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1.
Nature ; 588(7839): 712-716, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33328633

RESUMO

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.


Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Microscopia Crioeletrônica , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Mitocondriais/efeitos dos fármacos , Humanos , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Especificidade por Substrato/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Mol Cell ; 49(6): 1159-66, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23478441

RESUMO

F-box proteins and DCAF proteins are the substrate binding subunits of the Skp1-Cul1-F-box protein (SCF) and Cul4-RING protein ligase (CRL4) ubiquitin ligase complexes, respectively. Using affinity purification and mass spectrometry, we determined that the F-box protein FBXO11 interacts with CDT2, a DCAF protein that controls cell-cycle progression, and recruits CDT2 to the SCF(FBXO11)complex to promote its proteasomal degradation. In contrast to most SCF substrates, which exhibit phosphodegron-dependent binding to F-box proteins, CDK-mediated phosphorylation of Thr464 present in the CDT2 degron inhibits recognition by FBXO11. Finally, our results show that the functional interaction between FBXO11 and CDT2 is evolutionary conserved from worms to humans and plays an important role in regulating the timing of cell-cycle exit.


Assuntos
Ciclo Celular , Proteínas F-Box/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Diferenciação Celular , Sequência Conservada , Proteínas F-Box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , RNA Interferente Pequeno/genética , Ubiquitina-Proteína Ligases/genética
3.
Sci Rep ; 14(1): 20509, 2024 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-39227746

RESUMO

Natural compounds constitute a major resource for the development of medicines for multiple diseases. While many natural compounds show strong biological activity, the mechanisms that confer clinical benefits are often elusive and have been attributed to multiple pathways. Periplogenin (PPG), a natural compound isolated from Cortex periplocae, exhibits strong anti-tumor activities in several human cancer cell lines. However, its molecular mode of action remained unclear. In this study, we leveraged a forward genetic screening approach in DU145 prostate cancer cells to uncover the molecular target of PPG using chemical mutagenesis. Next generation sequencing revealed that a single amino acid substitution at amino acid 804 in ATP1A1 (ATPase Na + /K + Transporting Subunit Alpha 1) confers resistance to the cytotoxic activity of PPG. Mechanistically, ATP1A1 T804 forms a hydrogen bond with PPG which is abolished by the T804A substitution in ATP1A1, resulting in resistance to PPG treatment in vitro. Importantly, in vivo, PPG strongly suppressed tumor development in a DU145 xenograft model whereas DU145 xenograft tumors carrying a ATP1A1-T804A mutation were largely unaffected by the treatment. These findings demonstrate that PPG suppresses the growth of DU145 prostate cancer cells in vitro and in vivo by directly binding to ATP1A1 and highlight the power of our unbiased forward genetic screening approach to uncover direct drug target structures at single amino acid resolution.


Assuntos
Neoplasias da Próstata , ATPase Trocadora de Sódio-Potássio , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Animais , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Camundongos , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia
4.
Nat Microbiol ; 9(6): 1499-1512, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38548922

RESUMO

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.


Assuntos
Apolipoproteínas E , Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Receptores de LDL , Internalização do Vírus , Animais , Humanos , Camundongos , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/virologia , Febre Hemorrágica da Crimeia/metabolismo , Camundongos Knockout , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores Virais/metabolismo , Carrapatos/virologia , Carrapatos/metabolismo
5.
EMBO Mol Med ; 15(3): e16959, 2023 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-36740985

RESUMO

The natural compound Artemisinin is the most widely used antimalarial drug worldwide. Based on its cytotoxicity, it is also used for anticancer therapy. Artemisinin and its derivates are endoperoxides that damage proteins in eukaryotic cells; their definite mechanism of action and host cell targets, however, have remained largely elusive. Using yeast and haploid stem cell screening, we demonstrate that a single cellular pathway, namely porphyrin (heme) biosynthesis, is required for the cytotoxicity of Artemisinins. Genetic or pharmacological modulation of porphyrin production is sufficient to alter its cytotoxicity in eukaryotic cells. Using multiple model systems of human brain tumor development, such as cerebral glioblastoma organoids, and patient-derived tumor spheroids, we sensitize cancer cells to dihydroartemisinin using the clinically approved porphyrin enhancer and surgical fluorescence marker 5-aminolevulinic acid, 5-ALA. A combination treatment of Artemisinins and 5-ALA markedly and specifically killed brain tumor cells in all model systems tested, including orthotopic patient-derived xenografts in vivo. These data uncover the critical molecular pathway for Artemisinin cytotoxicity and a sensitization strategy to treat different brain tumors, including drug-resistant human glioblastomas.


Assuntos
Antimaláricos , Artemisininas , Neoplasias Encefálicas , Humanos , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Antimaláricos/farmacologia , Heme/metabolismo , Ácido Aminolevulínico , Neoplasias Encefálicas/tratamento farmacológico
6.
Nat Commun ; 14(1): 6785, 2023 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-37880247

RESUMO

Marburg and Ebola filoviruses are two of the deadliest infectious agents and several outbreaks have occurred in the last decades. Although several receptors and co-receptors have been reported for Ebola virus, key host factors remain to be elucidated. In this study, using a haploid cell screening platform, we identify the guanine nucleotide exchange factor CCZ1 as a key host factor in the early stage of filovirus replication. The critical role of CCZ1 for filovirus infections is validated in 3D primary human hepatocyte cultures and human blood-vessel organoids, both critical target sites for Ebola and Marburg virus tropism. Mechanistically, CCZ1 controls early to late endosomal trafficking of these viruses. In addition, we report that CCZ1 has a role in the endosomal trafficking of endocytosis-dependent SARS-CoV-2 infections, but not in infections by Lassa virus, which enters endo-lysosomal trafficking at the late endosome stage. Thus, we have identified an essential host pathway for filovirus infections in cell lines and engineered human target tissues. Inhibition of CCZ1 nearly completely abolishes Marburg and Ebola infections. Thus, targeting CCZ1 could potentially serve as a promising drug target for controlling infections caused by various viruses, such as SARS-CoV-2, Marburg, and Ebola.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Doença do Vírus de Marburg , Marburgvirus , Proteínas de Transporte Vesicular , Animais , Humanos , Ebolavirus/metabolismo , Lisossomos , Doença do Vírus de Marburg/genética , Doença do Vírus de Marburg/metabolismo , Marburgvirus/metabolismo , Proteínas de Transporte Vesicular/metabolismo
7.
Biol Chem ; 393(10): 1183-91, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23089536

RESUMO

The Wnt/ß-catenin and the Ras/mitogen-activated protein kinase (MAPK) pathways play important roles in cancer development. Both pathways have been studied discretely, but the mechanisms of possible crosstalk are still not fully understood. We have previously shown that ß-catenin and MAPK signaling interfere with each other in murine liver in vivo and in vitro. Here, we show that dual specificity phosphatases (Dusps) 6 and 14, known to play an essential role in regulating MAPK pathway activity via feedback mechanisms, are up-regulated by activation of ß-catenin in murine liver cells, whereas the epidermal growth factor receptor, an upstream effector in the Ras/MAPK cascade, is down-regulated by ß-catenin. In addition, we identified a ß-catenin-binding site within the Dusp6 promoter, which is responsible for the activation of the promoter by ß-catenin signaling, and demonstrated reduced inducibility of MAPK signaling in cultured mouse hepatoma cells following ß-catenin activation. Thus, ß-catenin is able to inhibit activation of the Egfr/Ras/MAPK signaling cascade, both at the receptor level and by interfering with MAPK activity via Dusps.


Assuntos
Fosfatase 6 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas ras/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Cães , Fosfatase 6 de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos
8.
Elife ; 112022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35229715

RESUMO

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM), and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function (LOF) is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 LOF resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Taken together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Hexosaminas , Animais , Vias Biossintéticas , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glicosilação , Hexosaminas/metabolismo , Camundongos
9.
EMBO Mol Med ; 13(1): e13426, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33179852

RESUMO

There is a critical need for safe and effective drugs for COVID-19. Only remdesivir has received authorization for COVID-19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS-CoV-2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS-CoV-2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS-CoV-2 in both models. By using single amino-acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub-toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID-19 clinical trials.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Enzima de Conversão de Angiotensina 2/farmacologia , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Animais , Células Cultivadas , Chlorocebus aethiops , Sinergismo Farmacológico , Humanos , Modelos Moleculares , Proteínas Recombinantes/farmacologia , SARS-CoV-2/fisiologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
10.
Nat Commun ; 11(1): 687, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019926

RESUMO

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/metabolismo , Retroalimentação Fisiológica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glicosilação , Hexosaminas/química , Humanos , Ligantes , Conformação Proteica , Proteostase , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/metabolismo
11.
FEBS Lett ; 594(1): 175-188, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31365120

RESUMO

UBR5 is an E3 ubiquitin ligase involved in distinct processes such as transcriptional regulation and development. UBR5 is highly upregulated in embryonic stem cells (ESCs), whereas its expression decreases with differentiation, suggesting a role for UBR5 in ESC function. However, little is known about how UBR5 regulates ESC identity. Here, we define the protein interactome of UBR5 in ESCs and find interactions with distinct components of the H/ACA ribonucleoprotein complex, which is required for proper maturation of ribosomal RNA (rRNA). Notably, loss of UBR5 induces an abnormal accumulation of rRNA processing intermediates, resulting in diminished ribosomal levels. Consequently, lack of UBR5 triggers an increase in p53 levels and a concomitant decrease in cellular proliferation rates. Thus, our results indicate a link between UBR5 and rRNA maturation.


Assuntos
RNA Ribossômico/metabolismo , Ribonucleoproteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Camundongos , Processamento Pós-Transcricional do RNA , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética
12.
iScience ; 23(3): 100887, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32086012

RESUMO

Activation of the hexosamine pathway (HP) through gain-of-function mutations in its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT-1) ameliorates proteotoxicity and increases lifespan in Caenorhabditis elegans. Here, we investigate the role of the HP in mammalian protein quality control. In mouse neuronal cells, elevation of HP activity led to phosphorylation of both PERK and eIF2α as well as downstream ATF4 activation, identifying the HP as a modulator of the integrated stress response (ISR). Increasing uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) levels through GFAT1 gain-of-function mutations or supplementation with the precursor GlcNAc reduces aggregation of the polyglutamine (polyQ) protein Ataxin-3. Blocking PERK signaling or autophagy suppresses this effect. In C. elegans, overexpression of gfat-1 likewise activates the ISR. Consistently, co-overexpression of gfat-1 and proteotoxic polyQ peptides in muscles reveals a strong protective cell-autonomous role of the HP. Thus, the HP has a conserved role in improving protein quality control through modulation of the ISR.

13.
Methods Mol Biol ; 1953: 23-31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912013

RESUMO

The steadily increasing throughput in next-generation sequencing technologies is revolutionizing a number of fields in biology. One application requiring massive parallel sequencing is forward genetic screening based on chemical mutagenesis. Such screens interrogate the entire genome in an entirely unbiased fashion and can be applied to a number of research questions. CRISPR/Cas9-based screens, which are largely limited to a gene's loss of function, have already been very successful in identifying drug targets and pathways related to the drug's mode of action. By inducing single nucleotide changes using an alkylating reagent, it is possible to generate amino acid changes that perturb the interaction between a drug and its direct target, resulting in drug resistance. This chemogenomic approach combined with latest sequencing technologies allows deconvolution of drug targets and characterization of drug-target binding interfaces at amino acid resolution, therefore nicely complementing existing biochemical approaches. Here we describe a general protocol for a chemical mutagenesis-based forward genetic screen applicable for drug-target deconvolution.


Assuntos
Sistemas CRISPR-Cas , Avaliação Pré-Clínica de Medicamentos/métodos , Edição de Genes/métodos , Mutagênese , Animais , Técnicas de Cultura de Células/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Descoberta de Drogas/métodos , Resistência a Medicamentos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Mutação , Análise de Sequência de DNA/métodos
14.
Oncotarget ; 9(11): 9838-9851, 2018 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-29515774

RESUMO

Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds. We compare and contrast chemical mutagenesis with insertional mutagenesis. Selecting mutagenized cells with thapsigargin, an inhibitor of the essential Ca2+ pump SERCA2, insertional mutagenesis retrieved cell clones overexpressing SERCA2. With chemical mutagenesis, we identify six single amino acid substitutions in the known SERCA2-thapsigargin binding interface that confer drug resistance. In a second screen, we used the anti-cancer drug MG132/bortezomib (Velcade), which inhibits proteasome activity. Using chemical mutagenesis, we found 7 point mutations in the essential subunit Psmb5 that map to the bortezomib binding surface. Importantly, 4 of these had previously been identified in human tumors with acquired bortezomib resistance. Insertional mutagenesis did not identify Psmb5 in this screen, demonstrating the unique ability of chemical mutagenesis to identify relevant point mutations in essential genes. Thus, chemical mutagenesis in haploid embryonic stem cells can define the interaction of toxic small molecules with essential proteins at amino acid resolution, fully mapping small molecule-protein binding interfaces.

16.
Dev Cell ; 28(6): 697-710, 2014 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-24613396

RESUMO

Developmental timing genes catalyze stem cell progression and animal maturation programs across taxa. Caenorhabditis elegans DRE-1/FBXO11 functions in an SCF E3-ubiquitin ligase complex to regulate the transition to adult programs, but its cognate proteolytic substrates are unknown. Here, we identify the conserved transcription factor BLMP-1 as a substrate of the SCF(DRE-1/FBXO11) complex. blmp-1 deletion suppressed dre-1 mutant phenotypes and exhibited developmental timing defects opposite to dre-1. blmp-1 also opposed dre-1 for other life history traits, including entry into the dauer diapause and longevity. BLMP-1 protein was strikingly elevated upon dre-1 depletion and dysregulated in a stage- and tissue-specific manner. The role of DRE-1 in regulating BLMP-1 stability is evolutionary conserved, as we observed direct protein interaction and degradation function for worm and human counterparts. Taken together, posttranslational regulation of BLMP-1/BLIMP-1 by DRE-1/FBXO11 coordinates C. elegans developmental timing and other life history traits, suggesting that this two-protein module mediates metazoan maturation processes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/citologia , Longevidade/genética , Animais , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Células Epidérmicas , Epiderme/metabolismo , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Inativação Gênica , Gônadas/citologia , Gônadas/metabolismo , Células HEK293 , Humanos , Técnicas Imunoenzimáticas , Larva/metabolismo , Especificidade de Órgãos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Interferente Pequeno/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação
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