RESUMEN
Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.
Asunto(s)
Glicoproteínas de la Zona Pelúcida , Humanos , Masculino , Semen , Espermatozoides/química , Espermatozoides/metabolismo , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/química , Glicoproteínas de la Zona Pelúcida/metabolismo , Óvulo/química , Óvulo/metabolismo , FemeninoRESUMEN
The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Bacterianas , ADN Helicasas , ADN Cruciforme , ATPasas Asociadas con Actividades Celulares Diversas/química , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/ultraestructura , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , ADN Cruciforme/química , ADN Cruciforme/metabolismo , ADN Cruciforme/ultraestructura , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/ultraestructura , Recombinación Homóloga , Hidrólisis , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/ultraestructura , Nucleótidos , Conformación Proteica , RotaciónRESUMEN
Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year1. Specialized protein transport systems-known as type VII secretion systems (T7SSs)-are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope2,3. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber that is formed by three EccB5 dimers, with the proteolytic sites of MycP5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, which highlights the importance of MycP5 for complex integrity. Beneath the EccB5-MycP5 chamber, dimers of the EccC5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.
Asunto(s)
Microscopía por Crioelectrón , Mycobacterium tuberculosis , Sistemas de Secreción Tipo VII/metabolismo , Sistemas de Secreción Tipo VII/ultraestructura , Citosol/química , Citosol/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/ultraestructura , Periplasma/química , Periplasma/metabolismo , Dominios Proteicos , Multimerización de Proteína , Estabilidad Proteica , Tuberculosis/virología , Sistemas de Secreción Tipo VII/químicaRESUMEN
E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Ciclina E/biosíntesis , Quinasa 2 Dependiente de la Ciclina/biosíntesis , Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Proteínas Oncogénicas/biosíntesis , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/genética , Ciclinas/biosíntesis , Ciclinas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Proteínas Oncogénicas/genéticaRESUMEN
Acute intestinal graft-versus-host disease (aGVHD) refractory to immunosuppressive treatment is a serious complication after allogenic hematopoietic stem cell transplantation (HSCT). The underlying mechanisms of refractory aGVHD of the gut are not fully understood. Although telomere length (TL) reflects the replicative history of a cell, critically short telomeres have been associated with replicative exhaustion and tissue failure. In this study, we demonstrate that enterocytes of patients with refractory intestinal aGVHD show significantly increased proliferation, which translates into significant and critical telomere attrition following HSCT as compared with unaffected patients undergoing HSCT. Calculated telomere loss in aGVHD patients is 190 bp/wk, thereby massively exceeding physiological steady-state TL shortening rates such as in lymphocytes (â¼50 bp/y). Our data support the hypothesis that increased compensatory proliferation following continued tissue damage can result in massive telomere loss in enterocytes of aGVHD patients. The present study introduces aGVHD-triggered increased cellular turnover and telomere loss with subsequent replicative exhaustion as a mechanism for refractory gut GVHD that is compatible with the long-term clinical aspect of the disease and provides a basis for stem cell protective therapies in the treatment of aGVHD.
Asunto(s)
Proliferación Celular , Enterocitos/metabolismo , Enfermedad Injerto contra Huésped/metabolismo , Trasplante de Células Madre Hematopoyéticas , Enfermedades Intestinales/metabolismo , Acortamiento del Telómero , Enfermedad Aguda , Aloinjertos , Enterocitos/patología , Femenino , Enfermedad Injerto contra Huésped/patología , Humanos , Enfermedades Intestinales/patología , Masculino , Estudios RetrospectivosRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is centrally involved in diverse processes including haematopoiesis, immunity and cancer progression. In response to cytokine stimulation, STAT3 is activated through phosphorylation of a single tyrosine residue. The phosphorylated STAT3 dimers are stabilized by intermolecular interactions between SH2 domains and phosphotyrosine. These activated dimers accumulate in the nucleus and bind to specific DNA sequences, resulting in target gene expression. We analysed and compared the structural organizations of the unphosphorylated latent and phosphorylated activated STAT3 dimers using Förster resonance energy transfer (FRET) in fixed and living cells. The latent dimers are stabilized by homotypic interactions between the N-terminal domains. A somatic mutation (L78R) found in inflammatory hepatocellular adenoma (IHCA), which is located in the N-terminal domain of STAT3 disturbs latent dimer formation. Applying intramolecular FRET, we verify a functional role of the SH2 domain in latent dimer formation suggesting that the protomers in the latent STAT3 dimer are in a parallel orientation, similar to activated STAT3 dimers but different from the antiparallel orientation of the latent dimers of STAT1 and STAT5. Our findings reveal unique structural characteristics of STAT3 within the STAT family and contribute to the understanding of the L78R mutation found in IHCA.
Asunto(s)
Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Western Blotting , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Microscopía Confocal , Mutación , Fosforilación , Multimerización de Proteína , Factor de Transcripción STAT3/química , Factor de Transcripción STAT5/química , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismoRESUMEN
BACKGROUND: Chronic myeloid leukemia (CML) is driven by the expression of the BCR-ABL oncoprotein. STAT5 is a BCR-ABL substrate and persistently activated by tyrosine phosphorylation in CML cells. Activated STAT5 (pSTAT5) drives proliferation and survival of leukemic cells and contributes to initial transformation and maintenance of the disease. In cytokine-induced STAT5 signaling, phosphorylation of STAT5A on Y694 leads to nuclear accumulation of the transcription factor, followed by DNA-binding and gene induction. However, Src-family kinases (SFK) mediate cytoplasmic retention of pSTAT5A leading to attenuated target gene expression and colony formation in CML cells. RESULTS: In this study we show that autophosphorylation of Y416 in the highly conserved activation loop of SFK generates a potent recruitment site for the SH2 domain of STAT5A. Binding of the SH2 domain to the activation loop is required for STAT5A(Y694) phosphorylation by SFK, but at the same time promotes the persistent cytoplasmic localization of the transcription factor as found in BCR-ABL(+) leukemia. As a consequence of the complex formation between tyrosine-phosphorylated SFK and the SH2 domain of STAT5A, the dimerization of STAT5A is impaired. We further demonstrate that constitutively active STAT5A(S710F) escapes from SFK-mediated cytoplasmic retention by enhancing STAT5A dimer stability. CONCLUSION: Our results reveal important structural aspects of cytoplasmic pSTAT5A found in myeloid leukemias and will contribute to the understanding of STAT5A mediated cytoplasmic signaling.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Multimerización de Proteína , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Familia-src Quinasas/metabolismo , Animales , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Células HeLa , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Fosforilación/genética , Transporte de Proteínas/genética , Factor de Transcripción STAT5/genética , Proteínas Supresoras de Tumor/genética , Dominios Homologos src , Familia-src Quinasas/genéticaRESUMEN
BACKGROUND: STAT3 is a transcription factor of central importance in chronic inflammation and cancer. In response to cytokine stimulation STAT3 is phosphorylated on a single tyrosine residue at position 705, dimerizes and accumulates in the nucleus to induce target gene expression. The substitution of tyrosine 705 to phenylalanine leads to a dominant-negative STAT3 mutant (STAT3-YF) which influences the activation of WT-STAT3 in stimulated cells through a mechanism that is not completely understood. In this study we analyzed the molecular mechanism of STAT3-YF dominant-negative activity in IL-6-induced STAT3 signaling and the relevance of the N-terminal domain. RESULTS: Expression of STAT3-YF-YFP impairs tyrosine phosphorylation, nuclear translocation and the transcriptional activity of WT-STAT3 in IL-6-stimulated cells. The fluorescently labelled STAT3-YF mutant binds to a phosphorylated gp130 receptor-peptide comparable to WT-STAT3-YFP. STAT3-YF-YFP forms homodimers as well as heterodimers with WT-STAT3 in the presence and absence of IL-6. The preformed heterodimers in unstimulated cells are detectable by colocalization of STAT3-CFP with STAT3-YF-YFP fused to a nuclear localization signal. STAT3/STAT3-YF heterodimers are not able to bind to DNA in stimulated cells, but the presence of the mutant reduces DNA-binding of WT-STAT3 homodimers. STAT3-YF-ΔN-YFP lacking the N-terminal domain forms no dimers and only marginally affects the activity of WT-STAT3. CONCLUSION: Our findings demonstrate that dominant-negative STAT3-YF affects the activation of WT-STAT3 at multiple levels. Unexpectedly, the N-terminal domain of STAT3-YF plays an important role for the dominant-negative effect. We show that (i) STAT3-YF competes with WT-STAT3 in binding to activated gp130-receptors, (ii) the formation of WT-STAT3/STAT3-YF heterodimers in IL-6-stimulated cells results in inactive, semiphosphorylated dimers which do not bind to DNA and thus fail to induce target gene expression, (iii) the N-terminal domain-mediated formation of preformed STAT3/STAT3-YF heterodimers in unstimulated cells which affects the IL-6-induced homodimerization of WT-STAT3 contributes to the dominant-negative effect of STAT3-YF. These findings will contribute to our understanding of naturally occuring dominant-negative STAT3 mutants that cause the hyper-IgE syndrome.
Asunto(s)
Factor de Transcripción STAT3/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Receptor gp130 de Citocinas/metabolismo , Humanos , Interleucina-6/metabolismo , Mutación , Fosforilación , Fosfotirosina/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Factor de Transcripción STAT3/genética , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte de Proteínas/fisiología , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/química , Sistemas de Secreción Tipo III/metabolismo , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica en Hélice alfa , Salmonella enterica/metabolismo , Salmonella typhimurium/genética , Sistemas de Secreción Tipo III/genéticaRESUMEN
Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of â¼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a â¼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.
Asunto(s)
Adenosina Trifosfatasas/química , Escherichia coli Enteropatógena/ultraestructura , Proteínas de Escherichia coli/química , Flagelos/ultraestructura , Canales de Translocación SEC/química , Sistemas de Secreción Tipo III/ultraestructura , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Regulación Alostérica , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Medición de Intercambio de Deuterio , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/genética , Flagelos/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Espectrometría de Masas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Translocación SEC/genética , Canales de Translocación SEC/metabolismo , Especificidad por Sustrato , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallo-peptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked 'CPDCP-trunk', and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWVXGP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel 'raised-elephant-trunk' mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWVXGP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.
RESUMEN
Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the subcellular localization of this mutant had not changed. Our findings support the concept that basal nucleocytoplasmic shuttling of STAT3 is different from active cytokine-induced nuclear import and does not require conserved N- or SH2-terminal domains, preformed dimer formation and GAS-element-specific DNA recognition.
Asunto(s)
Núcleo Celular/metabolismo , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Secuencia de Bases , Núcleo Celular/efectos de los fármacos , ADN/metabolismo , Humanos , Interleucina-6/farmacología , Ligandos , Ratones , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Eliminación de Secuencia , Relación Estructura-Actividad , alfa Carioferinas/metabolismoRESUMEN
As the size of nanoparticles (NPs) is in the range of biological molecules and subcellular structures, they provide new perspectives in biomedicine. This work presents studies concerning the cellular uptake and distribution of phosphine-stabilized cytotoxic 1.4 nm sized AuNPs and their probable degradation during this process. Therefore, ultrasmall phosphine-stabilized AuNPs are modified by linking a fluorophore covalently to the ligand shell. Monitoring the fluorescence on a cellular level by means of flow cytometry and confocal laser scanning microscopy allows determining the fate of the ligand shell during AuNP cell internalization, due to the fact that the fluorescence of a fluorophore bound near to the AuNP surface is quenched. Cell fractionation is conducted in order to quantify the AuNP content at the cell membrane, in the cytoplasm, and the cell nucleus. The incubation of cells with the fluorophore-modified AuNPs reveals a partial loss of the ligand shell upon AuNP cell interaction, evident by the emerging fluorescence signal. This loss is the precondition to unfold high AuNP cytotoxicity. Together with their significantly different biodistribution and enhanced circulation times compared to larger AuNPs, the findings demonstrate the high potential of ultrasmall AuNPs for drug development or therapy.
Asunto(s)
Oro/metabolismo , Nanopartículas del Metal/administración & dosificación , Fosfinas/metabolismo , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Fluorescencia , Células HeLa , Células Hep G2 , Humanos , Tamaño de la Partícula , Propiedades de Superficie , Distribución TisularRESUMEN
STAT5 is an essential transcription factor in hematopoiesis, which is activated through tyrosine phosphorylation in response to cytokine stimulation. Constitutive activation of STAT5 is a hallmark of myeloid and lymphoblastic leukemia. Using homology modeling and molecular dynamics simulations, a model of the STAT5 phosphotyrosine-SH2 domain interface was generated providing first structural information on the activated STAT5 dimer including a sequence, for which no structural information is available for any of the STAT proteins. We identified a novel intramolecular interaction mediated through F706, adjacent to the phosphotyrosine motif, and a unique hydrophobic interface on the surface of the SH2 domain. Analysis of corresponding STAT5 mutants revealed that this interaction is dispensable for Epo receptor-mediated phosphorylation of STAT5 but essential for dimer formation and subsequent nuclear accumulation. Moreover, the herein presented model clarifies molecular mechanisms of recently discovered leukemic STAT5 mutants and will help to guide future drug development.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Multimerización de Proteína , Factor de Transcripción STAT5/química , Secuencia de Aminoácidos , Sitios de Unión , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Relación Estructura-Actividad , Dominios Homologos srcRESUMEN
The tumor suppressor protein prostate apoptosis response-4 (PAR-4) is silenced in a subset of human cancers and its down-regulation serves as a mechanism for cancer cell survival following chemotherapy. PAR-4 re-expression selectively causes apoptosis in cancer cells but how its pro-apoptotic functions are controlled and executed precisely is currently unknown. We demonstrate here that UV-induced apoptosis results in a rapid caspase-dependent PAR-4 cleavage at EEPD131G, a sequence that was preferentially recognized by caspase-8. To investigate the effect on cell growth for this cleavage event we established stable cell lines that express wild-type-PAR-4 or the caspase cleavage resistant mutant PAR-4 D131G under the control of a doxycycline-inducible promoter. Induction of the wild-type protein but not the mutant interfered with cell proliferation, predominantly through induction of apoptosis. We further demonstrate that TNFα-induced apoptosis leads to caspase-8-dependent PAR-4-cleavage followed by nuclear accumulation of the C-terminal PAR-4 (132-340) fragment, which then induces apoptosis. Taken together, our results indicate that the mechanism by which PAR-4 orchestrates the apoptotic process requires cleavage by caspase-8.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Caspasa 8/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We characterized bone marrow stromal cells (BMSC) from patients with pre-fibrotic myeloproliferative neoplasms (MPN). MPN-BMSC showed decreased capacity to stimulate the proliferation of colony-forming units of normal hematopoietic stem and progenitor cells and displayed increased matrix remodelling (in particular fibronectin deposition) compared to control BMSC. This finding was confirmed in pre-fibrotic MPN bone marrow biopsies in a tissue microarray (n = 34), which stained positive for fibronectin in the absence of reticulin as a standard myelofibrosis marker. Fibronectin expression correlated significantly with reduced haemoglobin levels in MPN-patients (p = 0.007; R2 = 0.42). Our data show significant cell-intrinsic alterations in MPN-MSC and suggest that Fibronectin expression might be applicable as a biomarker for the identification of early myelofibrotic transformation in reticulin-negative MPN.
Asunto(s)
Fibronectinas/metabolismo , Células Madre Mesenquimatosas/patología , Trastornos Mieloproliferativos/patología , Humanos , Células Madre Mesenquimatosas/metabolismo , Trastornos Mieloproliferativos/genética , Fenotipo , Análisis de Matrices TisularesRESUMEN
Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (â¼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of â¼105 kDa and â¼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of â¼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (LâF) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration.