RESUMEN
BACKGROUND: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated. RESULTS: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein. CONCLUSION: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels.
Asunto(s)
Aedes , Animales , Flavonoides , Glutatión Transferasa/metabolismo , Humanos , Larva , Control de MosquitosRESUMEN
Cu-containing nitrite reductases (NiRs) are 110 kDa enzymes that play central roles in denitrification. Although the NiRs have been well studied, with over 100 Protein Data Bank entries, such issues as crystal packing, photoreduction, and lack of high pH cases have impeded structural analysis of their catalytic mechanisms. Here we show the cryogenic electron microscopy (cryo-EM) structures of Achromobacter cycloclastes NiR (AcNiR) at pH 6.2 and 8.1. The optimization of 3D-reconstruction parameters achieved 2.99 and 2.85 Å resolution. Comprehensive comparisons with cryo-EM and 56 AcNiR crystal structures suggested crystallographic artifacts in residues 185-215 and His255' due to packing and photoreduction, respectively. We used a newly developed map comparison method to detect structural change around the type 2 Cu site. While the theoretical estimation of coordinate errors of cryo-EM structures remains difficult, combined analysis using X-ray and cryo-EM structures will allow deeper insight into the local structural changes of proteins.
Asunto(s)
Cobre , Nitrito Reductasas , Achromobacter cycloclastes/metabolismo , Catálisis , Cobre/química , Microscopía por Crioelectrón/métodos , Nitrito Reductasas/química , Nitrito Reductasas/metabolismoRESUMEN
Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17ß-estradiol, a DmNobo inhibitor. 17ß-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17ß-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17ß-estradiol-mediated inhibition of DmNobo enzymatic activity, as 17ß-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.
Asunto(s)
Proteínas de Drosophila/química , Estradiol/química , Glutatión Transferasa/química , Aedes , Sustitución de Aminoácidos , Animales , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ecdisteroides/biosíntesis , Ecdisteroides/química , Ecdisteroides/genética , Estradiol/genética , Estradiol/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Mutación con Pérdida de Función , Mutación Missense , Relación Estructura-ActividadRESUMEN
The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.
Asunto(s)
Protones , Proteínas de la Matriz Viral/química , Secuencias de Aminoácidos , Enlace de Hidrógeno , Activación del Canal Iónico , Simulación de Dinámica Molecular , Dominios Proteicos , Electricidad Estática , Temperatura , Proteínas de la Matriz Viral/metabolismoRESUMEN
Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three ß-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner ß-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions.
Asunto(s)
Acinetobacter/química , Adhesinas Bacterianas/química , Sistemas de Secreción Tipo V/química , Acinetobacter/genética , Adhesinas Bacterianas/genética , Cristalografía por Rayos X , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sistemas de Secreción Tipo V/genéticaRESUMEN
TdIF1 was originally identified as a protein that directly binds to terminal deoxynucleotidyltransferase, TdT. Through in vitro selection assays (SELEX), we recently showed that TdIF1 recognizes both AT-tract and a specific DNA sequence motif, 5'-TGCATG-3', and can up-regulate the expression of RAB20 through the latter motif. However, whether TdIF1 binds to these sequences in the cells has not been clear and its other target genes remain to be identified. Here, we determined in vivo TdIF1-binding sequences (TdIF1-invivoBMs) on the human chromosomes through ChIP-seq analyses. The result showed a 160-base pair cassette containing 'AT-tract~palindrome (inverted repeat)~AT-tract' as a likely target sequence of TdIF1. Interestingly, the core sequence of the palindrome in the TdIF1-invivoBMs shares significant similarity to the above 5'-TGCATG-3' motif determined by SELEX in vitro. Furthermore, spacer sequences between AT-tract and the palindrome contain many potential transcription factor binding sites. In luciferase assays, TdIF1 can up-regulate transcription activity of the promoters containing the TdIF1-invivoBM, and this effect is mainly through the palindrome. Clusters of this motif were found in the potential target genes. Gene ontology analysis and RT-qPCR showed the enrichment of some candidate targets of TdIF1 among the genes involved in the regulation of ossification. Potential modes of transcription activation by TdIF1 are discussed.
Asunto(s)
Proteínas Portadoras/química , Proteínas Nucleares/química , Elementos de Respuesta/genética , Factores de Transcripción/química , Sitios de Unión , Proteínas Portadoras/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Mapeo Cromosómico/métodos , Proteínas de Unión al ADN , Humanos , Proteínas Nucleares/metabolismo , Osteogénesis , Factores de Transcripción/metabolismoRESUMEN
Mutations in the gene encoding bilirubin UDP-glucuronosyltransferase (UGT1A1) are known to cause Crigler-Najjar syndrome type II (CN-II). We previously encountered a patient with a nonsense mutation (Q331X) on one allele and with no other mutations in the promoter region or other exons, and proposed that CN-II is inherited as a dominant trait due to the formation of a heterologous subunit structure comprised of the altered UGT1A1 gene product (UGT1A1-p.Q331X) and the intact UGT1A1. Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells. UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively. Intact UGT1A1 was observed as a dimer using atomic force microscopy. Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation. These findings support the idea that UGT1A1-p.Q331X and UGT1A1 form a dimer and provide an increased mechanistic understanding of CN-II.
Asunto(s)
Síndrome de Crigler-Najjar/enzimología , Síndrome de Crigler-Najjar/metabolismo , Retículo Endoplásmico/enzimología , Proteínas Mutantes/metabolismo , Glucuronosiltransferasa/química , Glucuronosiltransferasa/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Fotoblanqueo , Unión Proteica , Transporte de Proteínas , Fracciones Subcelulares/metabolismoRESUMEN
DNA polymerase µ (pol µ) catalyzes nonhomologous end-joining in DNA double-stranded break repair. Pol µ consists of an amino-terminal BRCA1 carboxyl-terminal homology (BRCT) domain and a pol ß-like region, which contains the catalytic site. By DNA cellulose column chromatography, using full-length pol µ and five different deletion mutants, we found that the amino-terminal region has double-stranded DNA (dsDNA)-binding activity. Pol µ without BRCT domain reduces the DNA polymerization activity when compared to full-length pol µ. Observation by atomic force microscopy showed that full-length pol µ binds to the ends and middle part of dsDNA. Pol µ lacking the amino-terminal region or with a mutation within the BRCT domain bound only to DNA ends, whereas the amino-terminal region with the BRCT domain bound to both the ends and the middle part of dsDNA (mpdDNA). Terminal deoxynucleotidyltransferase, which, like pol µ, belongs to the X family DNA polymerases, also bound to mpdDNA through its amino-terminal region.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Polimerizacion , Secuencia de Aminoácidos , Dominio Catalítico , Cromatografía/métodos , Clonación Molecular , ADN/genética , ADN Nucleotidilexotransferasa/genética , ADN Nucleotidilexotransferasa/metabolismo , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Activación Enzimática , Biblioteca de Genes , Genoma Humano , Humanos , Microscopía de Fuerza Atómica , Mutación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Terminal deoxynucleotidyltransferase (TdT) interacting factor 2 (TdIF2) is an acidic protein that binds to TdT. TdIF2 binds to DNA and core histones and contains an acidic-amino acid-rich region in its C-terminus. It has therefore been suggested to function as a histone chaperone within the nucleus. TdIF2 localized within the nucleolus in HEK 293T cells, and its N-terminal (residues 1-234) and C-terminal (residues 606-756) regions were crucial for the nucleolar localization. A chromatin immunoprecipitation (ChIP) assay showed that TdIF2 associated with the promoter of human ribosomal RNA genes (hrDNAP), and an in vitro luciferase assay system showed that it promoted hrDNAP activity. Using the yeast two-hybrid system with TdIF2 as the bait, we isolated the cDNA encoding HIV Tat interactive protein 60 (Tip60), which has histone acetyltransferase (HAT) activity, as a TdIF2-binding protein. TdIF2 bound to Tip60 in vitro and in vivo, inhibited the Tip60 HAT activity in vitro and co-localized with Tip60 within the nucleolus. In addition, TdIF2 promotes upstream binding factor (UBF) acetylation in vivo. Thus, TdIF2 might promote hrDNAP activity by suppressing Tip60's HAT activity and promoting UBF acetylation.
Asunto(s)
Proteínas Portadoras/metabolismo , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , ARN Ribosómico/genética , Animales , Proteínas Portadoras/química , Citoplasma/metabolismo , ADN Nucleotidilexotransferasa/metabolismo , Regulación hacia Abajo , Células HEK293 , Histona Acetiltransferasas/metabolismo , Humanos , Lisina Acetiltransferasa 5 , Proteínas Nucleares/química , Unión Proteica , Transporte de Proteínas , Proteínas de Unión al ARN , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Insect growth regulators (IGRs) can be developed by elucidating the molecular mechanisms of insect-specific biological events. Because insect molting, and metamorphosis are controlled by ecdysteroids, their biosynthetic pathways can serve as targets for IGR development. The glutathione S-transferase Noppera-bo (Nobo), which is conserved in dipteran and lepidopteran species, plays an essential role in ecdysteroid biosynthesis. Our previous study using 17ß-estradiol as a molecular probe revealed that Asp113 of Drosophila melanogaster Nobo (DmNobo) is essential for its biological function. However, to develop IGRs with a greater Nobo inhibitory activity than 17ß-estradiol, further structural information is warranted. Here, we report five novel non-steroidal DmNobo inhibitors. Analysis of crystal structures of complexes revealed that DmNobo binds these inhibitors in an Asp113-independent manner. Among amino acid residues at the substrate-recognition site, conformation of conserved Phe39 was dynamically altered upon inhibitor binding. Therefore, these inhibitors can serve as seed compounds for IGR development.
RESUMEN
Oligomerization of Pr55Gag is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55Gag. However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55Gag to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles' membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain's 2D layers during assembly and budding.
Asunto(s)
Membrana Celular/metabolismo , Infecciones por VIH/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Ensamble de VirusRESUMEN
We isolated human cDNA clone encoding Bood POZ containing gene type 2 (BPOZ-2) as a gene with a product that binds to TdT interacting factor 1 (TdIF1) using a yeast two-hybrid system. BPOZ-2 is an adaptor for E3 ligase CUL3 and participates in developmental processes. The binding between BPOZ-2 and TdIF1 was confirmed by GST pull-down and immunoprecipitation assays using specific antibodies against BPOZ-2 and TdIF1 in vitro and in vivo. Although when BPOZ-2 solely was expressed in COS7 cells, BPOZ-2 was observed mainly within the cytoplasm, co-transfection of pEGFP-BPOZ-2 and pDsRed-TdIF1 into COS7 cells resulted in co-localization of EGFP-BPOZ-2 and DsRed-TdIF1 within the nucleus. TdIF1 may recruit BPOZ-2 into the nucleus from the cytoplasm by directly binding to BPOZ-2. BPOZ-2 enhanced TdT ubiquitylation when TdIF1 was expressed together with BPOZ-2 in 293T cells, strongly suggesting that the recruitment of BPOZ-2 into the nucleus from the cytoplasm is significant for the TdT ubiquitylation within the nucleus.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN Nucleotidilexotransferasa/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/genética , Chlorocebus aethiops , ADN Nucleotidilexotransferasa/genética , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Proteínas Nucleares/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción , Técnicas del Sistema de Dos Híbridos , UbiquitinaciónRESUMEN
Bood POZ containing gene type 2 (BPOZ-2), which contains ankyrin repeats, NLS, BTB/POZ domains and LXXLL motifs, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. We isolated a cDNA encoding eukaryotic elongation factor 1A1 (eEF1A1) as a BPOZ-2 binding protein by screening a human thymus cDNA library using a yeast two-hybrid system. eEF1A1 is essential for translation and is also involved in the 26S proteasome-dependent degradation of misfolded or unfolded proteins. The binding between BPOZ-2 and eEF1A1 was confirmed by pull-down and immunoprecipitation assays in vitro and in vivo, respectively. BPOZ-2 binds to eEF1A1 through the ankyrin repeats and both BTB/POZ domains in BPOZ-2 and Domains I and III in eEF1A1. BPOZ-2 and eEF1A1 over-expressed in HEK 293T cells co-localized as speckles within the cytoplasm. BPOZ-2 promoted eEF1A1 ubiquitylation and degradation, suggesting that eEF1A1 is a substrate of BPOZ-2. BPOZ-2 inhibited GTP binding to eEF1A1 and prevented translation in in vitro translation assay using rabbit reticulocytes.
Asunto(s)
Factor 1 de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Proteínas Represoras/metabolismo , Animales , Células COS , Bovinos , Línea Celular , Sistema Libre de Células , Chlorocebus aethiops , Humanos , Mutagénesis Sitio-Dirigida , Factor 1 de Elongación Peptídica/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Timo/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , UbiquitinaciónRESUMEN
Bood POZ containing gene type 2 (BPOZ-2) is involved in the growth suppressive effect of the phosphatase and tensin homologue (PTEN). We showed that BPOZ-2 is a human counterpart of yeast Btb3p, which is a putative adaptor for Pcu3p-based ubiquitin ligase. BPOZ-2 bound to E3 ligase CUL3 in vitro and in vivo. BPOZ-2 itself was ubiquitinated through the CUL3-based E3 ligase mainly within the nucleus and degraded by the 26S proteasome. Although BPOZ-2 was mainly expressed within the cytoplasm, it accumulated within the nucleus in the presence of the specific 26S proteasome inhibitor MG132. BPOZ-2 may be recruited to the nucleus from the cytoplasm. Terminal deoxynucleotidyltransferase (TdT) was detected as a BPOZ-2-binding protein using a yeast two-hybrid system by screening a human thymus cDNA library. TdT, BPOZ-2, and CUL3 formed a ternary complex in vivo. TdT was ubiquitinated only within the nucleus and degraded by the 26S proteasome. The ubiqutination or degradation of TdT was markedly promoted by co-expression of BPOZ-2 and CUL3 or BPOZ-2 in 293T cells, respectively.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Proteínas Represoras/metabolismo , Línea Celular , Proteínas Cullin/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Schizosaccharomyces pombe , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.
RESUMEN
AIM: Bilirubin, a final degradation product of heme produced mainly in the spleen, is carried to the liver through its binding to albumin in the blood circulation. After its transport to hepatocytes, ligandin (glutathione S-transferase; GST) carries bilirubin to the endoplasmic reticulum (ER). uridine 5'-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) glucuronidates bilirubin for solubilization in the ER. METHODS: By GST pull-down and co-immunoprecipitation assays, GSTA2, a member of the alpha-class of GST, was observed to directly bind to UGT1A1 through the region present inside the ER. RESULTS: GSTA2 was detected in the microsomal fraction together with the cytosolic fraction after hepatocyte fractionation. CONCLUSION: These results strongly suggest that bilirubin is directly delivered to UGT1A1 from ligandin for glucuronidation.
RESUMEN
We have constructed a novel, nonhomologous end-joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generated a double-strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs. DNA sequence analysis of the DSB junctions indicated that the repair was carried out mainly by using the closest homology sequence. Use of the NAV yielded rapid results within 3 days after transfection. We then used the NAV to analyse NHEJ in cells overexpressing terminal deoxynucleotidyltransferase (TdT). The results indicated that TdT suppresses DNA repair that is based on short (one- or two-base) homology regions, to efficiently add deoxynucleotides during VDJ recombination in lymphoid cells.
RESUMEN
TdIF1 was originally identified as a protein that directly binds to DNA polymerase TdT. TdIF1 is also thought to function in transcription regulation, because it binds directly to the transcriptional factor TReP-132, and to histone deacetylases HDAC1 and HDAC2. Here we show that TdIF1 recognizes a specific DNA sequence and regulates gene transcription. By constructing TdIF1 mutants, we identify amino acid residues essential for its interaction with DNA. An in vitro DNA selection assay, SELEX, reveals that TdIF1 preferentially binds to the sequence 5'-GNTGCATG-3' following an AT-tract, through its Helix-Turn-Helix and AT-hook motifs. We show that four repeats of this recognition sequence allow TdIF1 to regulate gene transcription in a plasmid-based luciferase reporter assay. We demonstrate that TdIF1 associates with the RAB20 promoter, and RAB20 gene transcription is reduced in TdIF1-knocked-down cells, suggesting that TdIF1 stimulates RAB20 gene transcription.
Asunto(s)
Secuencias AT-Hook/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , ADN/metabolismo , Secuencias Hélice-Giro-Hélice/fisiología , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Proteínas de Unión al GTP rab/genética , Secuencia de Bases , Sitios de Unión/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Especificidad por Sustrato/genética , Factores de Transcripción , Transcripción Genética/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system in vitro. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Ubiquitinación/genética , Animales , Bovinos , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , ADN Nucleotidilexotransferasa/genética , Biblioteca de Genes , Células HeLa , Humanos , Hígado/citología , Hígado/metabolismo , Plásmidos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Timo/citología , Timo/metabolismo , Transfección , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
TdT interacting factor 1 (TdIF1) was identified as a protein that binds to terminal deoxynucleotidyltransferase (TdT) to negatively regulate TdT activity. TdT is a template-independent DNA polymerase that catalyzes the incorporation of deoxynucleotides to the 3'-hydroxyl end of DNA templates to increase the junctional diversity of immunoglobulin or T-cell receptor (TcR) genes. Here, using bioinformatics analysis, we identified the TdT binding, DNA binding and dimerization regions, and nuclear localization signal (NLS) in TdIF1. TdIF1 bound to double-stranded DNA (dsDNA) through three DNA binding regions: residues 1-75, the AT-hook-like motif (ALM) and the predicted helix-turn-helix (HTH) motif. ALM in TdIF1 preferentially bound to AT-rich DNA regions. NLS was of the bipartite type and overlapped ALM. TdIF1 bound to the Pol beta-like region in TdT and blocked TdT access to DNA ends. In the presence of dsDNA, however, TdIF1 bound to dsDNA to release TdT from the TdIF1/TdT complex and to exhibit TdT activity, implying that active TdT released microenvironmentally concentrates around AT-rich DNA to synthesize DNA.