RESUMEN
RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).
Asunto(s)
Transducción de Señal , Quinasas raf , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Proteínas ras/química , Quinasas raf/metabolismo , Quinasas raf/genética , Animales , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genéticaRESUMEN
RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer4-6; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
Asunto(s)
Proteínas 14-3-3/antagonistas & inhibidores , Proteínas 14-3-3/química , Microscopía por Crioelectrón , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sitios de Unión , Transformación Celular Neoplásica/genética , Humanos , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Modelos Moleculares , Mutación , Fosforilación , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
The heart LIM protein (HLP) is a LIM-only protein family member that mediates protein-protein interactions. To date, no studies have yet been conducted regarding its function in the heart. In the present study, we have identified that HLP binds the cytosolic region of RyR2 in the heart using a bacterial two-hybrid system, LC-MS/MS, co-immunoprecipitation, and GST-pull down assays. Microscopy revealed that HLP forms a triple complex with RyR2 and caveolin-3. siRNA and adenovirus-mediated KD of HLP decreased the electrically evoked Ca(2+) release from the sarcoplasmic reticulum without directly affecting SERCA2 and RyR2 activities. Collectively, the HLP-RyR2 interaction in the cell surface caveolae region may be essential for efficient excitation-contraction coupling in the heart.
Asunto(s)
Calcio/metabolismo , Caveolina 3/metabolismo , Proteínas con Dominio LIM/metabolismo , Miocardio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Línea Celular , Masculino , Unión Proteica , Ratas , Ratas Sprague-Dawley , Rianodina/metabolismo , Espectrometría de Masas en TándemRESUMEN
Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys(48)-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Proteínas Reguladoras de la Apoptosis , Sitios de Unión/genética , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Poliubiquitina/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Ubiquitina/metabolismo , Proteína que Contiene ValosinaRESUMEN
Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca(2+)-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca(2+) that were decondensed upon Ca(2+) depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca(2+) depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca(2+) concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca(2+) and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca(2+) homeostasis.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Calsecuestrina/metabolismo , Proteínas de la Membrana/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteínas Musculares/metabolismo , Multimerización de Proteína/fisiología , Retículo Sarcoplasmático/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Calsecuestrina/genética , Línea Celular , Homeostasis/fisiología , Humanos , Proteínas de la Membrana/genética , Ratones , Oxigenasas de Función Mixta/genética , Proteínas Musculares/genética , Retículo Sarcoplasmático/genéticaRESUMEN
The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation-induced genotoxicity was investigated via gain-of-functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast-targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild-type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV-treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV-C radiation, the dark-grown E. coli RecA-overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation-induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV-C radiation-induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA-overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.
Asunto(s)
Daño del ADN , ADN de Cloroplastos/genética , Nicotiana/genética , Paraquat/farmacología , Rec A Recombinasas/metabolismo , Rayos Ultravioleta , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reparación del ADN , ADN de Cloroplastos/efectos de los fármacos , ADN de Cloroplastos/efectos de la radiación , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/efectos de la radiación , Rec A Recombinasas/genética , Alineación de Secuencia , Nicotiana/efectos de los fármacos , Nicotiana/efectos de la radiaciónRESUMEN
The structural proximity and functional coupling between the SR (sarcoplasmic reticulum) and mitochondria have been suggested to occur in the heart. However, the molecular architecture involved in the SR-mitochondrial coupling remains unclear. In the present study, we performed various genetic and Ca2+-probing studies to resolve the proteins involved in the coupling process. By using the bacterial 2-hybrid, glutathione transferase pull-down, co-immunoprecipitation and immunocytochemistry assays, we found that RyR2 (ryanodine receptor type 2), which is physically associated with VDAC2 (voltage-dependent anion channel 2), was co-localized in SR-mitochondrial junctions. Furthermore, a fractionation study revealed that VDAC2 was co-localized with RyR2 only in the subsarcolemmal region. VDAC2 knockdown by targeted short hairpin RNA led to an increased diastolic [Ca2+] (calcium concentration) and abolishment of mitochondrial Ca2+ uptake. Collectively, the present study suggests that the coupling of VDAC2 with RyR2 is essential for Ca2+ transfer from the SR to mitochondria in the heart.
Asunto(s)
Calcio/metabolismo , Mitocondrias Cardíacas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Técnicas In Vitro , Transporte Iónico , Masculino , Ratones , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Canal Aniónico 2 Dependiente del Voltaje/genéticaRESUMEN
At the apical tip of Drosophila testis, there is a stem cell niche known as the proliferation center, where the stem cells are maintained by hub cell cluster for the regulation of differentiation and proliferation. Germline stem cells go through mitosis four times from one primary spermatogonial cell to the 16-cell stage before the maturation. The cells derived from the same germline stem cell are located within one cyst, an enclosed system by two cyst cells, and they are connected by the intercellular bridges called ring canals. In this study, the three-dimensional (3D) structure of Drosophila testis tip was reconstructed from serial sections. The size of cells at each stage was compared in volume from the 3D structure. The stages of cells in a cyst could be distinguishable exactly by counting the cells linked with intercellular bridges in 3D-reconstructed structure. The cysts containing the same stage cells appeared in the horizontal plane. Both the germline stem cell directly attached to the hub cell and the spermatogonial cells detached from the hub cell were divided at the almost perpendicular direction to the spermatogonial cell layers. The dividing phase in one cyst was delayed gradually through the cytoplasmic region of intercellular bridge.
Asunto(s)
Drosophila/ultraestructura , Imagenología Tridimensional , Animales , Masculino , Microscopía Electrónica de Transmisión , Testículo/ultraestructuraRESUMEN
RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.
Asunto(s)
Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas p21(ras) , Microscopía por Crioelectrón , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Membrana Celular , Sistema de Señalización de MAP QuinasasRESUMEN
Many prokaryotic organisms acquire immunity against foreign genetic material by incorporating a short segment of foreign DNA called spacer into chromosomal loci, termed clustered regularly interspaced short palindromic repeats (CRISPRs). The encoded RNAs are processed into small fragments that guide the silencing of the invading genetic elements. The CRISPR-associated (Cas) proteins are the main executioners of these processes. Herein, we report the crystal structure of Stu0660 of Streptococcus thermophilus, a Cas protein involved in the acquisition of new spacers. By homotetramerization, Stu0660 forms a central channel which is decorated with basic amino acids and binds linear double-stranded DNA (dsDNA), but not circular dsDNA. Despite undetectably low sequence similarity, two N-terminal domains of Stu0660 are similar to the entire structure of an Enterococcus faecalis Csn2 protein, which also forms a homotetramer and binds dsDNA. Thus, this work identifies a previously unknown group of Stu0660-like Csn2 proteins (â¼350 residues), which are larger than the known canonical Csn2 proteins (â¼220 residues) by containing an extra C-terminal domain. The commonly present central channel in the two subgroups appears as a design to selectively interact with linear dsDNA.
Asunto(s)
Proteínas Bacterianas/química , Streptococcus thermophilus/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Alineación de Secuencia , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismoRESUMEN
Among various nanoparticles, mesoporous silica nanoparticles (MSNs) have attracted extensive attention for developing efficient drug-delivery systems, mostly due to their high porosity and biocompatibility. However, due to the small pore size, generally below 5 nm in diameter, potential drugs that are loaded into the pore have been limited to small molecules. Herein, a small interfering RNA (siRNA) delivery strategy based on MSNs possessing pores with an average diameter of 23 nm is presented. The siRNA is regarded as a powerful gene therapeutic agent for treatment of a wide range of diseases by enabling post-transcriptional gene silencing, so-called RNA interference. Highly efficient, sequence-specific, and technically very simple target gene knockdown is demonstrated using MSNs with ultralarge pores of size 23 nm in vitro and in vivo without notable cytotoxicity.
Asunto(s)
Nanopartículas/química , ARN Interferente Pequeño/administración & dosificación , Dióxido de Silicio/química , Porosidad , ARN Interferente Pequeño/química , TemperaturaRESUMEN
The condensed version: Thiolated glycol chitosan can form stable nanoparticles with polymerized siRNAs through charge-charge interactions and self-cross-linking (see scheme). This poly-siRNA/glycol chitosan nanoparticles (psi-TGC) provided sufficient inâ vivo stability for systemic delivery of siRNAs. Knockdown of tumor proteins by psi-TGC resulted in a reduction in tumor size and vascularization.
Asunto(s)
Quitosano/química , Nanopartículas/química , Neoplasias/terapia , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Animales , Línea Celular Tumoral , Terapia Genética , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , ARN Interferente Pequeño/genética , Compuestos de Sulfhidrilo/química , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
In Gram-negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid-cell division site. Its structure consists of an anti-MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long alpha-helix and two anti-parallel beta-strands, which mediate formation of a homodimeric alpha/beta structure. Here we report the crystal structure of full-length Helicobacter pylori MinE and redefine its TSD based on that structure. The N-terminal region of the TSD (residues 19-26), previously defined as part of the anti-MinCD domain, forms a beta-strand (betaA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti-parallel betaA-strands. Moreover, we observed serial dimer-dimer interactions within the crystal packing, resulting in the formation of a multimeric structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti-parallel beta-strand interactions.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Ciclo Celular/química , División Celular , Helicobacter pylori/química , Helicobacter pylori/citología , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Alineación de SecuenciaRESUMEN
Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4°C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10weeks at various storage temperatures (-20°C, 4°C, 25°C, and 37°C). During the period of 10weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Nanopartículas/química , Fluorescencia , Liofilización/métodos , Humanos , Microscopía Electrónica de Transmisión , Estabilidad ProteicaRESUMEN
Arabidopsis RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatases regulate stress-responsive gene expression and plant development via the dephosphorylation of serine (Ser) residues of the CTD. Some of these phosphatases (CTD phosphatase-like 1 (CPL1) to CPL3) negatively regulate ABA and stress responses. Here, we isolated AtCPL5, a cDNA encoding a protein containing two CTD phosphatase domains (CPDs). To characterize AtCPL5, we analyzed the gene expression patterns and subcellular protein localization, investigated various phenotypes of AtCPL5-overexpressors and knockout mutants involved in ABA and drought responses, performed microarray and RNA hybridization analyses using AtCPL5-overexpressors, and assessed the CTD phosphatase activities of the purified AtCPL5 and each CPD of the protein. Transcripts of the nucleus-localized AtCPL5 were induced by ABA and drought. AtCPL5-overexpressors exhibited ABA-hypersensitive phenotypes (increased inhibition of seed germination, seedling growth, and stomatal aperture), lower transpiration rates upon dehydration, and enhanced drought tolerance, while the knockout mutants showed weak ABA hyposensitivity. AtCPL5 overexpression changed the expression of numerous genes, including those involved in ABA-mediated responses. In contrast to Ser-5-specific phosphatase activity of the negative stress response regulators, purified AtCPL5 and each CPD of the protein specifically dephosphorylated Ser-2 in RNAPII CTD. We conclude that AtCPL5 is a unique CPL family protein that positively regulates ABA-mediated development and drought responses in Arabidopsis.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Sequías , Fosfoserina/metabolismo , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Clonación Molecular , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Técnicas de Inactivación de Genes , Genes de Plantas , Glucuronidasa/metabolismo , Datos de Secuencia Molecular , Filogenia , Estomas de Plantas/efectos de los fármacos , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Lactic acid bacteria (LAB) have been used for various food fermentations for thousands of years. Recently, LAB are receiving increased attention due to their great potential as probiotics for man and animals, and also as cell factories for producing enzymes, antibodies, vitamins, exopolysaccharides, and various feedstocks. LAB are safe organisms with GRAS (generally recognized as safe) status and possess relatively simple metabolic pathways easily subjected to modifications. However, relatively few studies have been carried out on LAB inhabiting plants compared to dairy LAB. Kimchi is a Korean traditional fermented vegetable, and its fermentation is carried out by LAB inhabiting plant raw materials of kimchi. Kimchi represents a model food with low pH and is fermented at low temperatures and in anaerobic environments. LAB have been adjusting to kimchi environments, and produce various metabolites such as bacteriocins, γ-aminobutyric acid, ornithine, exopolysaccharides, mannitol, etc. as products of metabolic efforts to adjust to the environments. The metabolites also contribute to the known health-promoting effects of kimchi. Due to the recent progress in multi-omics technologies, identification of genes and gene products responsible for the synthesis of functional metabolites becomes easier than before. With the aid of tools of metabolic engineering and synthetic biology, it can be envisioned that LAB strains producing valuable metabolites in large quantities will be constructed and used as starters for foods and probiotics for improving human health. Such LAB strains can also be useful as production hosts for value-added products for food, feed, and pharmaceutical industries. In this review, recent findings on the selected metabolites produced by kimchi LAB are discussed, and the potentials of metabolites will be mentioned.
RESUMEN
Fe65 and Dab1 are adaptor proteins that interact with the cytoplasmic domain of amyloid precursor protein (APP) via phosphotyrosine-binding (PTB) domain and that affect APP processing and Aß production. Co-expression of Dab1 with Fe65 and APP resumed nuclear translocation of Fe65 despite of its cytoplasmic anchor, APP. The decreased amount of Fe65 bound to APP was shown in co-immunoprecipitation assay from the cells with Dab1 which also displayed the effect on APP processing. These data suggested that Fe65 and Dab1 compete for binding to APP. Surprisingly, we found that Fe65 interacts with Dab1 via C-terminal region of Dab1 and unphosphorylated Dab1 is capable of binding Fe65. Dab1 interacts with the low-density lipoprotein receptor-related protein (LRP) as well as APP through its PTB domain. Dab1 significantly decreased the amount of APP bound to LRP and the level of secreted APP and APP-CTF in LRP expressing cells, unlike Fe65. It implies that overexpression of Dab1 diminish LRP-APP complex formation, resulting in altered APP processing. The competition for overlapped binding site among adaptor proteins may be related to the regulation mechanism of APP metabolism in various conditions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Unión Competitiva , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Sitios de Unión , Línea Celular , Regulación de la Expresión Génica , Humanos , Unión ProteicaRESUMEN
The design and synthesis of a novel reduction-sensitive, robust, and biocompatible vesicle (SSCB[6]VC) are reported, which is self-assembled from an amphiphilic cucurbit[6]uril (CB[6]) derivative that contains disulfide bonds between hexaethylene glycol units and a CB[6] core. The remarkable features of SSCB[6]VC include: 1) facile, non-destructive, non-covalent, and modular surface modification using exceptionally strong host-guest chemistry; 2) high structural stability; 3) facile internalization into targeted cells by receptor-mediated endocytosis, and 4) efficient triggered release of entrapped drugs in a reducing environment such as cytoplasm. Furthermore, a significantly increased cytotoxicity of the anticancer drug doxorubicin to cancer cells is demonstrated using doxorubicin-loaded SSCB[6]VC, the surface of which is decorated with functional moieties such as a folate-spermidine conjugate and fluorescein isothiocyanate-spermidine conjugate as targeting ligand and fluorescence imaging probe, respectively. SSCB[6]VC with such unique features can be used as a highly versatile multifunctional platform for targeted drug delivery, which may find useful applications in cancer therapy. This novel strategy based on supramolecular chemistry and the unique properties of CB[6] can be extended to design smart multifunctional materials for biomedical applications including gene delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Liposomas Unilamelares/química , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Citometría de Flujo , Fluoresceína-5-Isotiocianato/química , Ácido Fólico/química , Células HeLa , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Microscopía Confocal , Microscopía Electrónica de Transmisión , Oxidación-Reducción/efectos de los fármacos , Espectrometría de Fluorescencia , Espermidina/química , Propiedades de SuperficieRESUMEN
BACKGROUND: The transcription factor NF-AT plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-AT translocates from the cytoplasm to the nucleus then returns in response to the intracellular calcium level. METHODS: We have investigated NF-AT nucleocytoplasmic shuttling in real-time in living cells using NF-ATc1 tagged with the reversibly photoswitchable fluorescence protein, Dronpa. We monitored both nuclear import and export rate of Dronpa-tagged NF-AT in live cells upon stimulation with ionomycin plus calcium (I+Ca(2+)) or cyclosporin A (CsA). RESULTS: The results show that NF-AT moved into the nucleus within 3-9 min after stimulation and moved back out into the cytoplasm within 15-50 min after CsA addition. In the absence of stimulation, NF-AT stayed in the cytoplasm as in the cells overexpressing GSK-3beta, a calcineurin-opposing regulator. GENERAL SIGNIFICANCE: This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.