cell (IEC)-specific knockout of Mat2a exhibits impaired intestinal development and neonatal lethality. Mat2a deletion in the adult intestine reduces cell proliferation and triggers IEC apoptosis, leading to severe intestinal epithelial atrophy and intestinal inflammation. Mechanistically, we reveal that SAM maintains the integrity of differentiated epithelium and protects IECs from apoptosis by suppressing the expression of caspases 3 and 8 and their activation. SAM supplementation improves the defective intestinal epithelium and reduces inflammatory infiltration sequentially. In conclusion, our study demonstrates that methionine metabolism and its intermediate metabolite SAM play essential roles in intestinal development and homeostasis in mice.
Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Camundongos , Animais , S-Adenosilmetionina/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Mucosa Intestinal/metabolismo , Metionina , Suplementos NutricionaisRESUMO
GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington's disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Regulação Alostérica , Sítio Alostérico , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoproteínas/agonistas , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/ultraestruturaRESUMO
Nuclear respiratory factor 1 (NRF1) regulates the expression of genes that are vital for mitochondrial biogenesis, respiration, and various other cellular processes. While NRF1 has been reported to bind specifically to GC-rich promoters as a homodimer, the precise molecular mechanism governing its recognition of target gene promoters has remained elusive. To unravel the recognition mechanism, we have determined the crystal structure of the NRF1 homodimer bound to an ATGCGCATGCGCAT dsDNA. In this complex, NRF1 utilizes a flexible linker to connect its dimerization domain (DD) and DNA binding domain (DBD). This configuration allows one NRF1 monomer to adopt a U-turn conformation, facilitating the homodimer to specifically bind to the two TGCGC motifs in the GCGCATGCGC consensus sequence from opposite directions. Strikingly, while the NRF1 DBD alone could also bind to the half-site (TGCGC) DNA of the consensus sequence, the cooperativity between DD and DBD is essential for the binding of the intact GCGCATGCGC sequence and the transcriptional activity of NRF1. Taken together, our results elucidate the molecular mechanism by which NRF1 recognizes specific DNA sequences in the promoters to regulate gene expression.
Assuntos
DNA , Fator 1 Nuclear Respiratório , Humanos , Sequência de Bases , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Regiões Promotoras GenéticasRESUMO
Iron homeostasis is crucial for maintaining proper cellular function, and its disruption is considered one of the pathogenic mechanisms underlying musculoskeletal diseases. Under conditions of oxidative stress, the accumulation of cellular iron overload and lipid peroxidation can lead to ferroptosis. Extracellular vesicles (EVs), serving as mediators in the cell-to-cell communication, play an important role in regulating the outcome of cell ferroptosis. Growing evidence has proven that EV biogenesis and secretion are tightly associated with cellular iron export. Furthermore, different sources of EVs deliver diverse cargoes to bring about phenotypic changes in the recipient cells, either activating or inhibiting ferroptosis. Thus, delivering therapies targeting ferroptosis through EVs may hold significant potential for treating musculoskeletal diseases. This review aims to summarize current knowledge on the role of EVs in iron homeostasis and ferroptosis, as well as their therapeutic applications in musculoskeletal diseases, and thereby provide valuable insights for both research and clinical practice.
Assuntos
Vesículas Extracelulares , Ferroptose , Doenças Musculoesqueléticas , Humanos , Ferro , Doenças Musculoesqueléticas/terapia , HomeostaseRESUMO
The CCAAT/enhancer-binding proteins (C/EBPs) constitute a family of pivotal transcription factors involved in tissue development, cellular function, proliferation, and differentiation. NFIL3, as one of them, plays an important role in regulating immune cell differentiation, circadian clock system, and neural regeneration, yet its specific DNA recognition mechanism remains enigmatic. In this study, we showed by the ITC binding experiments that NFIL3 prefers to bind to the TTACGTAA DNA motif. Our structural studies revealed that the α-helical NFIL3 bZIP domain dimerizes through its leucine zipper region, and binds to DNA via its basic region. The two basic regions of the NFIL3 bZIP dimer were pushed apart upon binding to DNA, facilitating the snug accommodation of the two basic regions within the major grooves of the DNA. Remarkably, our binding and structural data also revealed that both NFIL3 and C/EBPα/ß demonstrate a shared preference for the TTACGTAA sequence. Furthermore, our study revealed that disease-associated mutations within the NFIL3 bZIP domain result in either reduction or complete disruption of its DNA binding ability. These discoveries not only provide valuable insights into the DNA binding mechanisms of NFIL3 but also elucidate the causal role of NFIL3 mutations in disease pathogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , DNA , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , HumanosRESUMO
RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.
Assuntos
Endorribonucleases , Evolução Molecular , Subunidades Proteicas , RNA Catalítico , Ribonuclease P , Coenzimas , Endorribonucleases/química , Endorribonucleases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ribonuclease P/química , Ribonuclease P/metabolismo , Processamento Pós-Transcricional do RNA , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Especificidade por Substrato , Eucariotos/enzimologiaRESUMO
BACKGROUND: First-line chemotherapy for advanced esophageal squamous-cell carcinoma results in poor outcomes. The monoclonal antibody nivolumab has shown an overall survival benefit over chemotherapy in previously treated patients with advanced esophageal squamous-cell carcinoma. METHODS: In this open-label, phase 3 trial, we randomly assigned adults with previously untreated, unresectable advanced, recurrent, or metastatic esophageal squamous-cell carcinoma in a 1:1:1 ratio to receive nivolumab plus chemotherapy, nivolumab plus the monoclonal antibody ipilimumab, or chemotherapy. The primary end points were overall survival and progression-free survival, as determined by blinded independent central review. Hierarchical testing was performed first in patients with tumor-cell programmed death ligand 1 (PD-L1) expression of 1% or greater and then in the overall population (all randomly assigned patients). RESULTS: A total of 970 patients underwent randomization. At a 13-month minimum follow-up, overall survival was significantly longer with nivolumab plus chemotherapy than with chemotherapy alone, both among patients with tumor-cell PD-L1 expression of 1% or greater (median, 15.4 vs. 9.1 months; hazard ratio, 0.54; 99.5% confidence interval [CI], 0.37 to 0.80; P<0.001) and in the overall population (median, 13.2 vs. 10.7 months; hazard ratio, 0.74; 99.1% CI, 0.58 to 0.96; P = 0.002). Overall survival was also significantly longer with nivolumab plus ipilimumab than with chemotherapy among patients with tumor-cell PD-L1 expression of 1% or greater (median, 13.7 vs. 9.1 months; hazard ratio, 0.64; 98.6% CI, 0.46 to 0.90; P = 0.001) and in the overall population (median, 12.7 vs. 10.7 months; hazard ratio, 0.78; 98.2% CI, 0.62 to 0.98; P = 0.01). Among patients with tumor-cell PD-L1 expression of 1% or greater, a significant progression-free survival benefit was also seen with nivolumab plus chemotherapy over chemotherapy alone (hazard ratio for disease progression or death, 0.65; 98.5% CI, 0.46 to 0.92; P = 0.002) but not with nivolumab plus ipilimumab as compared with chemotherapy. The incidence of treatment-related adverse events of grade 3 or 4 was 47% with nivolumab plus chemotherapy, 32% with nivolumab plus ipilimumab, and 36% with chemotherapy alone. CONCLUSIONS: Both first-line treatment with nivolumab plus chemotherapy and first-line treatment with nivolumab plus ipilimumab resulted in significantly longer overall survival than chemotherapy alone in patients with advanced esophageal squamous-cell carcinoma, with no new safety signals identified. (Funded by Bristol Myers Squibb and Ono Pharmaceutical; CheckMate 648 ClinicalTrials.gov number, NCT03143153.).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Esofágicas/tratamento farmacológico , Inibidores de Checkpoint Imunológico/administração & dosagem , Ipilimumab/administração & dosagem , Nivolumabe/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Antígeno B7-H1/antagonistas & inibidores , Carcinoma de Células Escamosas/mortalidade , Neoplasias Esofágicas/mortalidade , Feminino , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Ipilimumab/efeitos adversos , Masculino , Pessoa de Meia-Idade , Nivolumabe/efeitos adversos , Intervalo Livre de Progressão , Análise de SobrevidaRESUMO
N6-methyladenosine (m6A) in mRNA and 5-methylcytosine (5mC) in DNA have critical functions for regulating gene expression and modulating plant growth and development. However, the interplay between m6A and 5mC is an elusive territory and remains unclear mechanistically in plants. We reported an occurrence of crosstalk between m6A and 5mC in maize (Zea mays) via the interaction between mRNA adenosine methylase (ZmMTA), the core component of the m6A methyltransferase complex, and decrease in DNA methylation 1 (ZmDDM1), a key chromatin-remodeling factor that regulates DNA methylation. Genes with m6A modification were coordinated with a much higher level of DNA methylation than genes without m6A modification. Dysfunction of ZmMTA caused severe arrest during maize embryogenesis and endosperm development, leading to a significant decrease in CHH methylation in the 5' region of m6A-modified genes. Instead, loss of function of ZmDDM1 had no noteworthy effects on ZmMTA-related activity. This study establishes a direct link between m6A and 5mC during maize kernel development and provides insights into the interplay between RNA modification and DNA methylation.
Assuntos
Metilação de DNA , Zea mays , Metilação de DNA/genética , Zea mays/genética , Zea mays/metabolismo , Metilação de RNA , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA/metabolismoRESUMO
Methyltransferases of the mixed-lineage leukaemia (MLL) family-which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B-implement methylation of histone H3 on lysine 4 (H3K4), and have critical and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development1-6. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are associated with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities7-9. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 methyl marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favours open chromatin structures, but the underlying mechanism remains unknown10-12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules associated with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the association between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.
Assuntos
Proteínas de Ligação a DNA/química , Histona-Lisina N-Metiltransferase/metabolismo , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/química , Nucleossomos/química , Nucleossomos/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/química , Histonas/química , Histonas/metabolismo , Humanos , Metilação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Estrutura Quaternária de ProteínaRESUMO
Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2TRFH domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas Nucleares/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Aminopeptidases/genética , Aminopeptidases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Exodesoxirribonucleases , Fase G1 , Fase G2 , Células HCT116 , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fase S , Serina Proteases/genética , Serina Proteases/metabolismo , Complexo Shelterina , Relação Estrutura-Atividade , Telômero/genética , Telômero/patologia , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genéticaRESUMO
Telomeric repeat binding factor 1 (TRF1) is essential to the maintenance of telomere chromatin structure and integrity. However, how telomere integrity is maintained, especially in response to damage, remains poorly understood. Here, we identify Nek7, a member of the Never in Mitosis Gene A (NIMA) kinase family, as a regulator of telomere integrity. Nek7 is recruited to telomeres and stabilizes TRF1 at telomeres after damage in an ATM activation-dependent manner. Nek7 deficiency leads to telomere aberrations, long-lasting γH2AX and 53BP1 foci, and augmented cell death upon oxidative telomeric DNA damage. Mechanistically, Nek7 interacts with and phosphorylates TRF1 on Ser114, which prevents TRF1 from binding to Fbx4, an Skp1-Cul1-F box E3 ligase subunit, thereby alleviating proteasomal degradation of TRF1, leading to a stable association of TRF1 with Tin2 to form a shelterin complex. Our data reveal a mechanism of efficient protection of telomeres from damage through Nek7-dependent stabilization of TRF1.
Assuntos
Dano ao DNA , Quinases Relacionadas a NIMA/metabolismo , Estresse Oxidativo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/enzimologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Quinases Relacionadas a NIMA/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Interferência de RNA , Complexo Shelterina , Telômero/genética , Telômero/efeitos da radiação , Proteínas de Ligação a Telômeros/genética , Fatores de Tempo , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Telomerase , Proteínas de Transporte/genética , DNA de Cadeia Simples , Ligação Proteica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Complexo Shelterina , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Head and neck tumors (HNTs) constitute a multifaceted ensemble of pathologies that primarily involve regions such as the oral cavity, pharynx, and nasal cavity. The intricate anatomical structure of these regions poses considerable challenges to efficacious treatment strategies. Despite the availability of myriad treatment modalities, the overall therapeutic efficacy for HNTs continues to remain subdued. In recent years, the deployment of artificial intelligence (AI) in healthcare practices has garnered noteworthy attention. AI modalities, inclusive of machine learning (ML), neural networks (NNs), and deep learning (DL), when amalgamated into the holistic management of HNTs, promise to augment the precision, safety, and efficacy of treatment regimens. The integration of AI within HNT management is intricately intertwined with domains such as medical imaging, bioinformatics, and medical robotics. This article intends to scrutinize the cutting-edge advancements and prospective applications of AI in the realm of HNTs, elucidating AI's indispensable role in prevention, diagnosis, treatment, prognostication, research, and inter-sectoral integration. The overarching objective is to stimulate scholarly discourse and invigorate insights among medical practitioners and researchers to propel further exploration, thereby facilitating superior therapeutic alternatives for patients.
Assuntos
Inteligência Artificial , Neoplasias de Cabeça e Pescoço , Humanos , Aprendizado de Máquina , Redes Neurais de Computação , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/terapia , Diagnóstico por Imagem/métodosRESUMO
BACKGROUND: Telomere length is a critical metric linked to aging, health, and disease. Currently, the exploration of target proteins related to telomere length is usually limited to the context of aging and specific diseases, which limits the discovery of more relevant drug targets. This study integrated large-scale plasma cis-pQTLs data and telomere length GWAS datasets. We used Mendelian randomization(MR) to identify drug target proteins for telomere length, providing essential clues for future precision therapy and targeted drug development. METHODS: Using plasma cis-pQTLs data from a previous GWAS study (3,606 Pqtls associated with 2,656 proteins) and a GWAS dataset of telomere length (sample size: 472,174; GWAS ID: ieu-b-4879) from UK Biobank, using MR, external validation, and reverse causality testing, we identified essential drug target proteins for telomere length. We also performed co-localization, Phenome-wide association studies and enrichment analysis, protein-protein interaction network construction, search for existing intervening drugs, and potential drug/compound prediction for these critical targets to strengthen and expand our findings. RESULTS: After Bonferron correction (p < 0.05/734), RPN1 (OR: 0.96; 95%CI: (0.95, 0.97)), GDI2 (OR: 0.94; 95%CI: (0.92, 0.96)), NT5C (OR: 0.97; 95%CI: (0.95, 0.98)) had a significant negative causal association with telomere length; TYRO3 (OR: 1.11; 95%CI: (1.09, 1.15)) had a significant positive causal association with telomere length. GDI2 shared the same genetic variants with telomere length (coloc.abf-PPH 4 > 0.8). CONCLUSION: Genetically determined plasma RPN1, GDI2, NT5C, and TYRO3 have significant causal effects on telomere length and can potentially be drug targets. Further exploration of the role and mechanism of these proteins/genes in regulating telomere length is needed.