RESUMEN
Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.
Asunto(s)
Rabdomiosarcoma Alveolar , Rabdomiosarcoma , Niño , Humanos , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Rabdomiosarcoma Alveolar/genética , Línea Celular Tumoral , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas/metabolismoRESUMEN
Heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), spreads across large domains and can be epigenetically inherited in a self-propagating manner. Heterochromatin propagation depends upon a read-write mechanism, where the Clr4/Suv39h methyltransferase binds to preexisting trimethylated H3K9 (H3K9me3) and further deposits H3K9me. How the parental methylated histone template is preserved during DNA replication is not well understood. Here, we demonstrate using Schizosaccharomyces pombe that heterochromatic regions are specialized replication domains demarcated by their surrounding boundary elements. DNA replication throughout these domains is distinguished by an abundance of replisome components and is coordinated by Swi6/HP1. Although mutations in the replicative helicase subunit Mcm2 that affect histone binding impede the maintenance of a heterochromatin domain at an artificially targeted ectopic site, they have only a modest impact on heterochromatin propagation via the read-write mechanism at an endogenous site. Instead, our findings suggest a crucial role for the replication factor Mcl1 in retaining parental histones and promoting heterochromatin propagation via a mechanism involving the histone chaperone FACT. Engagement of FACT with heterochromatin requires boundary elements, which position the heterochromatic domain at the nuclear peripheral subdomain enriched for heterochromatin factors. Our findings highlight the importance of replisome components and boundary elements in creating a specialized environment for the retention of parental methylated histones, which facilitates epigenetic inheritance of heterochromatin.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Histonas/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ensamble y Desensamble de Cromatina , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Epigénesis GenéticaRESUMEN
Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase (MMP) family. MMPs extensively degrade core matrix components in addition to processing a range of other factors in the extracellular, plasma membrane, and intracellular compartments. The proteolytic activity of MMPs is modulated by the Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four multi-functional matrisome proteins with extensively characterized MMP inhibitory functions. Thus, a well-regulated balance between MMP activity and TIMP levels has been described as critical for healthy tissue homeostasis, and this balance can be chronically disturbed in pathological processes. The relationship between MMPs and TIMPs is complex and lacks the constraints of a typical enzyme-inhibitor relationship due to secondary interactions between various MMPs (specifically gelatinases) and TIMP family members. We illustrate a new complexity in this system by describing how MMP9 can cleave members of the TIMP family when in molar excess. Proteolytic processing of TIMPs can generate functionally altered peptides with potentially novel attributes. We demonstrate here that all TIMPs are cleaved at their C-terminal tails by a molar excess of MMP9. This processing removes the N-glycosylation site for TIMP3 and prevents the TIMP2 interaction with latent proMMP2, a prerequisite for cell surface MMP14-mediated activation of proMMP2. TIMP2/4 are further cleaved producing â¼14 kDa N-terminal proteins linked to a smaller C-terminal domain through residual disulfide bridges. These cleaved TIMP2/4 complexes show perturbed MMP inhibitory activity, illustrating that MMP9 may bear a particularly prominent influence upon the TIMP:MMP balance in tissues.
Asunto(s)
Metaloproteinasa 9 de la Matriz , Inhibidores Tisulares de Metaloproteinasas , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Proteolisis , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Gelatinasas/metabolismo , Proteínas/metabolismoRESUMEN
Elastomer-granule composites have been used to switch between soft and stiff states by applying negative pressure differentials that cause the membrane to squeeze the internal grains, inducing dilation and jamming. Applications of this phenomenon have ranged from universal gripping to adaptive mobility. Previously, the combination of this jamming phenomenon with the ability to transport grains across multiple soft actuators for shape morphing has not yet been demonstrated. In this paper, the authors demonstrate the use of hollow glass spheres as granular media that functions as a jammable "quasi-hydraulic" fluid in a fluidic elastomeric actuator that better mimics a key featur of animal musculature: independent control over i) isotonic actuation for motion; and ii) isometric actuation for stiffening without shape change. To best implement the quasi-hydraulic fluid, the authors design and build a fluidic device. Leveraging this combination of physical properties creates a new option for fluidic actuation that allows higher specific stiffness actuators using lower volumetric flow rates in addition to independent control over shape and stiffness. These features are showcased in a robotic catcher's mitt by stiffening the fluid in the glove's open configuration for catching, unjamming the media, then pumping additional fluid to the mitt to inflate and grasp.
Asunto(s)
Medios de Comunicación , Robótica , Animales , Medios de Cultivo , Elastómeros , Diseño de Equipo , Fuerza de la ManoRESUMEN
Regulated proteolysis by proteasomes involves ~800 enzymes for substrate modification with ubiquitin, including ~600 E3 ligases. We report here that E6AP/UBE3A is distinguished from other E3 ligases by having a 12 nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, the E6AP-binding domain in hRpn10 locks into a well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at proteasomes. Moreover, proteasome-associated ubiquitin is reduced following E6AP knockdown or displacement from proteasomes, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sitios de Unión , Células HCT116 , Humanos , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas de Unión al ARN/química , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1â¯×â¯100â¯mm, 3.5⯵m XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1â¯ng to 1000â¯ng in mouse plasma with the lower limit of detection for RP-182 at 1â¯ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Animales , Péptidos Catiónicos Antimicrobianos/sangre , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacocinética , Ácidos Carboxílicos/química , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Evaluación Preclínica de Medicamentos/métodos , Ghrelina/sangre , Ghrelina/química , Ghrelina/aislamiento & purificación , Límite de Detección , Fenómenos Magnéticos , Ratones , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
This corrects the article DOI: 10.1038/ncomms15034.
RESUMEN
Genome wide association studies (GWAS) have mapped multiple independent cancer susceptibility loci to chr5p15.33. Here, we show that fine-mapping of pancreatic and testicular cancer GWAS within one of these loci (Region 2 in CLPTM1L) focuses the signal to nine highly correlated SNPs. Of these, rs36115365-C associated with increased pancreatic and testicular but decreased lung cancer and melanoma risk, and exhibited preferred protein-binding and enhanced regulatory activity. Transcriptional gene silencing of this regulatory element repressed TERT expression in an allele-specific manner. Proteomic analysis identifies allele-preferred binding of Zinc finger protein 148 (ZNF148) to rs36115365-C, further supported by binding of purified recombinant ZNF148. Knockdown of ZNF148 results in reduced TERT expression, telomerase activity and telomere length. Our results indicate that the association with chr5p15.33-Region 2 may be explained by rs36115365, a variant influencing TERT expression via ZNF148 in a manner consistent with elevated TERT in carriers of the C allele.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Melanoma/genética , Neoplasias Pancreáticas/genética , Neoplasias Cutáneas/genética , Telomerasa/genética , Neoplasias Testiculares/genética , Factores de Transcripción/genética , Alelos , Línea Celular Tumoral , Mapeo Cromosómico , Cromosomas Humanos Par 5 , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Femenino , Sitios Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Melanoma/metabolismo , Melanoma/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Polimorfismo de Nucleótido Simple , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismo , Homeostasis del Telómero , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Regulación hacia Abajo/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Receptor ErbB-3/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Humanos , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Ingeniería de Proteínas/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismoRESUMEN
Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.
Asunto(s)
Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Exones , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/antagonistas & inhibidores , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína Proto-Oncogénica c-fli-1/antagonistas & inhibidores , Proteína Proto-Oncogénica c-fli-1/genética , Interferencia de ARN , Precursores del ARN/metabolismo , Empalme del ARN , Factores de Empalme de ARN , ARN Interferente Pequeño/metabolismo , Proteína EWS de Unión a ARN/antagonistas & inhibidores , Proteína EWS de Unión a ARN/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/antagonistas & inhibidores , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Sarcoma de Ewing/patología , Transactivadores , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Genome-wide association studies (GWAS) have identified multiple common susceptibility loci for pancreatic cancer. Here we report fine-mapping and functional analysis of one such locus residing in a 610 kb gene desert on chr13q22.1 (marked by rs9543325). The closest candidate genes, KLF5, KLF12, PIBF1, DIS3 and BORA, range in distance from 265-586 kb. Sequencing three sub-regions containing the top ranked SNPs by imputation P-value revealed a 30 bp insertion/deletion (indel) variant that was significantly associated with pancreatic cancer risk (rs386772267, P = 2.30 × 10-11, OR = 1.22, 95% CI 1.15-1.28) and highly correlated to rs9543325 (r2 = 0.97 in the 1000 Genomes EUR population). This indel was the most significant cis-eQTL variant in a set of 222 histologically normal pancreatic tissue samples (ß = 0.26, P = 0.004), with the insertion (risk-increasing) allele associated with reduced DIS3 expression. DIS3 encodes a catalytic subunit of the nuclear RNA exosome complex that mediates RNA processing and decay, and is mutated in several cancers. Chromosome conformation capture revealed a long range (570 kb) physical interaction between a sub-region of the risk locus, containing rs386772267, and a region â¼6 kb upstream of DIS3 Finally, repressor regulatory activity and allele-specific protein binding by transcription factors of the TCF/LEF family were observed for the risk-increasing allele of rs386772267, indicating that expression regulation at this risk locus may be influenced by the Wnt signaling pathway. In conclusion, we have identified a putative functional indel variant at chr13q22.1 that associates with decreased DIS3 expression in carriers of pancreatic cancer risk-increasing alleles, and could therefore affect nuclear RNA processing and/or decay.
Asunto(s)
Cromosomas Humanos Par 13 , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Neoplasias Pancreáticas/genética , Alelos , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Mapeo Cromosómico/métodos , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Sitios Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Mutación INDEL , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/genéticaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/genética , ARN no Traducido/metabolismo , Enfermedad Aguda , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Hemo/metabolismo , Proteínas de Unión al Hemo , Hemoproteínas/genética , Hemoproteínas/metabolismo , Homeostasis , Humanos , Inmunización , Pulmón/microbiología , Pulmón/patología , Ratones , Datos de Secuencia Molecular , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , ARN no Traducido/administración & dosificación , ARN no Traducido/genética , ARN no Traducido/inmunología , Eliminación de Secuencia , VirulenciaRESUMEN
Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.
Asunto(s)
Fibrosis Quística/microbiología , Homeostasis/fisiología , Hierro/metabolismo , Oligopéptidos/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Adaptación Fisiológica , Adolescente , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Niño , Preescolar , Regulación Bacteriana de la Expresión Génica/fisiología , Homeostasis/genética , Humanos , Mutación , Oligopéptidos/genética , Pigmentos Biológicos , Pseudomonas aeruginosa/genética , Adulto JovenRESUMEN
Iron is an essential element to all living organisms and is an important determinant of bacterial virulence. Bacteria have evolved specialized systems to sequester and transport iron from the environment or host. Pseudomonas aeruginosa, an opportunistic pathogen, uses two outer membrane receptor mediated systems (Phu and Has) to utilize host heme as a source of iron. PhuS is a 39 kDa soluble cytoplasmic heme binding protein which interacts and transports heme from the inner membrane heme transporter to the cytoplasm where it is degraded by heme oxygenase thus releasing iron. PhuS is unique among other cytoplasmic heme transporter proteins owing to the presence of three histidines in the heme binding pocket which can potentially serve as heme ligands. Out of the three histidine residues on the heme binding helix, His 209 is conserved among heme trafficking proteins while His 210 and His 212 are unique to PhuS. Here we report the crystal structure of PhuS at 1.98Å resolution which shows a unique heme binding pocket and oligomeric structure compared to other known cytoplasmic heme transporter and accounts for some of the unusual biochemical properties of PhuS.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Hemoproteínas/química , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Citoplasma/metabolismo , Proteínas de Unión al Hemo , Hemoproteínas/genética , Hemoproteínas/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/genética , Homología de Secuencia de AminoácidoRESUMEN
The Pseudomonas aeruginosa heme utilization (Phu) system encodes several proteins involved in the acquisition of heme as an iron source. Once internalized, heme is degraded by the iron-regulated heme oxygenase, HemO to biliverdin (BV) IXδ and ß. In vitro studies have shown holo-PhuS transfers heme to the iron-regulated HemO. This protein-protein interaction is specific for HemO as PhuS does not interact with the α-regioselective heme oxygenase, BphO. Bacterial genetics and isotopic labeling ((13)C-heme) studies confirmed extracellular heme is converted to (13)C-BVIX δ and ß through the catalytic action of HemO. In an effort to further understand the role of PhuS, similar studies were performed on the P. aeruginosa PAO1 ΔphuS and ΔphuS/ΔhemO strains. In contrast to wild-type strain, the absence of PhuS results in extracellular heme uptake and degradation via the catalytic action of HemO and BphO. At low heme concentrations, loss of PhuS leads to inefficient extracellular heme uptake supported by the fact the mRNA levels of PhuR, HemO, and BphO remain elevated when compared to the wild-type PAO1. On increasing extracellular heme concentrations, the elevated levels of PhuR, HemO, and BphO allow "leaky uptake" and degradation of heme via HemO and BphO. Similarly, in the ΔphuS/ΔhemO strain, the higher heme concentrations combined with elevated levels of PhuR and BphO leads to nonspecific heme uptake and degradation by BphO. Thus we propose heme flux into the cell is driven by the catalytic action of HemO with PhuS acting as a "control valve" to regulate extracellular heme flux.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Citoplasma/química , Hemo Oxigenasa (Desciclizante)/química , Hemo/farmacocinética , Hemoproteínas/química , Pseudomonas aeruginosa , Cromatografía Liquida , Hemo Oxigenasa (Desciclizante)/farmacocinética , Proteínas de Unión al Hemo , Espectrometría de Masas , Modelos Biológicos , Estructura Molecular , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/enzimologíaRESUMEN
Iron, an essential nutrient with limited bioavailability, requires specialized cellular mechanisms for uptake. Although iron uptake into the cytoplasm in the form of heme has been well characterized in many bacteria, the subsequent trafficking is poorly understood. The cytoplasmic heme-binding proteins belong to a structurally related family thought to have evolved as "induced fit" ligand-binding macromolecules. One member, Pseudomonas aeruginosa cytoplasmic protein (PhuS), has previously been shown to be important for delivering heme to the iron regulated heme oxygenase (HemO). Spectroscopic investigations of the holo-PhuS complex revealed a dynamic heme environment with overlapping but distinct heme-binding sites with alternative coordinating heme ligands, His-209 or His-212. In the present work we establish a mechanism for how heme is transferred from PhuS to its partner, HemO. Using surface plasmon resonance and isothermal titration calorimetry, we have discovered that holo-PhuS, but not apo-PhuS, forms a 1:1 complex with HemO. Sedimentation velocity and limited proteolysis experiments suggest that heme binding to PhuS induces a conformational rearrangement that drives the protein interaction with HemO. Hydrodynamic analysis reveals that the holo-PhuS displays a more expanded hydrodynamic envelope compared with apo-PhuS, and we propose that this conformational change drives the interaction with HemO. We further demonstrate that replacement of His-212 by Ala disrupts the interaction of holo-PhuS with HemO; in contrast, the His-209-Ala variant can still complex with HemO, albeit more weakly. Together, the present studies reveal a mechanism that couples a heme-dependent conformational switch in PhuS to protein-protein interaction, the subsequent free energy of which drives heme release to HemO.