RESUMEN
Type IVa pili (T4aP) function as bacterial virulence factors. T4aP pass through the outer membranes of Gram-negative bacteria via homo-oligomeric secretins. We present a 7.4 Å cryoelectron microscopy structure of the Pseudomonas aeruginosa PilQ secretin. Peripheral and internal features show that the secretin is composed of 14 subunits with C7 symmetry. The channel is a ribbed cylinder with central peripheral spokes and a central gate closed on the periplasmic side. The structure suggests that during pilus extrusion, the central gate is displaced to the interior walls and that no additional conformational changes are required, as the internal diameter can accommodate the pilus. The N1 domain was resolved, while the N0 and the N-terminal ß-domains proposed to bind peptidoglycan were absent in class average images and the final 3D map, indicating a high flexibility. These data provide the highest-resolution structure to date of a T4aP secretin.
Asunto(s)
Proteínas Fimbrias/química , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/metabolismo , Microscopía por Crioelectrón , Proteínas Fimbrias/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pseudomonas aeruginosa/químicaRESUMEN
Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1-12)). Structural analysis, multiangle light scattering coupled with size exclusion chromatography, and bacterial two-hybrid data revealed that PilM forms dimers mediated by the binding of a novel conserved motif in the N terminus of PilM, and binding PilN abrogates this binding interface, resulting in PilM monomerization. Structural comparison of PilM with PilM·PilN(1-12) revealed that upon PilN binding, there is a large domain closure in PilM that alters its ATP binding site. Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. Bacterial two-hybrid data suggested the connectivity of the cytoplasmic and inner membrane components of the type IVa pilus machinery in P. aeruginosa, with PilM binding to PilB, PilT, and PilC in addition to PilN. Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. We propose a working model in which dynamic binding of PilN facilitates functionally relevant structural changes in PilM.
Asunto(s)
Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Pseudomonas aeruginosa/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Proteínas Fimbrias/genética , Fimbrias Bacterianas/química , Fimbrias Bacterianas/clasificación , Fimbrias Bacterianas/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
RUNX3 functions as a tumor suppressor in the gastric epithelium, where its inactivation is frequently observed during carcinogenesis. We identified IL23A as a RUNX3 target gene in gastric epithelial cells. This was confirmed in a series of in vitro analyses in gastric epithelial cell lines. In elucidating the underlying regulatory network, we uncovered a prominent role for the TNF-α/NF-κB pathway in activating IL23A transcription. Moreover, the activating effect of TNF-α was markedly augmented by the infection of Helicobacter pylori, the primary cause of human gastritis. Of note, H. pylori utilized the CagA/SHP2 pathway to activate IL23A, as well as the induction of the NOD1 pathway by iE-DAP. Importantly, RUNX3 synergized strongly with these physiologically relevant stimuli to induce IL23A. Lastly, we present evidence for the secretion of IL23A by gastric epithelial cells in a form that is distinct from canonical IL-23 (IL23A/IL12B).
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Mucosa Gástrica/metabolismo , Subunidad p19 de la Interleucina-23/metabolismo , Activación Transcripcional , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Línea Celular , Línea Celular Tumoral , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Mucosa Gástrica/microbiología , Helicobacter pylori/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Subunidad p19 de la Interleucina-23/genética , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Recent studies have revealed that differentiated epithelial cells would acquire stem cell-like and tumorigenic properties following an Epithelial-Mesenchymal Transition (EMT). However, the signaling pathways that participate in this novel mechanism of tumorigenesis have not been fully characterized. In Runx3 (-/-) p53 (-/-) murine gastric epithelial (GIF-14) cells, EMT-induced plasticity is reflected in the expression of the embryonal proto-oncogene Hmga2 and Lgr5, an exclusive gastrointestinal stem cell marker. Here, we report the concurrent activation of an EGFR/Ras gene expression signature during TGF-ß1-induced EMT in GIF-14 cells. Amongst the altered genes was the induction of Egfr, which corresponded with a delayed sensitization to EGF treatment in GIF-14. Co-treatment with TGF-ß1 and EGF or the expression of exogenous KRas led to increased Hmga2 or Lgr5 expression, sphere initiation and colony formation in soft agar assay. Interestingly, the gain in cellular plasticity/tumorigenicity was not accompanied by increased EMT. This uncoupling of EMT and the induction of plasticity reveals an involvement of distinct signaling cues, whereby the EGFR/Ras pathway specifically promotes stemness and tumorigenicity in EMT-altered GIF-14 cells. These data show that the EGFR/Ras pathway requisite for the sustenance of gastric stem cells in vivo and in vitro is involved in the genesis and promotion of EMT-induced tumor-initiating cells.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Transición Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Células Madre/metabolismo , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Análisis por Conglomerados , Factor de Crecimiento Epidérmico/farmacología , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Modelos Biológicos , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Células Madre/efectos de los fármacos , Transcriptoma , Factor de Crecimiento Transformador beta1/farmacología , Células Tumorales CultivadasRESUMEN
Pseudomonas aeruginosa uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The P. aeruginosa T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein-protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from Pseudomonas cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined.
Asunto(s)
Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas , Pseudomonas aeruginosa/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Prueba de Complementación Genética , Modelos Moleculares , MutagénesisRESUMEN
Pseudomonas aeruginosa type IV pili (T4P) are virulence factors that promote infection of cystic fibrosis and immunosuppressed patients. As the absence of T4P impairs colonization, they are attractive targets for the development of novel therapeutics. Genes in the pilMNOPQ operon are important for both T4P assembly and a form of bacterial movement, called twitching motility, that is required for pathogenicity. The type II membrane proteins, PilN and PilO, dimerize via their periplasmic domains and anchor this complex in the inner membrane. Our earlier work showed that PilNO binds PilP, a periplasmic lipoprotein (S. Tammam, L. M. Sampaleanu, J. Koo, P. Sundaram, M. Ayers, P. A. Chong, J. D. Forman-Kay, L. L. Burrows, and P. L. Howell, Mol. Microbiol. 82:1496-1514, 2011). Here, we show that PilP interacts with the N0 segment of the outer membrane secretin PilQ via its C-terminal domain, and that the N-terminal cytoplasmic tail of PilN binds to the actin-like protein PilM, thereby connecting all cellular compartments via the PilMNOPQ protein interaction network. We show that PilA, the major pilin subunit, interacts with PilNOPQ. The results allow us to propose a model whereby PilA makes extensive contacts with the transenvelope complex, possibly to increase local concentrations of PilA monomers for polymerization. The PilNOP complex could provide a stable anchor in the inner membrane, while the PilMNOPQ transenvelope complex facilitates transit of the pilus through the periplasm and clamps the pilus in the cell envelope. The PilMN interaction is proposed to be responsible for communicating signals from the cytoplasmic to periplasmic components of this complex macromolecular machine.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas Fimbrias/metabolismo , Pseudomonas aeruginosa/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Western Blotting , Proteínas Fimbrias/genética , Unión Proteica , Pseudomonas aeruginosa/genética , Factores de Virulencia/genéticaRESUMEN
The transcription factor RUNX3 functions as a tumor suppressor in the gastrointestinal epithelium, where its loss is an early event in carcinogenesis. While RUNX3 acts concurrently as a mediator of TGF-ß signaling and an antagonist of Wnt, the cellular changes that follow its loss and their contribution to tumorigenicity are not fully understood. Here, we report that the loss of Runx3 in gastric epithelial cells results in spontaneous epithelial-mesenchymal transition (EMT). This produces a tumorigenic stem cell-like subpopulation, which remarkably expresses the gastric stem cell marker Lgr5. This phenomenon is due to the compounding effects of the dysregulation of the TGF-ß and Wnt pathways. Specifically, Runx3(-/-) p53(-/-) gastric epithelial cells were unexpectedly sensitized for TGF-ß-induced EMT, during which the resultant induction of Lgr5 was enhanced by an aberrantly activated Wnt pathway. These data demonstrate a protective role for RUNX3 in safeguarding gastric epithelial cells against aberrant growth factor signaling and the resultant cellular plasticity and stemness.
Asunto(s)
Transformación Celular Neoplásica/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Neoplasias Gastrointestinales/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Animales , Diferenciación Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transfección , Factor de Crecimiento Transformador beta/farmacología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Secretins are channels that allow translocation of macromolecules across the outer membranes of Gram-negative bacteria. Virulence, natural competence, and motility are among the functions mediated by these large oligomeric protein assemblies. Filamentous phage also uses secretins to exit their bacterial host without causing cell lysis. However, the secretin is only a part of a larger membrane-spanning complex, and additional proteins are often required for its formation. A class of outer membrane lipoproteins called pilotins has been implicated in secretin assembly and/or localization. Additional accessory proteins may also be involved in secretin stability. Significant progress has recently been made toward deciphering the complex interactions required for functional secretin assembly. To allow for easier comparison between different systems, we have classified the secretins into five different classes based on their requirements for proteins involved in their assembly, localization, and stability. An overview of pilotin and accessory protein structures, functions, and characterized modes of interaction with the secretin is presented.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacterias Gramnegativas/fisiología , Proteínas de Transporte de Membrana/metabolismo , Competencia de la Transformación por ADN , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/patogenicidad , Locomoción , Sustancias Macromoleculares/metabolismo , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Liberación del VirusRESUMEN
In intestinal epithelial cells, inactivation of APC, a key regulator of the Wnt pathway, activates beta-catenin to initiate tumorigenesis. However, other alterations may be involved in intestinal tumorigenesis. Here we found that RUNX3, a gastric tumor suppressor, forms a ternary complex with beta-catenin/TCF4 and attenuates Wnt signaling activity. A significant fraction of human sporadic colorectal adenomas and Runx3(+/-) mouse intestinal adenomas showed inactivation of RUNX3 without apparent beta-catenin accumulation, indicating that RUNX3 inactivation independently induces intestinal adenomas. In human colon cancers, RUNX3 is frequently inactivated with concomitant beta-catenin accumulation, suggesting that adenomas induced by inactivation of RUNX3 may progress to malignancy. Taken together, these data demonstrate that RUNX3 functions as a tumor suppressor by attenuating Wnt signaling.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/fisiología , Neoplasias Intestinales/patología , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Pólipos Adenomatosos/genética , Pólipos Adenomatosos/metabolismo , Pólipos Adenomatosos/patología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Western Blotting , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Ciclina D , Ciclinas/genética , Ciclinas/metabolismo , Células HCT116 , Humanos , Hiperplasia , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/genética , Neoplasias Intestinales/metabolismo , Intestinos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Transcripción TCF/genética , Factor de Transcripción 4 , Proteína 2 Similar al Factor de Transcripción 7 , beta Catenina/genéticaRESUMEN
Type IV pili (T4P) are retractile appendages that contribute to the virulence of bacterial pathogens. PilF is a Pseudomonas aeruginosa lipoprotein that is essential for T4P biogenesis. Phenotypic characterization of a pilF mutant confirmed that T4P-mediated functions are abrogated: T4P were no longer present on the cell surface, twitching motility was abolished, and the mutant was resistant to infection by T4P retraction-dependent bacteriophage. The results of cellular fractionation studies indicated that PilF is the outer membrane pilotin required for the localization and multimerization of the secretin, PilQ. Mutation of the putative PilF lipidation site untethered the protein from the outer membrane, causing secretin assembly in both inner and outer membranes. T4P-mediated twitching motility and bacteriophage susceptibility were moderately decreased in the lipidation site mutant, while cell surface piliation was substantially reduced. The tethering of PilF to the outer membrane promotes the correct localization of PilQ and appears to be required for the formation of stable T4P. Our 2.0-A structure of PilF revealed a superhelical arrangement of six tetratricopeptide protein-protein interaction motifs that may mediate the contacts with PilQ during secretin assembly. An alignment of pseudomonad PilF sequences revealed three highly conserved surfaces that may be involved in PilF function.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Bacteriófagos/fisiología , Western Blotting , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/virología , Homología Estructural de ProteínaRESUMEN
Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimidazole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly alpha-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall beta-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP.FUM) complexes have been determined to 2.0, 1.85, and 2.0 A resolution, respectively. The H171A-ADS and H171N-AMP.FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.
Asunto(s)
Adenilosuccinato Liasa/química , Adenilosuccinato Liasa/metabolismo , Escherichia coli/enzimología , Estructura Terciaria de Proteína , Adenilosuccinato Liasa/genética , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , MutaciónRESUMEN
Delta-crystallin, the major soluble protein component in the avian eye lens, is homologous to argininosuccinate lyase (ASL). Two delta-crystallin isoforms exist in ducks, delta1- and delta2-crystallin, which are 94% identical in amino acid sequence. While duck delta2-crystallin (ddeltac2) has maintained ASL activity, evolution has rendered duck delta1-crystallin (ddeltac1) enzymatically inactive. Previous attempts to regenerate ASL activity in ddeltac1 by mutating the residues in the 20s (residues 22-31) and 70s (residues 74-89) loops to those found in ddeltac2 resulted in a double loop mutant (DLM) which was enzymatically inactive (Tsai, M. et al. (2004) Biochemistry 43, 11672-82). This result suggested that one or more of the remaining five amino acid substitutions in domain 1 of the DLM contributes to the loss of ASL activity in ddeltac1. In the current study, residues Met-9, Val-14, Ala-41, Ile-43, and Glu-115 were targeted for mutagenesis, either alone or in combination, to the residues found in ddeltac2. ASL activity was recovered in the DLM by changing Met-9 to Trp, and this activity is further potentiated in the DLM-M9W mutant when Glu-115 is changed to Asp. The roles of Trp-9 and Asp-115 were further investigated by site-directed mutagenesis in wild-type ddeltac2. Changing the identity of either Trp-9 or Asp-115 in ddeltac2 resulted in a dramatic drop in enzymatic activity. The loss of activity in Trp-9 mutants indicates a preference for an aromatic residue at this position. Truncation mutants of ddeltac2 in which the first 8, 9, or 14 N-terminal residues were removed displayed either decreased or no ASL activity, suggesting residues 1-14 are crucial for enzymatic activity in ddeltac2. Our kinetic studies combined with available structural data suggest that the N-terminal arm in ASL/delta2-crystallin is involved in stabilizing regions of the protein involved in substrate binding and catalysis, and in completely sequestering the substrate from the solvent.