RESUMO
Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.
RESUMO
Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As it is an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of IDO1 inhibitors with various mechanisms of inhibition is of great interest. Comparison of an apo-form-binding IDO1 inhibitor (GSK5628) to the heme-coordinating compound, epacadostat (Incyte), allows us to explore the details of the apo-binding inhibition of IDO1. Herein, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1), whereas epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, previously undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition might help design superior inhibitors or could confer a unique competitive advantage over other IDO1 inhibitors vis-à-vis specificity and pharmacokinetic parameters.
Assuntos
Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Inibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Conformação MolecularRESUMO
UNLABELLED: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies nuclear and cytoplasmic proteins via O-linked addition of a single N-acetylglucosamine (GlcNAc) moiety. Among the many targets of OGT is host cell factor 1 (HCF-1), a transcriptional regulator that is required for transactivation of the immediate-early genes of herpes simplex virus (HSV). HCF-1 is synthesized as a large precursor that is proteolytically cleaved by OGT, which may regulate its biological function. In this study, we tested whether inhibition of the enzymatic activity of OGT with a small molecule inhibitor, OSMI-1, affects initiation of HSV immediate-early gene expression and viral replication. We found that inhibiting OGT's enzymatic activity significantly decreased HSV replication. The major effect of the inhibitor occurred late in the viral replication cycle, when it reduced the levels of late proteins and inhibited capsid formation. However, depleting OGT levels with small interfering RNA (siRNA) reduced the expression of HSV immediate-early genes, in addition to reducing viral yields. In this study, we identified OGT as a novel cellular factor involved in HSV replication. Our results obtained using a small molecule inhibitor and siRNA depletion suggest that OGT's glycosylation and scaffolding functions play distinct roles in the replication cycle of HSV. IMPORTANCE: Antiviral agents can target viral or host gene products essential for viral replication. O-GlcNAc transferase (OGT) is an important cellular enzyme that catalyzes the posttranslational addition of GlcNAc sugar residues to hundreds of nuclear and cytoplasmic proteins, and this modification regulates their activity and function. Some of the known OGT targets are cellular proteins that are critical for the expression of herpes simplex virus (HSV) genes, suggesting a role for OGT in the replication cycle of HSV. In this study, we found that OGT is required for efficient expression of viral genes and for assembly of new virions. Thus, we identify OGT as a novel host factor involved in the replication of HSV and a potential target for antiviral therapy.
Assuntos
Citomegalovirus/fisiologia , Inibidores Enzimáticos/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Fator C1 de Célula Hospedeira/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Simplexvirus/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Genes Precoces/genética , Humanos , Immunoblotting , Microscopia Eletrônica , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Células VeroRESUMO
O-GlcNAc transferase (OGT) is an essential mammalian enzyme that regulates numerous cellular processes through the attachment of O-linked N-acetylglucosamine (O-GlcNAc) residues to nuclear and cytoplasmic proteins. Its targets include kinases, phosphatases, transcription factors, histones, and many other intracellular proteins. The biology of O-GlcNAc modification is still not well understood, and cell-permeable inhibitors of OGT are needed both as research tools and for validating OGT as a therapeutic target. Here, we report a small molecule OGT inhibitor, OSMI-1, developed from a high-throughput screening hit. It is cell-permeable and inhibits protein O-GlcNAcylation in several mammalian cell lines without qualitatively altering cell surface N- or O-linked glycans. The development of this molecule validates high-throughput screening approaches for the discovery of glycosyltransferase inhibitors, and further optimization of this scaffold may lead to yet more potent OGT inhibitors useful for studying OGT in animal models.
Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células CHO , Permeabilidade da Membrana Celular , Cricetulus , Inibidores Enzimáticos/síntese química , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Lectinas/química , Lectinas/metabolismo , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Difosfato de Uridina/química , Difosfato de Uridina/metabolismo , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
O-GlcNAc transferase (OGT) is a serine/threonine glycosyltransferase that is essential for development and continues to be critically important throughout life. Understanding OGT's complex biology requires identifying its substrates. Here we demonstrate the utility of a microarray approach for discovering novel OGT substrates. We also report a rapid method to validate OGT substrates that combines in vitro transcription-translation with O-GlcNAc mass tagging. Among the validated new OGT targets is Orthodenticle homeobox 2 (OTX2), a transcription factor critical for brain development, which is primarily expressed only during early embryogenesis and in medulloblastomas, where it functions as an oncogene. We show that endogenous OTX2 from a medulloblastoma cell line is O-GlcNAcylated at several sites. Our results demonstrate that protein microarray technology, combined with the target validation strategy we report, is useful for identifying biologically important OGT substrates, including substrates not present in most tissue types or cell lines.
Assuntos
Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fatores de Transcrição Otx/metabolismo , Acilação , Linhagem Celular Tumoral , Humanos , Análise Serial de ProteínasRESUMO
Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Conformação Molecular , RNA Mensageiro/genética , RNA Mensageiro/ultraestrutura , RNA de Transferência/genética , RNA de Transferência/ultraestrutura , Ribossomos/ultraestrutura , TermodinâmicaRESUMO
Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.
Assuntos
Conformação de Ácido Nucleico , RNA Ribossômico 23S/química , RNA Ribossômico 23S/metabolismo , Ribossomos/metabolismo , Códon de Terminação/genética , Códon de Terminação/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Mutação , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/genética , Especificidade por SubstratoRESUMO
The structural basis of the tRNA selection process is investigated by cryo-electron microscopy of ribosomes programmed with UGA codons and incubated with ternary complex (TC) containing the near-cognate Trp-tRNA(Trp) in the presence of kirromycin. Going through more than 350 000 images and employing image classification procedures, we find â¼8% in which the TC is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near-cognate aa-tRNA with respect to elongation factor Tu (EF-Tu) and the ribosome, as well as the domain movements of the ribosome. One of the interesting findings is that near-cognate tRNA's acceptor stem region is flexible and CCA end becomes disordered. The data bring direct structural insights into the induced-fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa-tRNA and EF-Tu and comparison with the cognate case (UGG codon) offers clues on how the conformational signals conveyed to the GTPase differ in the two cases.
Assuntos
Códon , Fator Tu de Elongação de Peptídeos/química , Biossíntese de Proteínas , RNA de Transferência/química , Ribossomos/química , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestruturaRESUMO
The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA(Trp) miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.
Assuntos
RNA Ribossômico/química , RNA Ribossômico/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Códon/química , Códon/genética , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Elongação Traducional da Cadeia Peptídica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/genética , RNA de Transferência/genética , RNA de Transferência de Triptofano/química , RNA de Transferência de Triptofano/genética , RNA de Transferência de Triptofano/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , TermodinâmicaRESUMO
The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.
Assuntos
Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Estrutura Quaternária de Proteína , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Inibidores da Síntese de Proteínas/farmacologia , Piridonas/farmacologiaRESUMO
A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.
Assuntos
RNA de Transferência/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Elongação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Subunidades Proteicas/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/fisiologia , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/fisiologiaRESUMO
A novel class of DNA cleavage agents are reported that derive activity from amino acids tethered to a photoactive intercalator.