RESUMO
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.
Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Polimerase II/genética , UbiquitinaçãoRESUMO
Identifying specific markers of adipose stem and progenitor cells (ASPCs) in vivo is crucial for understanding the biology of white adipose tissues (WAT). PDGFRα-positive perivascular stromal cells represent the best candidates for ASPCs. This cell lineage differentiates into myofibroblasts that contribute to the impairment of WAT function. However, ASPC marker protein(s) that are functionally crucial for maintaining WAT homeostasis are unknown. We previously identified Meflin as a marker of mesenchymal stem cells (MSCs) in bone marrow and tissue-resident perivascular fibroblasts in various tissues. We also demonstrated that Meflin maintains the undifferentiated status of MSCs/fibroblasts. Here, we show that Meflin is expressed in WAT ASPCs. A lineage-tracing experiment showed that Meflin+ ASPCs proliferate in the WAT of obese mice induced by a high-fat diet (HFD), while some of them differentiate into myofibroblasts or mature adipocytes. Meflin knockout mice fed an HFD exhibited a significant fibrotic response as well as increases in adipocyte cell size and the number of crown-like structures in WAT, accompanied by impaired glucose tolerance. These data suggested that Meflin expressed by ASPCs may have a role in reducing disease progression associated with WAT dysfunction.
RESUMO
Gibberellins (GAs) are plant hormones with a tetracyclic diterpenoid structure that are involved in various important developmental processes. Two GA-deficient mutants were isolated: a semidwarf mutant "sd1", which was found to have a defective GA20ox2 gene and was introduced to the world in a green revolution cultivar, and a severe dwarf allele of "d18", with a defective GA3ox2 gene. Based on the phenotypic similarity of d18, rice dwarf mutants were screened, further classifying them into GA-sensitive and GA-insensitive by applying exogenous GA3. Finally, GA-deficient rice mutants at 6 different loci and 3 GA signaling mutants (gid1, gid2, and slr1) were isolated. The GID1 gene encodes a GA nuclear receptor, and the GID1-DELLA (SLR1) system for GA perception is widely used in vascular plants. The structural characteristics of GID1 and GA metabolic enzymes have also been reviewed.
Assuntos
Giberelinas , Oryza , Giberelinas/metabolismo , Proteínas de Plantas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais/genética , Oryza/genética , Oryza/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Elucidation of the genetic control of rice architecture is crucial due to the global demand for high crop yields. Rice architecture is a complex trait affected by plant height, tillering, and panicle morphology. In this study, principal component analysis (PCA) on 8 typical traits related to plant architecture revealed that the first principal component (PC), PC1, provided the most information on traits that determine rice architecture. A genome-wide association study (GWAS) using PC1 as a dependent variable was used to isolate a gene encoding rice, SPINDLY (OsSPY), that activates the gibberellin (GA) signal suppression protein SLR1. The effect of GA signaling on the regulation of rice architecture was confirmed in 9 types of isogenic plant having different levels of GA responsiveness. Further population genetics analysis demonstrated that the functional allele of OsSPY associated with semidwarfism and small panicles was selected in the process of rice breeding. In summary, the use of PCA in GWAS will aid in uncovering genes involved in traits with complex characteristics.
Assuntos
Oryza/genética , Genes de Plantas/genética , Estudo de Associação Genômica Ampla/métodos , Giberelinas/metabolismo , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Componente Principal/métodos , Locos de Características Quantitativas/genéticaRESUMO
The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Evolução Molecular , Filogenia , Receptores de Superfície Celular/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Receptores de Superfície Celular/metabolismo , Especificidade da EspécieRESUMO
Translocation and long-distance transport of phytohormones are considered important processes for phytohormone responses, as well as their synthesis and signaling. Here, we report on the dual function of OsSWEET3a, a bidirectional sugar transporter from clade I of the rice SWEET family of proteins, as both a gibberellin (GA) and a glucose transporter. OsSWEET3a efficiently transports GAs in the C13-hydroxylation pathway of GA biosynthesis. Both knockout and overexpression lines of OsSWEET3a showed defects in germination and early shoot development, which were partially restored by GA, especially GA20. Quantitative reverse transcription PCR, GUS staining and in situ hybridization revealed that OsSWEET3a was expressed in vascular bundles in basal parts of the seedlings. OsSWEET3a expression was co-localized with OsGA20ox1 expression in the vascular bundles but not with OsGA3ox2, whose expression was restricted to leaf primordia and young leaves. These results suggest that OsSWEET3a is expressed in the vascular tissue of basal parts of seedlings and is involved in the transport of both GA20 and glucose to young leaves, where GA20 is possibly converted to the bioactive GA1 form by OsGA3ox2, during early plant development. We also indicated that such GA transport activities of SWEET proteins have sporadically appeared in the evolution of plants: GA transporters in Arabidopsis have evolved from sucrose transporters, while those in rice and sorghum have evolved from glucose transporters.
Assuntos
Giberelinas/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/fisiologia , Oryza/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Brotos de Planta/crescimento & desenvolvimento , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Oryza/metabolismo , Oryza/fisiologia , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/metabolismo , Brotos de Planta/metabolismo , Brotos de Planta/fisiologia , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Plântula/fisiologiaRESUMO
Metabolic syndrome is a common metabolic disorder that involves multiple organs and is predominantly influenced by obesity, especially the accumulation of visceral fat. It is also known that macrophages that infiltrate obese adipose tissue play an important role in inflammation of the adipose tissue. Macrophage-inducible C-type lectin (Mincle), a new inflammatory regulator found in obese adipose tissue, is expressed in pro-inflammatory M1 macrophages in adipose tissue. In addition, Mincle is expressed in macrophages that form a crown-like structure, where dead or dying adipocytes are surrounded by pro-inflammatory M1 macrophages; within this crown-like structure, adipocyte-macrophage crosstalk may occur in close proximity. Although there is no significant difference in body weight between Mincle-deficient and wild-type mice under high-fat diet, the epididymal fat weight is significantly higher and the liver weight is significantly lower in Mincle-deficient mice than those in wild-type mice. It has been shown that adipose tissue inflammation and fibrosis are attenuated in Mincle-deficient mice when compared with wild-type mice. In addition, Mincle-deficient mice have reduced hepatic lipid accumulation and better glucose metabolism. These results suggest that Mincle signaling in adipose tissue macrophages activates adipose tissue fibroblasts, which leads to adipose tissue fibrosis.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Inflamação/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Síndrome Metabólica/genética , Obesidade/metabolismo , Receptores Imunológicos/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Animais , Fibrose , Humanos , Inflamação/patologia , Lectinas Tipo C/genética , Fígado/metabolismo , Fígado/patologia , Macrófagos/patologia , Proteínas de Membrana/genética , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Obesidade/patologia , Transdução de SinaisRESUMO
BACKGROUND: Patients presenting with edema, skin redness, pain, and itching in their lower legs are common and encountered often in daily practice. However, although commonly recognized diseases such as deep venous thrombosis, stasis dermatitis due to varicose veins, lymphedema, and cellulitis are diagnosed correctly in most cases, micro-arteriovenous fistulas (AVFs) may often be overlooked due to low awareness and rarity. This study was carried out to evaluate the prevalence of micro-AVF in patients presenting with foot skin symptoms. METHODS: A total of 134 patients (184 limbs) visited the Department of Vascular Surgery at Kesennuma City Hospital with edema, skin redness, pain, and itching in their lower legs from January to September 2017 and were enrolled and followed up until November 2017. All patients received ultrasonic inspection of their symptomatic limb, and a blood test (white blood cell count, C-reactive protein, and d-dimer) was performed if needed. When micro-AVF was detected in one limb, the other limb was routinely inspected by ultrasonography. A computed tomography scan was performed with the patient's consent. Patients diagnosed with micro-AVF started compression therapy immediately and were followed up for at least 2 months. A surgical procedure was considered if the symptoms worsened. RESULTS: Micro-AVFs were detected in 24 limbs (13%, 24/184) of 14 patients (7 males and 7 females; age 70 ± 11.7 years). Four patients had unilateral skin symptoms with unilateral micro-AVFs and 7 patients had unilateral skin symptoms and bilateral micro-AVFs. Three patients had bilateral skin symptoms and bilateral micro-AVFs. Asymptomatic micro-AVFs were detected in 7 limbs. Subjective symptoms disappeared and skin appearance normalized in 14 limbs of 12 patients during the first 2 months with compression therapy only. Compression therapy was not effective in 3 limbs of 2 patients and they underwent vein ligation surgery. None of the patients had a surgical history or history of trauma in their lower legs. CONCLUSIONS: Among the lower legs presenting with skin symptoms, we detected micro-AVFs in 13% of limbs; therefore, micro-AVF of the lower leg is not as rare as previously thought. In addition, 10 of 14 patients (71%) had micro-AVFs of the lower leg bilaterally.
Assuntos
Fístula Arteriovenosa/epidemiologia , Extremidade Inferior/irrigação sanguínea , Adulto , Idoso , Idoso de 80 Anos ou mais , Fístula Arteriovenosa/diagnóstico por imagem , Fístula Arteriovenosa/fisiopatologia , Fístula Arteriovenosa/terapia , Angiografia por Tomografia Computadorizada , Edema/epidemiologia , Eritema/epidemiologia , Feminino , Humanos , Incidência , Japão/epidemiologia , Masculino , Microcirculação , Pessoa de Meia-Idade , Dor/epidemiologia , Prevalência , Prognóstico , Prurido/epidemiologia , Fluxo Sanguíneo Regional , Estudos Retrospectivos , Fatores de Tempo , UltrassonografiaRESUMO
Strigolactones (SLs) are a class of plant hormones that regulate diverse physiological processes, including shoot branching and root development. They also act as rhizosphere signaling molecules to stimulate the germination of root parasitic weeds and the branching of arbuscular mycorrhizal fungi. Although various types of cross talk between SLs and other hormones have been reported in physiological analyses, the cross talk between gibberellin (GA) and SLs is poorly understood. We screened for chemicals that regulate the level of SLs in rice (Oryza sativa) and identified GA as, to our knowledge, a novel SL-regulating molecule. The regulation of SL biosynthesis by GA is dependent on the GA receptor GID1 and F-box protein GID2. GA treatment also reduced the infection of rice plants by the parasitic plant witchers weed (Striga hermonthica). These data not only demonstrate, to our knowledge, the novel plant hormone cross talk between SL and GA, but also suggest that GA can be used to control parasitic weed infections.
Assuntos
Giberelinas/metabolismo , Lactonas/metabolismo , Transdução de Sinais , Genes de Plantas , Germinação/efeitos dos fármacos , Mutação/genética , Oryza/genética , Oryza/metabolismo , Oryza/parasitologia , Doenças das Plantas/parasitologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/parasitologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Striga/fisiologiaRESUMO
Previously, we found 123 transcription factors (TFs) as candidate regulators of secondary cell wall (SCW) formation in rice by using phylogenetic and co-expression network analyses. Among them, we examined in this work the role of OsIDD2, a zinc finger and indeterminate domain (IDD) family TF. Its overexpressors showed dwarfism, fragile leaves, and decreased lignin content, which are typical phenotypes of plants defective in SCW formation, whereas its knockout plants showed slightly increased lignin content. The RNA-seq and quantitative reverse transcription polymerase chain reaction analyses confirmed that some lignin biosynthetic genes were downregulated in the OsIDD2-overexpressing plants, and revealed the same case for other genes involved in cellulose synthesis and sucrose metabolism. The transient expression assay using rice protoplasts revealed that OsIDD2 negatively regulates the transcription of genes involved in lignin biosynthesis, cinnamyl alcohol dehydrogenase 2 and 3 (CAD2 and 3), and sucrose metabolism, sucrose synthase 5 (SUS5), whereas an AlphaScreen assay, which can detect the interaction between TFs and their target DNA sequences, directly confirmed the interaction between OsIDD2 and the target sequences located in the promoter regions of CAD2 and CAD3. Based on these observations, we conclude that OsIDD2 is negatively involved in SCW formation and other biological events by downregulating its target genes.
Assuntos
Parede Celular/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Dedos de Zinco , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , Células do Mesofilo/metabolismo , Oryza/genética , Fenótipo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Protoplastos/metabolismo , Interferência de RNA , Transcrição GênicaRESUMO
Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.
Assuntos
Antígenos de Superfície/imunologia , Inflamação/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos CD/imunologia , Antígenos de Superfície/sangue , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Dexametasona/farmacologia , Feminino , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
DELLA protein is a key negative regulator of gibberellin (GA) signaling. Although how DELLA regulates downstream gene expression remains unclear, DELLA has been proposed to function as a transcriptional activator. However, because DELLA lacks a DNA-binding domain, intermediate protein(s) mediating the DELLA/DNA interaction are believed to be necessary for activating DELLA target genes. Here, using yeast hybrid screenings, we identified five members of indeterminate domain (IDD) protein family which bind physically to both DELLA and the promoter sequence of the GA-positive regulator SCARECROW-LIKE 3 (SCL3), which previously was characterized as a DELLA direct target gene. Transient assays using Arabidopsis protoplasts demonstrated that a luciferase reporter controlled by the SCL3 promoter was additively transactivated by REPRESSOR of ga1-3 (RGA) and IDDs. Phenotypic analysis of transgenic plants expressing AtIDD3 (one of the 16 IDDs in the Arabidopsis genome) fused with the plant-specific repression domain (SRDX) supported the possibility that AtIDD3 is positively involved in GA signaling. In addition, we found that SCL3 protein also interacts with IDDs, resulting in the suppression of its target gene expression. In this context, DELLA and SCL3 interact competitively with IDD proteins to regulate downstream gene expression. These results suggest that the coregulators DELLA and SCL3, using IDDs as transcriptional scaffolds for DNA binding, antagonistically regulate the expression of their downstream targets to control the GA signaling pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Proteínas de Arabidopsis/genética , Proteínas Correpressoras/genética , Primers do DNA , Regulação da Expressão Gênica de Plantas/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells.
Assuntos
Endosperma/citologia , Endosperma/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Oryza/embriologia , Proteínas de Plantas/metabolismo , Biolística , Análise por Conglomerados , Simulação por Computador , Regulação para Baixo/genética , Genes de Plantas , Modelos Biológicos , Mutação/genética , Oryza/genética , Proteínas de Plantas/genética , Transdução de Sinais/genética , Transcriptoma/genética , alfa-Amilases/genética , alfa-Amilases/metabolismoRESUMO
The organ size of flowering plants is determined by two post-embryonic developmental events: cell proliferation and cell expansion. In this study, we identified a new rice loss-of-function mutant, small organ size1 (smos1), that decreases the final size of various organs due to decreased cell size and abnormal microtubule orientation. SMOS1 encodes an unusual APETALA2 (AP2)-type transcription factor with an imperfect AP2 domain, and its product belongs to the basal AINTEGUMENTA (ANT) lineage, including WRINKLED1 (WRI1) and ADAP. SMOS1 expression was induced by exogenous auxin treatment, and the auxin response element (AuxRE) of the SMOS1 promoter acts as a cis-motif through interaction with auxin response factor (ARF). Furthermore, a functional fluorophore-tagged SMOS1 was localized to the nucleus, supporting the role of SMOS1 as a transcriptional regulator for organ size control. Microarray analysis showed that the smos1 mutation represses expression of several genes involved in microtubule-based movement and DNA replication. Among the down-regulated genes, we demonstrated by gel-shift and chromatin immunoprecipitation (ChIP) experiments that OsPHI-1, which is involved in cell expansion, is a target of SMOS1. SMOS1 homologs in early-diverged land plants partially rescued the smos1 phenotype of rice. We propose that SMOS1 acts as an auxin-dependent regulator for cell expansion during organ size control, and that its function is conserved among land plants.
Assuntos
Ácidos Indolacéticos/farmacologia , Proteínas de Plantas/genética , Transdução de Sinais , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Contagem de Células , Tamanho Celular , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células do Mesofilo/citologia , Células do Mesofilo/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Oryza/citologia , Oryza/genética , Oryza/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in Escherichia coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.
Assuntos
Oryza/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fragmentos de Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TripsinaRESUMO
Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. A nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). Here we analyse the crystal structure of Oryza sativa GID1 (OsGID1) bound with GA(4) and GA(3) at 1.9 A resolution. The overall structure of both complexes shows an alpha/beta-hydrolase fold similar to that of HSLs except for an amino-terminal lid. The GA-binding pocket corresponds to the substrate-binding site of HSLs. On the basis of the OsGID1 structure, we mutagenized important residues for GA binding and examined their binding activities. Almost all of them showed very little or no activity, confirming that the residues revealed by structural analysis are important for GA binding. The replacement of Ile 133 with Leu or Val-residues corresponding to those of the lycophyte Selaginella moellendorffii GID1s-caused an increase in the binding affinity for GA(34), a 2beta-hydroxylated GA(4). These observations indicate that GID1 originated from HSL and was further modified to have higher affinity and more strict selectivity for bioactive GAs by adapting the amino acids involved in GA binding in the course of plant evolution.
Assuntos
Giberelinas/química , Giberelinas/metabolismo , Oryza/química , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Hidrolases/química , Hidrolases/metabolismo , Hidroxilação , Modelos Moleculares , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
Obesity is often associated with aging. However, the mechanism of age-related obesity is unknown. The melanocortin-4 receptor (MC4R) mediates leptin-melanocortin anti-obesity signaling in the hypothalamus. Here, we discovered that MC4R-bearing primary cilia of hypothalamic neurons progressively shorten with age in rats, correlating with age-dependent metabolic decline and increased adiposity. This "age-related ciliopathy" is promoted by overnutrition-induced upregulation of leptin-melanocortin signaling and inhibited or reversed by dietary restriction or the knockdown of ciliogenesis-associated kinase 1 (CILK1). Forced shortening of MC4R-bearing cilia in hypothalamic neurons by genetic approaches impaired neuronal sensitivity to melanocortin and resulted in decreased brown fat thermogenesis and energy expenditure and increased appetite, finally developing obesity and leptin resistance. Therefore, despite its acute anti-obesity effect, chronic leptin-melanocortin signaling increases susceptibility to obesity by promoting the age-related shortening of MC4R-bearing cilia. This study provides a crucial mechanism for age-related obesity, which increases the risk of metabolic syndrome.
Assuntos
Cílios , Leptina , Neurônios , Obesidade , Receptor Tipo 4 de Melanocortina , Animais , Receptor Tipo 4 de Melanocortina/metabolismo , Receptor Tipo 4 de Melanocortina/genética , Cílios/metabolismo , Cílios/patologia , Obesidade/metabolismo , Obesidade/patologia , Neurônios/metabolismo , Neurônios/patologia , Leptina/metabolismo , Ratos , Masculino , Transdução de Sinais , Hipotálamo/metabolismo , Envelhecimento/metabolismo , Envelhecimento/patologia , Ratos Sprague-Dawley , Camundongos , Metabolismo Energético , Tecido Adiposo Marrom/metabolismo , TermogêneseRESUMO
When the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF 1 (GID1) binds to GA, GID1 interacts with DELLA proteins, repressors of GA signaling. This interaction inhibits the suppressive function of DELLA protein and thereby activates the GA response. However, how DELLA proteins exert their suppressive function and how GID1s inhibit suppressive function of DELLA proteins is unclear. By yeast one-hybrid experiments and transient expression of the N-terminal region of rice DELLA protein (SLR1) in rice callus, we established that the N-terminal DELLA/TVHYNP motif of SLR1 possesses transactivation activity. When SLR1 proteins with various deletions were over-expressed in rice, the severity of dwarfism correlated with the transactivation activity observed in yeast, indicating that SLR1 suppresses plant growth through transactivation activity. This activity was suppressed by the GA-dependent GID1-SLR1 interaction, which may explain why GA responses are induced in the presence of GA. The C-terminal GRAS domain of SLR1 also exhibits a suppressive function on plant growth, possibly by directly or indirectly interacting with the promoter region of target genes. Our results indicate that the N-terminal region of SLR1 has two roles in GA signaling: interaction with GID1 and transactivation activity.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Giberelinas/metabolismo , Oryza/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Ativação Transcricional/genética , Motivos de Aminoácidos , Modelos Moleculares , Mutação , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/crescimento & desenvolvimento , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Transdução de Sinais/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
To investigate gibberellin (GA) signaling using the rice (Oryza sativa) GA receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) mutant gid1-8, we isolated a suppressor mutant, Suppressor of gid1-1 (Sgd-1). Sgd-1 is an intragenic mutant containing the original gid1-8 mutation (L45F) and an additional amino acid substitution (P99S) in the loop region. GID1(P99S) interacts with the rice DELLA protein SLENDER RICE1 (SLR1), even in the absence of GA. Substitution of the 99th Pro with other amino acids revealed that substitution with Ala (P99A) caused the highest level of GA-independent interaction. Physicochemical analysis using surface plasmon resonance revealed that GID1(P99A) has smaller K(a) (association) and K(d) (dissociation) values for GA(4) than does wild-type GID1. This suggests that the GID1(P99A) lid is at least partially closed, resulting in both GA-independent and GA-hypersensitive interactions with SLR1. One of the three Arabidopsis thaliana GID1s, At GID1b, can also interact with DELLA proteins in the absence of GA, so we investigated whether GA-independent interaction of At GID1b depends on a mechanism similar to that of rice GID1(P99A). Substitution of the loop region or a few amino acids of At GID1b with those of At GID1a diminished its GA-independent interaction with GAI while maintaining the GA-dependent interaction. Soybean (Glycine max) and Brassica napus also have GID1s similar to At GID1b, indicating that these unique GID1s occur in various dicots and may have important functions in these plants.
Assuntos
Giberelinas/metabolismo , Oryza/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Giberelinas/genética , Dados de Sequência Molecular , Mutação , Oryza/anatomia & histologia , Oryza/genética , Filogenia , Reguladores de Crescimento de Plantas/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/anatomia & histologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1(G576V)). The GA-dependent degradation of SLR1(G576V) was reduced in Slr1-d4, and compared with SLR1, SLR1(G576V) showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1(G576V) interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.