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1.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445335

RESUMO

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK-Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.


Assuntos
Compostos de Amônio/metabolismo , Ácidos Cetoglutáricos/metabolismo , Methanococcales/metabolismo , Proteínas PII Reguladoras de Nitrogênio , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Transporte de Íons , Redes e Vias Metabólicas , Modelos Moleculares , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/química , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Análise de Sequência de Proteína
2.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445356

RESUMO

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.


Assuntos
Ferritinas/genética , Poríferos/genética , Animais , Sequência Conservada , Ferritinas/química , Ferritinas/metabolismo , Ferro/metabolismo , Redes e Vias Metabólicas/genética , Modelos Moleculares , Filogenia , Poríferos/classificação , Poríferos/metabolismo , Domínios Proteicos/genética , Análise de Sequência de DNA , Transcriptoma/fisiologia
3.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
4.
BMC Plant Biol ; 21(1): 376, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399701

RESUMO

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.


Assuntos
Genoma de Planta , Manihot/enzimologia , Manihot/genética , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Cloroplastos/enzimologia , Mapeamento Cromossômico , Cromossomos de Plantas , Sequência Conservada , Citoplasma/enzimologia , Éxons , Flores/enzimologia , Íntrons , Família Multigênica , Oxigênio/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Transcriptoma
5.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360976

RESUMO

The Eph receptor tyrosine kinase member EphB6 is a pseudokinase, and similar to other pseudoenzymes has not attracted an equivalent amount of interest as its enzymatically-active counterparts. However, a greater appreciation for the role pseudoenzymes perform in expanding the repertoire of signals generated by signal transduction systems has fostered more interest in the field. EphB6 acts as a molecular switch that is capable of modulating the signal transduction output of Eph receptor clusters. Although the biological effects of EphB6 activity are well defined, the molecular mechanisms of EphB6 function remain enigmatic. In this review, we use a comparative approach to postulate how EphB6 acts as a scaffold to recruit adaptor proteins to an Eph receptor cluster and how this function is regulated. We suggest that the evolutionary repurposing of EphB6 into a kinase-independent molecular switch in mammals has involved repurposing the kinase activation loop into an SH3 domain-binding site. In addition, we suggest that EphB6 employs the same SAM domain linker and juxtamembrane domain allosteric regulatory mechanisms that are used in kinase-positive Eph receptors to regulate its scaffold function. As a result, although kinase-dead, EphB6 remains a strategically active component of Eph receptor signaling.


Assuntos
Receptor EphB6/metabolismo , Transdução de Sinais , Regulação Alostérica , Animais , Sequência Conservada , Humanos , Receptor EphB6/química , Receptor EphB6/genética , Domínios de Homologia de src
6.
BMC Plant Biol ; 21(1): 366, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380425

RESUMO

BACKGROUND: Small RNAs are short non-coding RNAs that are key gene regulators controlling various biological processes in eukaryotes. Plants may regulate discrete sets of sRNAs in response to pathogen attack. Sclerotinia sclerotiorum is an economically important pathogen affecting hundreds of plant species, including the economically important oilseed B. napus. However, there are limited studies on how regulation of sRNAs occurs in the S. sclerotiorum and B. napus pathosystem. RESULTS: We identified different classes of sRNAs from B. napus using high throughput sequencing of replicated mock and infected samples at 24 h post-inoculation (HPI). Overall, 3999 sRNA loci were highly expressed, of which 730 were significantly upregulated during infection. These 730 up-regulated sRNAs targeted 64 genes, including disease resistance proteins and transcriptional regulators. A total of 73 conserved miRNA families were identified in our dataset. Degradome sequencing identified 2124 cleaved mRNA products from these miRNAs from combined mock and infected samples. Among these, 50 genes were specific to infection. Altogether, 20 conserved miRNAs were differentially expressed and 8 transcripts were cleaved by the differentially expressed miRNAs miR159, miR5139, and miR390, suggesting they may have a role in the S. sclerotiorum response. A miR1885-triggered disease resistance gene-derived secondary sRNA locus was also identified and verified with degradome sequencing. We also found further evidence for silencing of a plant immunity related ethylene response factor gene by a novel sRNA using 5'-RACE and RT-qPCR. CONCLUSIONS: The findings in this study expand the framework for understanding the molecular mechanisms of the S. sclerotiorum and B. napus pathosystem at the sRNA level.


Assuntos
Ascomicetos/fisiologia , Brassica napus/genética , Brassica napus/microbiologia , Doenças das Plantas/microbiologia , RNA de Plantas , Pequeno RNA não Traduzido , Sequência Conservada , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Análise de Sequência de RNA , Regulação para Cima
7.
FASEB J ; 35(8): e21681, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196428

RESUMO

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.


Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
8.
MAbs ; 13(1): 1953683, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34313527

RESUMO

The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in widespread social and economic disruption. Effective interventions are urgently needed for the prevention and treatment of COVID-19. Neutralizing monoclonal antibodies (mAbs) have demonstrated their prophylactic and therapeutic efficacy against SARS-CoV-2, and several have been granted authorization for emergency use. Here, we discover and characterize a fully human cross-reactive mAb, MW06, which binds to both SARS-CoV-2 and SARS-CoV spike receptor-binding domain (RBD) and disrupts their interaction with angiotensin-converting enzyme 2 (ACE2) receptors. Potential neutralization activity of MW06 was observed against both SARS-CoV-2 and SARS-CoV in different assays. The complex structure determination and epitope alignment of SARS-CoV-2 RBD/MW06 revealed that the epitope recognized by MW06 is highly conserved among SARS-related coronavirus strains, indicating the potential broad neutralization activity of MW06. In in vitro assays, no antibody-dependent enhancement (ADE) of SARS-CoV-2 infection was observed for MW06. In addition, MW06 recognizes a different epitope from MW05, which shows high neutralization activity and has been in a Phase 2 clinical trial, supporting the development of the cocktail of MW05 and MW06 to prevent against future escaping variants. MW06 alone and the cocktail show good effects in preventing escape mutations, including a series of variants of concern, B.1.1.7, P.1, B.1.351, and B.1.617.1. These findings suggest that MW06 recognizes a conserved epitope on SARS-CoV-2, which provides insights for the development of a universal antibody-based therapy against SARS-related coronavirus and emerging variant strains, and may be an effective anti-SARS-CoV-2 agent.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Vírus da SARS/imunologia , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Anticorpos Facilitadores , COVID-19/tratamento farmacológico , COVID-19/terapia , Sequência Conservada , Reações Cruzadas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Modelos Moleculares , Testes de Neutralização , Pandemias , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/química , Vírus da SARS/genética , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
9.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203933

RESUMO

Natural resistance-associated macrophage proteins (Nramps) are specific metal transporters in plants with different functions among various species. The evolutionary and functional information of the Nramp gene family in Spirodela polyrhiza has not been previously reported in detail. To identify the Nramp genes in S. polyrhiza, we performed genome-wide identification, characterization, classification, and cis-elements analysis among 22 species with 138 amino acid sequences. We also conducted chromosomal localization and analyzed the synteny relationship, promoter, subcellular localization, and expression patterns in S. polyrhiza. ß-Glucuronidase staining indicated that SpNramp1 and SpNramp3 mainly accumulated in the root and joint between mother and daughter frond. Moreover, SpNramp1 was also widely displayed in the frond. SpNramp2 was intensively distributed in the root and frond. Quantitative real-time PCR results proved that the SpNramp gene expression level was influenced by Cd stress, especially in response to Fe or Mn deficiency. The study provides detailed information on the SpNramp gene family and their distribution and expression, laying a beneficial foundation for functional research.


Assuntos
Araceae/genética , Cádmio/toxicidade , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Araceae/efeitos dos fármacos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Cromossomos de Plantas/genética , Sequência Conservada , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Estresse Fisiológico/efeitos dos fármacos , Sintenia/genética
10.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34201937

RESUMO

The AVRPPHB SUSCEPTIBLE1 (PBS1) and RESISTANCE TO PSEUDOMONAS SYRINGAE 5 (RPS5) proteins are involved in signal transduction to evoke innate plant immune response. In Arabidopsis, PBS1 is cleaved by the AvrPphB (Pseudomonas phaseolicola Avirulence protein B) protease, activating RPS5 and turning in a hypersensitive response (HR). We searched for PBS1 orthologs to trace their origin and evolution. PBS1 orthologs were found in embryophytes and in other plant taxa but with lower similarity. PBS1 phylogenetic analysis indicates high divergence, suggesting that the decoy function described for Arabidopsis PBS1 might be associated with a small fraction of orthologs. Ancestral reconstruction analysis suggests an elevated diversity in the amino acid sequence within the described motifs. All the orthologs contain the conserved PBS1 kinase subdomains, whereas the cleavage motif is present in several embryophyte orthologs but absent in most other taxa. The putative resistance recognition motifs in PBS1 orthologs are highly diverse. PBS1 cleavage site motif is exposed in some 3D structure predictions, whereas it is not in others, suggesting different modes of regulation and functions in PBS1 orthologs. Our findings suggest that PBS1 originated in the lineage that gave rise to embryophytes, with the angiosperm sequences forming a separate clade from pteridophyte proteins.


Assuntos
Evolução Biológica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Filogenia , Fenômenos Fisiológicos Vegetais , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais , Relação Estrutura-Atividade
11.
Nat Commun ; 12(1): 4254, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253723

RESUMO

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/biossíntese , Acilação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Cisteína/metabolismo , Análise Mutacional de DNA , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Especificidade por Substrato
12.
Genomics ; 113(5): 3174-3184, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34293476

RESUMO

As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver named CoVrimer by which users can search for and align existing or newly designed conserved/degenerate primer pair sequences against the viral genome and assess the mutation load of both primers and amplicons. CoVrimer uses mutation data obtained from an online platform established by NGDC-CNCB (12 May 2021) to identify genomic regions, either conserved or with low levels of mutations, from which potential primer pairs are designed and provided to the user for filtering based on generalized and SARS-CoV-2 specific parameters. Alignments of primers and probes can be visualized with respect to the reference genome, indicating variant details and the level of conservation. Consequently, CoVrimer is likely to help researchers with the challenges posed by viral evolution and is freely available at http://konulabapps.bilkent.edu.tr:3838/CoVrimer/.


Assuntos
Primers do DNA/química , SARS-CoV-2/genética , Análise de Sequência de DNA/métodos , Software , Sequência Conservada , Primers do DNA/genética , Genoma Viral , Mutação
13.
Nature ; 596(7871): 281-284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290409

RESUMO

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.


Assuntos
Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Leucina/deficiência , Longevidade/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/agonistas , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
BMC Plant Biol ; 21(1): 288, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34167468

RESUMO

BACKGROUND: B-box (BBX) genes play important roles in plant growth regulation and responses to abiotic stresses. The plant growth and yield production of allotetraploid rapeseed is usually hindered by diverse nutrient stresses. However, no systematic analysis of Brassicaceae BBXs and the roles of BBXs in the regulation of nutrient stress responses have not been identified and characterized previously. RESULTS: In this study, a total of 536 BBXs were identified from nine brassicaceae species, including 32 AtBBXs, 66 BnaBBXs, 41 BoBBXs, 43 BrBBXs, 26 CrBBXs, 81 CsBBXs, 52 BnBBXs, 93 BjBBXs, and 102 BcBBXs. Syntenic analysis showed that great differences in the gene number of Brassicaceae BBXs might be caused by genome duplication. The BBXs were respectively divided into five subclasses according to their phylogenetic relationships and conserved domains, indicating their diversified functions. Promoter cis-element analysis showed that BBXs probably participated in diverse stress responses. Protein-protein interactions between BnaBBXs indicated their functions in flower induction. The expression profiles of BnaBBXs were investigated in rapeseed plants under boron deficiency, boron toxicity, nitrate limitation, phosphate shortage, potassium starvation, ammonium excess, cadmium toxicity, and salt stress conditions using RNA-seq data. The results showed that different BnaBBXs showed differential transcriptional responses to nutrient stresses, and some of them were simultaneously responsive to diverse nutrient stresses. CONCLUSIONS: Taken together, the findings investigated in this study provided rich resources for studying Brassicaceae BBX gene family and enriched potential clues in the genetic improvement of crop stress resistance.


Assuntos
Brassica napus/genética , Brassicaceae/genética , Genes de Plantas/genética , Fatores de Transcrição/genética , Brassica napus/fisiologia , Sequência Conservada , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/fisiologia , Estudo de Associação Genômica Ampla , Filogenia , Mapas de Interação de Proteínas , Estresse Fisiológico , Sintenia , Tetraploidia , Fatores de Transcrição/fisiologia
15.
BMC Plant Biol ; 21(1): 295, 2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34174836

RESUMO

BACKGROUND: Drought is a common phenomenon worldwide. It is also one of the main abiotic factors that affect the growth and quality of strawberry. The dehydration-responsive element binding protein (DREB) members that belong to the APETALA2/ethylene-responsive element binding protein (AP2/EREBP) superfamily are unique transcription factors in plants that play important roles in the abiotic stress response. RESULTS: Here, a total of 119 AP2/EREBP genes were identified in Fragaria vesca, and the AP2/EREBP superfamily was divided into AP2, RAV, ERF, DREB, and soloist subfamilies, containing 18, 7, 61, 32, and one member(s), respectively. The DREB subfamily was further divided into six subgroups (A-1 to A-6) based on phylogenetic analysis. Gene structure, conserved motifs, chromosomal location, and synteny analysis were conducted to comprehensively investigate the characteristics of FvDREBs. Furthermore, transcriptome analysis revealed distinctive expression patterns among the FvDREB genes in strawberry plants exposed to drought stress. The expression of FvDREB6 of the A-2 subgroup was down-regulated in old leaves and up-regulated in young leaves in response to drought. Furthermore, qRT-PCR analysis found that FvDREB8 from the A-2 subgroup had the highest expression level under drought stress. Together, analyses with the expression pattern, phylogenetic relationship, motif, and promoter suggest that FvDREB18 may play a critical role in the regulation of FvDREB1 and FvDREB2 expression. CONCLUSIONS: Our findings provide new insights into the characteristics and potential functions of FvDREBs. These FvDREB genes should be further studied as they appear to be excellent candidates for drought tolerance improvement of strawberry.


Assuntos
Fragaria/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Transcriptoma , Sequência Conservada , Desidratação , Fragaria/metabolismo , Fragaria/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/fisiologia , Estudo de Associação Genômica Ampla , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/fisiologia
16.
BMC Plant Biol ; 21(1): 296, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34182934

RESUMO

BACKGROUND: Moso bamboo, the fastest growing plant on earth, is an important source for income in large areas of Asia, mainly cultivated in China. Lateral organ boundaries domain (LBD) proteins, a family of transcription factors unique to plants, are involved in multiple transcriptional regulatory pathways and play important roles in lateral organ development, pathogen response, secondary growth, and hormone response. The LBD gene family has not previously been characterized in moso bamboo (Phyllostachys edulis). RESULTS: In this study, we identified 55 members of the LBD gene family from moso bamboo and found that they were distributed non-uniformly across its 18 chromosomes. Phylogenetic analysis showed that the moso bamboo LBD genes could be divided into two classes. LBDs from the same class share relatively conserved gene structures and sequences encoding similar amino acids. A large number of hormone response-associated cis-regulatory elements were identified in the LBD upstream promoter sequences. Synteny analysis indicated that LBDs in the moso bamboo genome showed greater collinearity with those of O. sativa (rice) and Zea mays (maize) than with those of Arabidopsis and Capsicum annuum (pepper). Numerous segmental duplicates were found in the moso bamboo LBD gene family. Gene expression profiles in four tissues showed that the LBD genes had different spatial expression patterns. qRT-PCR assays with the Short Time-series Expression Miner (STEM) temporal expression analysis demonstrated that six genes (PeLBD20, PeLBD29, PeLBD46, PeLBD10, PeLBD38, and PeLBD06) were consistently up-regulated during the rapid growth and development of bamboo shoots. In addition, 248 candidate target genes that function in a variety of pathways were identified based on consensus LBD binding motifs. CONCLUSIONS: In the current study, we identified 55 members of the moso bamboo transcription factor LBD and characterized for the first time. Based on the short-time sequence expression software and RNA-seq data, the PeLBD gene expression was analyzed. We also investigated the functional annotation of all PeLBDs, including PPI network, GO, and KEGG enrichment based on String database. These results provide a theoretical basis and candidate genes for studying the molecular breeding mechanism of rapid growth of moso bamboo.


Assuntos
Genes de Plantas/genética , Poaceae/genética , Fatores de Transcrição/genética , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Sequência Conservada , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Estudo de Associação Genômica Ampla , Filogenia , Alinhamento de Sequência , Transcriptoma
17.
Viruses ; 13(6)2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073222

RESUMO

African swine fever virus (ASFV) causes a devastating disease of swine that has caused outbreaks in Central Europe since 2007, spreading into Asia in 2018. ASFV is a large, structurally complex virus with a large dsDNA genome encoding for more than 160 genes, most of them still uncharacterized. p22, encoded by the ASFV gene KP177R, is an early transcribed, structural virus protein located in the ASFV particle. Although its exact function is unknown, p22 has recently been identified as an interacting partner of several host proteins. Here, we describe the development of a recombinant ASFV (ASFV-G-∆KP177R) lacking the KP177R gene as a tool to evaluate the role of p22 in virus replication and virulence in swine. The recombinant ASFV-G-∆KP177R demonstrated that the KP177R gene is non-essential for ASFV replication in primary swine macrophages, with virus yields similar to those of the parental, highly virulent field isolate Georgia2010 (ASFV-G). In addition, experimental infection of domestic pigs with ASFV-G-∆KP177R produced a clinical disease similar to that caused by the parental ASFV-G. Therefore, and surprisingly, p22 does not seem to be involved in virus replication or virulence in swine.


Assuntos
Vírus da Febre Suína Africana/genética , Febre Suína Africana/virologia , Proteínas Estruturais Virais/genética , Replicação Viral , Vírus da Febre Suína Africana/patogenicidade , Sequência de Aminoácidos , Animais , Células Cultivadas , Sequência Conservada , Deleção de Genes , Macrófagos/virologia , Mutação , Suínos , Carga Viral , Virulência , Fatores de Virulência/genética
18.
J Biol Chem ; 297(1): 100901, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34157281

RESUMO

Alx1, a homeodomain-containing transcription factor, is a highly conserved regulator of skeletogenesis in echinoderms. In sea urchins, Alx1 plays a central role in the differentiation of embryonic primary mesenchyme cells (PMCs) and positively regulates the transcription of most biomineralization genes expressed by these cells. The alx1 gene arose via duplication and acquired a skeletogenic function distinct from its paralog (alx4) through the exonization of a 41-amino acid motif (the D2 domain). Alx1 and Alx4 contain glutamine-50 paired-type homeodomains, which interact preferentially with palindromic binding sites in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) studies have shown, however, that Alx1 binds both to palindromic and half sites in vivo. To address this apparent discrepancy and explore the function of the D2 domain, we used an endogenous cis-regulatory module associated with Sp-mtmmpb, a gene that encodes a PMC-specific metalloprotease, to analyze the DNA-binding properties of Alx1. We find that Alx1 forms dimeric complexes on TAAT-containing half sites by a mechanism distinct from the well-known mechanism of dimerization on palindromic sites. We used transgenic reporter assays to analyze the functional roles of half sites in vivo and demonstrate that two sites with partially redundant functions are essential for the PMC-specific activity of the Sp-mtmmpb cis-regulatory module. Finally, we show that the D2 domain influences the DNA-binding properties of Alx1 in vitro, suggesting that the exonization of this motif may have facilitated the acquisition of new transcriptional targets and consequently a novel developmental function.


Assuntos
Biomineralização , Equinodermos/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Sequência Conservada , DNA/metabolismo , Equinodermos/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Ligação Proteica , Multimerização Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genética
19.
Commun Biol ; 4(1): 795, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172814

RESUMO

The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of TSHR alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate TSHR mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2 kb retrotransposon insertion upstream of the TSHR transcription start site, near an open chromatin region, which is likely to affect TSHR expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the TSHR locus may both contribute to the regulation of seasonal reproduction in herring.


Assuntos
Peixes/fisiologia , Receptores da Tireotropina/genética , Alelos , Animais , Oceano Atlântico , Sequência Conservada , Haplótipos , Mutação , Receptores da Tireotropina/fisiologia , Reprodução/fisiologia , Estações do Ano , Transdução de Sinais , Tireotropina Subunidade beta/genética
20.
Nucleic Acids Res ; 49(13): 7644-7664, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181727

RESUMO

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.


Assuntos
Proteínas do Complexo SMN/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Dimerização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Humanos , Locomoção , Modelos Moleculares , Mutação , Mutação Puntual , Domínios Proteicos , Multimerização Proteica , Proteínas do Complexo SMN/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
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