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1.
Nature ; 596(7871): 296-300, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349264

RESUMO

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Spliceossomos/enzimologia , Actinas/genética , Adenosina/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , RNA Helicases DEAD-box/ultraestrutura , Modelos Moleculares , Mutação , Domínios Proteicos , Precursores de RNA/metabolismo , Precursores de RNA/ultraestrutura , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Spliceossomos/química , Spliceossomos/metabolismo
2.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361714

RESUMO

α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.


Assuntos
Antracenos/química , Proteínas Fúngicas/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/química , Hipoglicemiantes/química , Perileno/análogos & derivados , alfa-Glucosidases/química , Antracenos/metabolismo , Sítios de Ligação , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Inibidores de Glicosídeo Hidrolases/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Hipoglicemiantes/metabolismo , Cinética , Simulação de Acoplamento Molecular , Nitrofenilgalactosídeos/química , Nitrofenilgalactosídeos/metabolismo , Perileno/química , Perileno/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/enzimologia , Ressonância de Plasmônio de Superfície , alfa-Glucosidases/metabolismo
3.
Molecules ; 26(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299598

RESUMO

In this work we introduce a novel filtering and molecular modeling pipeline based on a fingerprint and descriptor similarity procedure, coupled with molecular docking and molecular dynamics (MD), to select potential novel quoinone outside inhibitors (QoI) of cytochrome bc1 with the aim of determining the same or different chromophores to usual. The study was carried out using the yeast cytochrome bc1 complex with its docked ligand (stigmatellin), using all the fungicides from FRAC code C3 mode of action, 8617 Drugbank compounds and 401,624 COCONUT compounds. The introduced drug repurposing pipeline consists of compound similarity with C3 fungicides and molecular docking (MD) simulations with final QM/MM binding energy determination, while aiming for potential novel chromophores and perserving at least an amide (R1HN(C=O)R2) or ester functional group of almost all up to date C3 fungicides. 3D descriptors used for a similarity test were based on the 280 most stable Padel descriptors. Hit compounds that passed fingerprint and 3D descriptor similarity condition and had either an amide or an ester group were submitted to docking where they further had to satisfy both Chemscore fitness and specific conformation constraints. This rigorous selection resulted in a very limited number of candidates that were forwarded to MD simulations and QM/MM binding affinity estimations by the ORCA DFT program. In this final step, stringent criteria based on (a) sufficiently high frequency of H-bonds; (b) high interaction energy between protein and ligand through the whole MD trajectory; and (c) high enough QM/MM binding energy scores were applied to further filter candidate inhibitors. This elaborate search pipeline led finaly to four Drugbank synthetic lead compounds (DrugBank) and seven natural (COCONUT database) lead compounds-tentative new inhibitors of cytochrome bc1. These eleven lead compounds were additionally validated through a comparison of MM/PBSA free binding energy for new leads against those obtatined for 19 QoIs.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Avaliação Pré-Clínica de Medicamentos , Complexo III da Cadeia de Transporte de Elétrons/química , Proteínas de Saccharomyces cerevisiae/química
4.
Am J Hum Genet ; 108(9): 1735-1751, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34314704

RESUMO

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Mutação de Sentido Incorreto , Medicamentos sob Prescrição/metabolismo , Saccharomyces cerevisiae/enzimologia , Xenobióticos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/genética , Ensaios Enzimáticos , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenitoína/química , Polimorfismo Genético , Medicamentos sob Prescrição/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Transgenes , Varfarina/química , Varfarina/metabolismo , Xenobióticos/química
5.
Mol Biochem Parasitol ; 244: 111393, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34197864

RESUMO

Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.


Assuntos
Di-Hidrolipoamida Desidrogenase/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Sistemas de Translocação de Proteínas/genética , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Di-Hidrolipoamida Desidrogenase/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Tetra-Hidrofolato Desidrogenase/metabolismo , Trypanosoma brucei brucei/enzimologia
6.
J Enzyme Inhib Med Chem ; 36(1): 1509-1520, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34238110

RESUMO

In the present study, a series of azo derivatives (TR-1 to TR-9) have been synthesised via the diazo-coupling approach between substituted aromatic amines with phenol or naphthol derivatives. The compounds were evaluated for their therapeutic applications against alpha-glucosidase (anti-diabetic) and pathogenic bacterial strains E. coli (gram-negative), S. aureus (gram-positive), S. aureus (gram-positive) drug-resistant strain, P. aeruginosa (gram-negative), P. aeruginosa (gram-negative) drug-resistant strain and P. vulgaris (gram-negative). The IC50 (µg/mL) of TR-1 was found to be most effective (15.70 ± 1.3 µg/mL) compared to the reference drug acarbose (21.59 ± 1.5 µg/mL), hence, it was further selected for the kinetic studies in order to illustrate the mechanism of inhibition. The enzyme inhibitory kinetics and mode of binding for the most active inhibitor (TR-1) was performed which showed that the compound is a non-competitive inhibitor and effectively inhibits the target enzyme by binding to its binuclear active site reversibly.


Assuntos
Antibacterianos/farmacologia , Compostos Azo/farmacologia , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Simulação de Acoplamento Molecular , alfa-Glucosidases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Compostos Azo/síntese química , Compostos Azo/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Cinética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Staphylococcus aureus/efeitos dos fármacos
7.
Chem Commun (Camb) ; 57(60): 7445-7448, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34232232

RESUMO

G-quadruplex DNA interacts with the N-terminal intrinsically disordered domain of the DEAD-box helicase Ded1p, diminishing RNA unwinding activity but enhancing liquid-liquid phase separation of Ded1p in vitro and in cells. The data highlight multifaceted effects of quadruplex DNA on an enzyme with intrinsically disordered domains.


Assuntos
RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Quadruplex G , Proteínas de Saccharomyces cerevisiae/metabolismo , Citoplasma/química , Citoplasma/metabolismo , RNA Helicases DEAD-box/química , DNA/genética , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Transição de Fase , Domínios Proteicos , RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química
8.
Gene ; 793: 145745, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34077774

RESUMO

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.


Assuntos
Aspergillus oryzae/enzimologia , Ácidos Graxos/biossíntese , Proteínas Fúngicas/genética , Lipase/genética , Esporos Fúngicos/enzimologia , Triglicerídeos/biossíntese , Aspergillus oryzae/classificação , Aspergillus oryzae/genética , Sistemas CRISPR-Cas , Ácidos Graxos/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Humanos , Microbiologia Industrial , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lipase/metabolismo , Metabolismo dos Lipídeos/genética , Micélio/enzimologia , Micélio/genética , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , Triglicerídeos/genética
9.
Mol Cell ; 81(13): 2778-2792.e4, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33932350

RESUMO

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.


Assuntos
DNA Polimerase II/química , Exonucleases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Substituição de Aminoácidos , Domínio Catalítico , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Fúngico/biossíntese , DNA Fúngico/química , DNA Fúngico/genética , Exonucleases/genética , Exonucleases/metabolismo , Mutação de Sentido Incorreto , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Mol Cell ; 81(11): 2460-2476.e11, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33974913

RESUMO

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.


Assuntos
Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteólise , Proteômica/métodos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
11.
Biomolecules ; 11(4)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923845

RESUMO

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2'3'-cGAMP) formation was successfully developed. The 2'3'-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2'3'-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2'3'-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.


Assuntos
Adenosina Quinase/metabolismo , Proteínas de Bactérias/metabolismo , Nucleotídeos Cíclicos/síntese química , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acinetobacter/enzimologia , Nucleotídeos de Adenina/metabolismo , Biocatálise , Biotecnologia/métodos , Saccharomyces cerevisiae/enzimologia , Sinorhizobium meliloti/enzimologia
12.
J Biol Chem ; 296: 100703, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895134

RESUMO

The vacuolar H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V1 domain from the integral membrane Vo domain, accompanied by release of the V1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H+-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V1 complexes. Both purified RAVE and RAVE-V1 contain substoichiometric levels of subunit C. RAVE-V1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V1 interacts with the N-terminal domain of Vo subunit Vph1 in vitro. RAVE-V1 complexes, like isolated V1, have no MgATPase activity, suggesting that RAVE cannot reverse V1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V1 carrying a mutant subunit H incapable of inhibition with Vo complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , ATPases Vacuolares Próton-Translocadoras/genética
13.
Nature ; 594(7861): 124-128, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33902107

RESUMO

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (Pol II) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast1-9 and human10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH12-14. Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus Pol II, which is 99.9% identical to human Pol II. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8 Å resolution, and resolve the structure of TFIIH at 2.9-4.0 Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.


Assuntos
DNA/química , DNA/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Animais , Pareamento de Bases , DNA/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Mamíferos/genética , Modelos Moleculares , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , TATA Box/genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética
14.
Nucleic Acids Res ; 49(8): 4629-4642, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823543

RESUMO

H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.


Assuntos
Hidroliases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Catálise , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Hidroliases/genética , Técnicas In Vitro , Sequências Repetidas Invertidas , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Proteínas Nucleares/genética , Domínios Proteicos , Dobramento de RNA , RNA Guia , RNA Nucleolar Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
Biochemistry (Mosc) ; 86(Suppl 1): S96-S108, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33827402

RESUMO

Inorganic polyphosphates (polyP) are the linear polymers of orthophosphoric acid varying in the number of phosphate residues linked by the energy-rich phosphoanhydride bonds. PolyP is an essential component in living cells. Knowledge of polyP metabolizing enzymes in eukaryotes is necessary for understanding molecular mechanisms of polyP metabolism in humans and development of new approaches for treating bone and cardiovascular diseases associated with impaired mineral phosphorus metabolism. Yeast cells represent a rational experimental model for this research due to availability of the methods for studying phosphorus metabolism and construction of knockout mutants and strains overexpressing target proteins. Multicomponent system of polyP metabolism in Saccharomyces cerevisiae cells is presented in this review discussing properties, functioning, and practical significance of the enzymes involved in the synthesis and degradation of this important metabolite.


Assuntos
Polifosfatos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Biochem Biophys Res Commun ; 551: 33-37, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33714757

RESUMO

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides and considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to DHOase remains unknown. Here, we report the crystal structures of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with malate, 5-FU, and 5-aminouracil (5-AU). ScDHOase shares structural similarity with Escherichia coli DHOase. We also characterized the binding of 5-FU and 5-AU to ScDHOase by using the fluorescence quenching method. These complexed structures revealed that residues Arg18, Asn43, Thr106, and Ala275 of ScDHOase were involved in the 5-FU (PDB entry 6L0B) and 5-AU binding (PDB entry 6L0F). Overall, these results provide structural insights that may facilitate the development of new inhibitors targeting DHOase and constitute the 5-FU and 5-AU interactomes for further clinical chemotherapies.


Assuntos
Antineoplásicos/química , Di-Hidro-Orotase/química , Fluoruracila/química , Saccharomyces cerevisiae/enzimologia , Uracila/análogos & derivados , Antineoplásicos/farmacologia , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Di-Hidro-Orotase/metabolismo , Escherichia coli/enzimologia , Fluoruracila/farmacologia , Malatos/química , Modelos Moleculares , Ligação Proteica , Uracila/química , Uracila/farmacologia
17.
Mol Cell ; 81(9): 1920-1934.e9, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33689748

RESUMO

Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery.


Assuntos
RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/enzimologia , Elongação da Transcrição Genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HeLa , Humanos , Células K562 , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética , Fatores de Tempo
18.
Mol Cell ; 81(9): 1879-1889.e6, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33743194

RESUMO

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.


Assuntos
Aminoácidos/deficiência , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Commun Biol ; 4(1): 363, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742106

RESUMO

ATM and ATR are conserved regulators of the DNA damage response linked to cancer. Comprehensive DNA sequencing efforts identified ~4,000 cancer-associated mutations in ATM/ATR; however, their cancer implications remain largely unknown. To gain insights, we identify functionally important conserved residues in ATM, ATR and budding yeast Mec1ATR via cancer genome datamining and a functional genetic analysis, respectively. Surprisingly, only a small fraction of the critical residues is in the active site of the respective enzyme complexes, implying that loss of the intrinsic kinase activity is infrequent in carcinogenesis. A number of residues are solvent accessible, suggestive of their involvement in interacting with a protein-partner(s). The majority, buried inside the respective enzyme complexes, might play a structural or regulatory role. Together, these findings identify evolutionarily conserved ATM, ATR, and Mec1ATR residues involved in diverse aspects of the enzyme function and provide fresh insights into the elusive genotype-phenotype relationships in ATM/ATR and their cancer-associated variants.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Transformação Celular Neoplásica/genética , Mineração de Dados , Mutação de Sentido Incorreto , Neoplasias/genética , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Transformação Celular Neoplásica/metabolismo , Sequência Conservada , Bases de Dados Genéticas , Evolução Molecular , Predisposição Genética para Doença , Genoma Humano , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Neoplasias/enzimologia , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
20.
Biosci Biotechnol Biochem ; 85(5): 1252-1265, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33728459

RESUMO

ω3 Polyunsaturated fatty acids are currently obtained mainly from fisheries; thus, sustainable alternative sources such as oleaginous microorganisms are required. Here, we describe the isolation, characterization, and application of 3 novel ω3 desaturases with ω3 polyunsaturated fatty acid-producing activity at ordinary temperatures (28 °C). First, we selected Pythium sulcatum and Plectospira myriandra after screening for oomycetes with high eicosapentaenoic acid/arachidonic acid ratios and isolated the genes psulω3 and pmd17, respectively, which encode ω3 desaturases. Subsequent characterization showed that PSULω3 exhibited ω3 desaturase activity on both C18 and C20 ω6 polyunsaturated fatty acids while PMD17 exhibited ω3 desaturase activity exclusively on C20 ω6 polyunsaturated fatty acids. Expression of psulω3 and pmd17 in the arachidonic acid-producer Mortierella alpina resulted in transformants that produced eicosapentaenoic acid/total fatty acid values of 38% and 40%, respectively, at ordinary temperatures. These ω3 desaturases should facilitate the construction of sustainable ω3 polyunsaturated fatty acid sources.


Assuntos
Ácido Eicosapentaenoico/biossíntese , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Insaturados/biossíntese , Mortierella/genética , Oomicetos/genética , Pythium/genética , Ácido Araquidônico/biossíntese , Clonagem Molecular , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/classificação , Expressão Gênica , Biblioteca Gênica , Engenharia Metabólica/métodos , Mortierella/enzimologia , Oomicetos/classificação , Oomicetos/enzimologia , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Pythium/classificação , Pythium/enzimologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transformação Genética , Transgenes
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