RESUMO
Pentamidine and melarsoprol are primary drugs used to treat the lethal human sleeping sickness caused by the parasite Trypanosoma brucei. Cross-resistance to these two drugs has recently been linked to aquaglyceroporin 2 of the trypanosome (TbAQP2). TbAQP2 is the first member of the aquaporin family described as capable of drug transport; however, the underlying mechanism remains unclear. Here, we present cryo-electron microscopy structures of TbAQP2 bound to pentamidine or melarsoprol. Our structural studies, together with the molecular dynamic simulations, reveal the mechanisms shaping substrate specificity and drug permeation. Multiple amino acids in TbAQP2, near the extracellular entrance and inside the pore, create an expanded conducting tunnel, sterically and energetically allowing the permeation of pentamidine and melarsoprol. Our study elucidates the mechanism of drug transport by TbAQP2, providing valuable insights to inform the design of drugs against trypanosomiasis.
Assuntos
Aquagliceroporinas , Microscopia Crioeletrônica , Melarsoprol , Simulação de Dinâmica Molecular , Pentamidina , Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Aquagliceroporinas/metabolismo , Aquagliceroporinas/química , Melarsoprol/metabolismo , Melarsoprol/química , Pentamidina/química , Pentamidina/metabolismo , Transporte Biológico , Tripanossomicidas/química , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , HumanosRESUMO
Staphylococcal aureus (S. aureus) infection can lead to a wide range of diseases such as sepsis and pneumonia. Staphylococcal superantigen-like (SSL) proteins, expressed by all known S. aureus strains, are shown to be involved in immune evasion during S. aureus infection. Here, we show that SSL10, an SSL family protein, exhibits potent cytotoxicity against human cells (HEK293T and HUVEC) by inducing necroptosis upon binding to its receptor TNFR1 on the cell membrane. After binding, two distinct signaling pathways are activated downstream of TNFR1 in a RIPK3-dependent manner, i.e., the RIPK1-RIPK3-MLKL and RIPK3-CaMKII-mitochondrial permeability transition pore (mPTP) pathways. Knockout of ssl10 in S. aureus significantly reduces cytotoxicity of the culture supernatants of S. aureus, indicating that SSL10 is involved in extracellular cytotoxicity during infection. We determined the crystal structure of SSL10 at 1.9 Å resolution and identified a positively charged surface of SSL10 responsible for TNFR1 binding and cytotoxic activity. This study thus provides the description of cytotoxicity through induction of necroptosis by the SSL10 protein, and a potential target for clinical treatment of S. aureus-associated diseases.
Assuntos
Necroptose , Receptores Tipo I de Fatores de Necrose Tumoral , Antígenos de Bactérias , Proteínas de Bactérias , Células HEK293 , Humanos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Staphylococcus aureus/metabolismoRESUMO
Serotonylation of histone H3Q5 (H3Q5ser) is a recently identified posttranslational modification of histones that acts as a permissive marker for gene activation in synergy with H3K4me3 during neuronal cell differentiation. However, any proteins that specifically recognize H3Q5ser remain unknown. Here, we found that WDR5 interacts with the N-terminal tail of histone H3 and functions as a "reader" for H3Q5ser. Crystal structures of WDR5 in complex with H3Q5ser and H3K4me3Q5ser peptides revealed that the serotonyl group is accommodated in a shallow surface pocket of WDR5. Experiments in neuroblastoma cells demonstrate that H3K4me3 modification is hampered upon disruption of WDR5-H3Q5ser interaction. WDR5 colocalizes with H3Q5ser in the promoter regions of cancer-promoting genes in neuroblastoma cells, where it promotes gene transcription to induce cell proliferation. Thus, beyond revealing a previously unknown mechanism through which WDR5 reads H3Q5ser to activate transcription, our study suggests that this WDR5-H3Q5ser-mediated epigenetic regulation apparently promotes tumorigenesis.
RESUMO
Background: Rhabdomyosarcoma (RMS) is one of the most common types of soft-tissue sarcomas in children, and it exhibits a low 5-years survival rate. The survival outcome has shown no significant improvements in the past 30 years miRNA profiling of RMS might therefore provide a novel insight into uncovering new molecular targets for therapy. Methods: We analyzed miRNA and RNA sequencing data from patients and the TARGET database to reveal the potential miRNA-mRNA axes and validated them in patients' samples. After the miRNA antagomirs were used to silence the target miRNAs in the cell model, qRT-PCR, western immunoblotting analysis, and proliferation assays were performed to explore the interaction between miR-130a/b and peroxisome proliferator-activated receptor gamma (PPARG) and their effects. Results: In RMS patients, the expression of miR-130a/b was augmented, and its related PPARG gene was suppressed. Bioinformatics analysis showed that miR-130a/b targeted the PPARG gene and inhibited the proliferation of human RMS cell lines. In addition, rosiglitazone maleate activated the expression of PPARG in human RMS cell lines to suppress proliferation. Conclusion: miR-130a/b regulates the malignant process in RMS by targeting PPARG. Furthermore, the PPARG agonist rosiglitazone maleate attenuated the proliferation of RD cells and might therefore be of benefit to RMS patients.
RESUMO
Staphylococcus aureus is an anaerobic facultative microorganism that features the NreABC system for nitrate respiration. NreB is the sensor histidine kinase that phosphorylates the response regulator NreC to stimulate the expression of target genes. NreA is a nitrate sensor which dissociates from NreB in the present of nitrate and relieves its inhibition on NreB. However, the molecular basis of how NreA regulate NreB remains unknown. In this study, we determined the crystal structures of nitrate-bound NreA from S. aureus (SaNreA/NO3-) and its apoNreA-like mutant SaNreAY94A in complex with ethanediol (SaNreAY94A/EDO). Structural comparison reveals that the C-terminal loop in SaNreA/NO3- rearranges to an α-helix (α7) in SaNreAY94A/EDO, which converts an acidic pocket on the surface to a positively charged region. This conformational change of SaNreA C-terminus might play a role in SaNreB binding.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Histidina Quinase/química , Histidina Quinase/genética , Nitratos/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Histidina Quinase/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Staphylococcus aureus/químicaRESUMO
Tetrathiomolybdate (TM) is used in the clinic for the treatment of Wilson's disease by targeting the cellular copper efflux protein ATP7B (WLN). Interestingly, both TM and WLN are associated with the efficacy of cisplatin, a widely used anticancer drug. Herein, we show that TM induces dimerization of the metal-binding domain of ATP7B (WLN4) through a unique sulfur-bridged Mo2S6O2 cluster. TM expels copper ions from Cu-WLN4 and forms a copper-free dimer. The binding of Mo to cysteine residues of WLN4 inhibits platination of the protein. Reaction with multi-domain proteins indicates that TM can also connect two domains in the same molecule, forming Mo-bridged intramolecular crosslinks. These results provide structural and chemical insight into the mechanism of action of TM against ATPase, and reveal the molecular mechanism by which TM attenuates the cisplatin resistance mediated by copper efflux proteins.
Assuntos
Antineoplásicos/farmacologia , Quelantes/farmacologia , Cisplatino/farmacologia , ATPases Transportadoras de Cobre/metabolismo , Molibdênio/farmacologia , Antineoplásicos/uso terapêutico , Quelantes/uso terapêutico , Cisplatino/uso terapêutico , Cobre/metabolismo , ATPases Transportadoras de Cobre/antagonistas & inibidores , ATPases Transportadoras de Cobre/química , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Reagentes de Ligações Cruzadas/uso terapêutico , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Molibdênio/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Platina/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de ProteínaRESUMO
Histone modification is a ubiquitous regulatory mechanism involved in a variety of biological processes, including gene expression, DNA damage repair, cell differentiation, and ontogenesis. Succinylation sites on histones have been identified and may have functional consequences. Here, we demonstrate that human sirtuin 5 (Sirt5) catalyzes the sequence-selective desuccinylation of numerous histone succinyl sites. Structural studies of Sirt5 in complex with four succinyl peptides indicate an essential role for the conserved main chain hydrogen bonds formed by the succinyl lysine (0), +1, and +3 sites for substrate-enzyme recognition. Furthermore, biochemical assays reveal that the proline residue at the +1 site of the histone succinylation substrate is unfavorable for Sirt5 interaction. Our findings illustrate the molecular mechanism underlying the sequence-selective desuccinylase activity of Sirt5 and provide insights for further studies of the biological functions associated with histone succinylation and Sirt5.
Assuntos
Histonas/química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Sirtuínas/química , Ácido Succínico/química , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos/genética , Peptídeos/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Relação Estrutura-Atividade , Ácido Succínico/metabolismoRESUMO
Epigenetic modifiers have emerged as critical factors governing the biology of different cancers. Herein we show that FBXL10 (also called KDM2B or JHDM1B), an important member of Polycomb repressive complexes, is overexpressed in human diffuse large B-cell lymphoma (DLBCL) tissues and the derived cell lines. Knocking down FBXL10 by specific short hairpin RNAs in DLBCL cells inhibits cell proliferation and induces apoptosis in vitro. Moreover, FBXL10 depletion in DLBCL cells abrogates tumor growth in mouse xenograft models. Through the analysis of RNA sequencing, we find that one of the key derepressed genes by depletion of FBXL10 is DUSP6, encoding a phosphatase for ERK1/2. Mechanistically FBXL10 maintains the silencing of DUSP6 expression via recruitment of Polycomb group proteins and deposition of repressive histone modifications at the DUSP6 promoter. Consistently, FBXL10 is required for ERK1/2 phosphorylation in DLBCL cells. Furthermore, we show that ERK1/2 activation and the proliferation rate of FBXL10-depleted cells can be rescued by downregulation of DUSP6 expression. These findings indicate that FBXL10 may be a promising therapeutic target in DLBCL and establish a link of epigenetic regulators to kinase signaling pathways.
Assuntos
Proteínas F-Box/genética , Histona Desmetilases com o Domínio Jumonji/genética , Linfoma Difuso de Grandes Células B/genética , Sistema de Sinalização das MAP Quinases/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Epigênese Genética , Proteínas F-Box/biossíntese , Proteínas F-Box/metabolismo , Xenoenxertos , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/biossíntese , Histona Desmetilases com o Domínio Jumonji/metabolismo , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
UHRF2 (Ubiquitin-like with PHD and ring finger domains 2) is an E3 ubiquitin ligase that plays important roles in DNA methylation, histone modifications and cell cycle regulation by interacting with multiple epigenetic or cell-cycle related proteins. Previous studied have identified PCNA (Proliferating cell nuclear antigen) as an interacting partner of UHRF2 by using the antibody microarray. However, the molecular mechanism and the function of UHRF2-PCNA interaction remains unclear. Here, we report the complex structure of PCNA and the peptide (784NEILQTLLDLFFPGYSK800) derived from UHRF2 that contains a PIP box. Structural analysis combined with mutagenesis experiments provide the molecular basis for the recognition of UHRF2 by PCNA via PIP-box.