RESUMO
The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.
Assuntos
Peptídeos , S-Adenosilmetionina , Humanos , S-Adenosilmetionina/metabolismo , Peptídeos/química , Biologia Computacional , Carbono , EspasmoRESUMO
Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.
Assuntos
Biologia Computacional , S-Adenosilmetionina , Sequência de Aminoácidos , Carbono/metabolismo , Peptídeos/química , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/metabolismoRESUMO
Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.
Assuntos
Biologia Computacional/métodos , Enzimas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Produtos Biológicos/química , Produtos Biológicos/classificação , Produtos Biológicos/metabolismo , Enzimas/química , Hidroxilação , Metilação , Peptídeos/classificação , Peptídeos/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/fisiologia , Ribossomos/metabolismoRESUMO
Electron paramagnetic resonance (EPR) inversion recovery curves for vanadium catecholates and ironsulfur clusters were analyzed with three models: the sum of two exponentials, a stretched exponential, and a model-free distribution of exponentials (UPEN). For all data sets studied fits with a stretched exponential were statistically indistinguishable from the sum of two exponentials, and were significantly better than for single exponentials. UPEN provides insights into the structures of the distributions. For a vanadium(IV) tris catecholate the distribution of relaxation rates calculated with UPEN shows the contribution from spectral diffusion at low temperatures. The energy of the local mode for this complex, found from the temperature dependence of the spin lattice relaxation, is consistent with values expected for a metal-ligand vibration. For the [2Fe-2S]+ cluster in pyruvate formate lyase activating enzyme (PFL-AE) the small stretched exponential ß values (0.3) at low temperature and the distributions calculated with UPEN reflect the contribution from a second rapidly relaxing species that could be difficult to detect by continuous wave EPR. The distributions in 1/T1 for the [4Fe-4S]+ clusters in Mycofactocin maturase were about a factor of four wider than for the three other systems studied. The very broad distribution of relaxation rates may be due to protein mobility and distributions in electronic energies and local environments for the clusters. UPEN provides insight into several situations that can result in low values of stretch parameter ß including contributions from spectral diffusion, overlapping signals from distinguishable clusters, or very wide distributions.
Assuntos
Catecóis/química , Proteínas Ferro-Enxofre/química , Compostos Organometálicos/química , Vanádio/química , Acetiltransferases/química , Espectroscopia de Ressonância de Spin EletrônicaRESUMO
Mycofactocin (MFT) is a putative ribosomally synthesized and post-translationally modified (RiPP) redox cofactor. The biosynthesis of MFT is encoded by the gene cluster mftABCDEF. While processing of the precursor peptide by MftB, MftC, and MftE has been shown to result in the formation of the small molecule 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP), no activity has been shown for the putative dehydrogenase MftD and the putative glycosyltransferase MftF. In addition, evidence demonstrating that MFT is a redox cofactor has only been limited to the requirement of mft genes for ethanol assimilation in Mycobacterium smegmatis mc2155. Here, we demonstrate that MftD catalyzes the oxidative deamination of AHDP, forming an α-keto moiety on the resulting molecule, which we call pre-mycofactocin (PMFT). We characterize PMFT by 1D and 2D NMR spectroscopy techniques and by high-resolution mass spectrometry data to solve its structure. We further characterized PMFT by cyclic voltammetry and found its midpoint potential to be â¼255 mV. Lastly, we demonstrate that PMFT is a biologically active redox cofactor that oxidizes NADH bound by M. smegmatis carveol dehydrogenase (MsCDH) and can be used by MsCDH in the oxidation of carveol. These data demonstrate for the first time that PMFT functions as a biologically active redox mediator and provides the most direct evidence to date that MFT is a RiPP-derived redox cofactor.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Oxirredutases/metabolismo , Desaminação , Humanos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Oxirredução , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismoRESUMO
Mycofactocin is a member of the rapidly growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Although the mycofactocin biosynthetic pathway is widely distributed among Mycobacterial species, the structure, function, and biosynthesis of the pathway product remain unknown. This mini-review will discuss the current state of knowledge regarding the mycofactocin biosynthetic pathway. In particular, we focus on the architecture and distribution of the mycofactocin biosynthetic cluster, mftABCDEF, among the Actinobacteria phylum. We discuss the potential molecular and physiological role of mycofactocin. We review known biosynthetic steps involving MftA, MftB, MftC, and MftE and relate them to pyrroloquinoline quinone biosynthesis. Lastly, we propose the function of the remaining putative biosynthetic enzymes, MftD and MftF.
Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Actinobacteria/genética , Proteínas de Bactérias/biossíntese , Vias Biossintéticas , Mycobacterium/enzimologia , Mycobacterium/genética , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismoRESUMO
Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo ß-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.
Assuntos
Proteínas de Bactérias/química , Di-Hidroxifenilalanina/análogos & derivados , Oxigenases de Função Mista/química , Catálise , Di-Hidroxifenilalanina/química , Hidroxilação , Ferro/química , Methylobacterium extorquens/enzimologia , Modelos Químicos , Zinco/químicaRESUMO
Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dose-dependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Animais , Especificidade de Anticorpos , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Humanos , Cinética , Masculino , Transtornos de Enxaqueca/imunologia , Transtornos de Enxaqueca/prevenção & controle , Células PC12 , Ratos , Ratos Sprague-DawleyRESUMO
Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico , Cisteína/química , Técnicas Eletroquímicas , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Ferro-Enxofre/genética , Mycobacterium ulcerans/química , Oxirredução , S-Adenosilmetionina/metabolismo , Espectroscopia de MossbauerRESUMO
The structure of the ribosomally synthesized and post-translationally modified peptide product mycofactocin is unknown. Recently, the first step in mycofactocin biosynthesis was shown to be catalyzed by MftC in two S-adenosylmethionine-dependent steps. In the first step, MftC catalyzes the oxidative decarboxylation of the MftA peptide to produce the styrene-containing intermediate MftA**, followed by a subsequent C-C bond formation to yield the lactam-containing MftA*. Here, we demonstrate the subsequent biosynthetic step catalyzed by MftE is specific for MftA*. The hydrolysis of MftA* leads to the formation of MftA(1-28) and 3-amino-5-[( p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP). The hydrolysis reaction is Fe2+-dependent, and addition of the metal to the reaction mixture leads to a kobs of â¼0.2 min-1. Lastly, we validate the structure of AHDP by 1H, 13C, and COSY nuclear magnetic resonance techniques as well as mass spectrometry.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium/metabolismo , Proteína O-Metiltransferase/metabolismo , Pirrolidinonas/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.
Assuntos
Proteínas de Bactérias/química , Endopeptidases/química , Proteínas Ferro-Enxofre/química , Methylobacterium extorquens/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Conformação Proteica , TemperaturaRESUMO
Radical S-adenosylmethionine (RS) enzymology has emerged as a major biochemical strategy for the homolytic cleavage of unactivated C-H bonds. At the same time, the post-translational modification of ribosomally synthesized peptides is a rapidly expanding area of investigation. We discuss the functional cross-section of these two disciplines, highlighting the recently uncovered importance of protein-protein interactions, especially between the peptide substrate and its chaperone, which functions either as a stand-alone protein or as an N-terminal fusion to the respective RS enzyme. The need for further work on this class of enzymes is emphasized, given the poorly understood roles performed by multiple, auxiliary iron-sulfur clusters and the paucity of protein X-ray structural data.
Assuntos
Proteínas Ferro-Enxofre , Chaperonas Moleculares , Processamento de Proteína Pós-Traducional/fisiologia , S-Adenosilmetionina , Radicais Livres/química , Radicais Livres/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismoRESUMO
Ribosomally synthesized and posttranslationally modified peptide (RiPP) pathways produce a diverse array of natural products. A subset of these pathways depends on radical S-adenosylmethionine proteins to modify the RiPP-produced peptide. Mycofactocin biosynthesis is one example of an S-adenosylmethionine protein-dependent RiPP pathway. Recently, it has been shown that MftC catalyzes the oxidative decarboxylation of the C-terminal tyrosine (Tyr-30) on the mycofactocin precursor peptide MftA; however, this product has not been verified by techniques other than MS. Herein, we provide a more detailed study of MftC catalysis and report a revised mechanism for MftC chemistry. We show that MftC catalyzes the formation of two isomeric products. Using a combination of MS, isotope labeling, and 1H and 13C NMR techniques, we established that the major product, MftA*, is a tyramine-valine-cross-linked peptide formed by MftC through two S-adenosylmethionine-dependent turnovers. In addition, we show that the hydroxyl group on MftA Tyr-30 is required for MftC catalysis. Furthermore, we show that a substitution in the penultimate MftA Val-29 position causes the accumulation of an MftA** minor product. The 1H NMR spectrum indicates that this minor product contains an αß-unsaturated bond that likely arises from an aborted intermediate of MftA* synthesis. The finding that MftA* is the major product formed during MftC catalysis could have implications for the further elucidation of mycofactocin biosynthesis.
Assuntos
Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium ulcerans/enzimologia , Precursores de Proteínas/metabolismo , S-Adenosilmetionina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Carboxiliases/química , Carboxiliases/genética , Cromatografia Líquida de Alta Pressão , Descarboxilação , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Mutação , Mycobacterium ulcerans/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Espectrometria de Massas em Tandem , Tiramina/química , Tiramina/metabolismo , Tirosina/química , Tirosina/metabolismo , Valina/química , Valina/metabolismoRESUMO
Adrenocorticotropic hormone (ACTH) is the primary regulator of adrenal glucocorticoid production. Elevated levels of ACTH play a critical role in disease progression in several indications, including congenital adrenal hyperplasia and Cushing disease. We have generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1613) to ACTH. In vitro, ALD1613 neutralizes ACTH-induced signaling via all 5 melanocortin receptors and inhibited ACTH-induced cyclic adenosine monophosphate accumulation in a mouse adrenal cell line (Y1). ALD1613 administration to wild-type rats significantly reduced plasma corticosterone levels in a dose-dependent manner. In rodent models with either chronic infusion of ACTH or acute restraint stress-induced ACTH, corticosterone levels were significantly reduced by ALD1613. Administration of ALD1613 to nonhuman primates on days 1 and 7 stably reduced plasma cortisol levels >50% for 57 days. ALD1613 demonstrates the potential of a monoclonal antibody to be an effective therapeutic for conditions with elevated ACTH levels.
Assuntos
Hormônio Adrenocorticotrópico/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Hidrocortisona/sangue , Hiperplasia Suprarrenal Congênita/tratamento farmacológico , Hormônio Adrenocorticotrópico/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Células CHO , Corticosterona/sangue , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Humanos , Macaca fascicularis , Masculino , Hipersecreção Hipofisária de ACTH/tratamento farmacológico , Coelhos , Ratos , Ratos Endogâmicos Lew , Receptor Tipo 2 de Melanocortina/metabolismo , Estresse Psicológico/sangueRESUMO
Mycofactocin is a putative, peptide derived, cofactor that is associated primarily with the Mycobacterium genera including the pathogen M. tuberculosis. The pathway consists of the three genes mftA, mftB, and mftC that encode for the peptide substrate, peptide chaperone, and a radical S-adenosylmethionine protein (RS), respectively. Here, we show that the MftB acts as a peptide chaperone, binding MftA with a submicromolar KD (~ 100 nm) and MftC with a low micromolar KD (~ 2 µm). Moreover, we demonstrate that MftC is a radical S-adenosylmethionine (SAM) enzyme. Finally, we show that MftC catalyzes the oxidative decarboxylation of the peptide MftA.
Assuntos
Proteínas Ferro-Enxofre/genética , Mycobacterium ulcerans/enzimologia , Proteína O-Metiltransferase/genética , S-Adenosilmetionina/metabolismo , Catálise , Humanos , Proteínas Ferro-Enxofre/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Mycobacterium ulcerans/química , Mycobacterium ulcerans/genética , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Proteína O-Metiltransferase/química , S-Adenosilmetionina/química , Especificidade por SubstratoRESUMO
The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin- and flavodoxin reductase-based reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.
Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Mycobacterium/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Endopeptidases/química , Endopeptidases/genética , Mycobacterium/química , Mycobacterium/genéticaRESUMO
Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be â¼12 µm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD â¼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be â¼5 µm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein.
Assuntos
Proteínas de Bactérias/química , Endopeptidases/química , Methylobacterium extorquens/química , Chaperonas Moleculares/química , Complexos Multiproteicos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Espectrometria de Massas , Methylobacterium extorquens/genética , Methylobacterium extorquens/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Human THEM4 (hTHEM4) is comprised of a catalytically active hotdog-fold acyl-CoA thioesterase domain and an N-terminal domain of unknown fold and function. hTHEM4 has been linked to Akt1 regulation and cell apoptosis. Herein, we report the X-ray structure of hHTEM4 bound with undecan-2-one-CoA. Structure guided mutagenesis was carried out to confirm the catalytic residues. The N-terminal domain is shown to be partially comprised of irregular and flexible secondary structure, reminiscent of a protein-binding domain. We demonstrate direct hTHEM4-Akt1 binding by immunoprecipitation and by inhibition of Akt1 kinase activity, thus providing independent evidence that hTHEM4 is an Akt1 negative regulator.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Membrana/química , Tioléster Hidrolases/química , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismoRESUMO
Histone H3K4 trimethylation by the Set1/MLL family of proteins provides a hallmark for transcriptional activity from yeast to humans. In S. cerevisiae, H3K4 methylation is mediated by the Set1-containing COMPASS complex and is regulated in trans by prior ubiquitination of histone H2BK123. All of the events that regulate H2BK123ub and H3K4me are thought to occur at gene promoters. Here we report that this pathway is indispensable for methylation of the only other known substrate of Set1, K233 in Dam1, at kinetochores. Deletion of RAD6, BRE1, or Paf1 complex members abolishes Dam1 methylation, as does mutation of H2BK123. Our results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription. Moreover, our data identify a node of regulatory crosstalk in trans between a histone modification and modification on a nonhistone protein, demonstrating that changing chromatin states can signal functional changes in other essential cellular proteins and machineries.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Animais , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Mitose , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Histones undergo several different post-translational modifications that control a variety of physiological processes. These covalent modifications show substantial cross-regulation, providing a wealth of regulatory potential. New insights into the communication between modifications on histones have emerged in recent years. This review assesses the current understanding of cross-regulation of histone modifications and identifies future questions to be addressed in this field.