RESUMO
Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.
Assuntos
Carcinogênese , Ependimoma , Regulação Neoplásica da Expressão Gênica , Histonas , Fatores de Transcrição , Histonas/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Carcinogênese/metabolismo , Camundongos , Humanos , Ependimoma/genética , Ependimoma/metabolismo , Ependimoma/patologia , Fatores de Transcrição/metabolismo , Masculino , Feminino , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Epigênese Genética , Cromatina/metabolismo , Cromatina/genética , Microambiente Tumoral , Linhagem Celular Tumoral , Progressão da DoençaRESUMO
Epigenetic dysregulation is a universal feature of cancer that results in altered patterns of gene expression that drive malignancy. Brain tumors exhibit subtype-specific epigenetic alterations; however, the molecular mechanisms responsible for these diverse epigenetic states remain unclear. Here, we show that the developmental transcription factor Sox9 differentially regulates epigenomic states in high-grade glioma (HGG) and ependymoma (EPN). Using our autochthonous mouse models, we found that Sox9 suppresses HGG growth and expands associated H3K27ac states, while promoting ZFTA-RELA (ZRFUS) EPN growth and diminishing H3K27ac states. These contrasting roles for Sox9 correspond with protein interactions with histone deacetylating complexes in HGG and an association with the ZRFUS oncofusion in EPN. Mechanistic studies revealed extensive Sox9 and ZRFUS promoter co-occupancy, indicating functional synergy in promoting EPN tumorigenesis. Together, our studies demonstrate how epigenomic states are differentially regulated in distinct subtypes of brain tumors, while revealing divergent roles for Sox9 in HGG and EPN tumorigenesis.
Assuntos
Neoplasias Encefálicas , Ependimoma , Epigênese Genética , Fatores de Transcrição SOX9 , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Ependimoma/genética , Ependimoma/patologia , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/fisiologiaRESUMO
Astrocytes play central roles in normal brain function and are critical components of synaptic networks that oversee behavioral outputs. Despite their close affiliation with neurons, how neuronal-derived signals influence astrocyte function at the gene expression level remains poorly characterized, largely due to difficulties associated with dissecting neuron- versus astrocyte-specific effects. Here, we use an in vitro system of stem cell-derived astrocytes to identify gene expression profiles in astrocytes that are influenced by neurons and regulate astrocyte development. Furthermore, we show that neurotransmitters and neuromodulators induce distinct transcriptomic and chromatin accessibility changes in astrocytes that are unique to each of these neuroactive compounds. These findings are highlighted by the observation that noradrenaline has a more profound effect on transcriptional profiles of astrocytes compared to glutamate, gamma-aminobutyric acid (GABA), acetylcholine, and serotonin. This is demonstrated through enhanced noradrenaline-induced transcriptomic and chromatin accessibility changes in vitro and through enhanced calcium signaling in vivo. Taken together, our study reveals distinct transcriptomic and chromatin architecture signatures in astrocytes in response to neuronal-derived neuroactive compounds. Since astrocyte function is affected in all neurological disorders, this study provides a new entry point for exploring genetic mechanisms of astrocyte-neuron communication that may be dysregulated in disease.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Transcriptoma/genética , Acetilcolina/genética , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Ácido Glutâmico/genética , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Norepinefrina/genética , Serotonina/genética , Transdução de Sinais/efeitos dos fármacos , Ácido gama-Aminobutírico/genéticaRESUMO
A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are short-lived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.
Assuntos
Metabolismo , Modelos Biológicos , Escherichia coli/metabolismo , Ácido Mevalônico/metabolismo , Prenilação de ProteínaRESUMO
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are ubiquitous natural products. Bioactive RiPPs are produced from a precursor peptide, which is modified by enzymes. Usually, a single product is encoded in a precursor peptide. However, in cyanobactins and several other RiPP pathways, a single precursor peptide encodes multiple bioactive products flanking with recognition sequences known as "cassettes". The role of multiple cassettes in one peptide is mysterious, but in general their presence is a marker of biosynthetic plasticity. Here, we show that in cyanobactin biosynthesis the presence of multiple cassettes confers distributive enzyme processing to multiple steps of the pathway, a feature we propose to be a hallmark of multicassette RiPPs. TruD heterocyclase is stochastic and distributive. Although a canonical biosynthetic route is favored with certain substrates, every conceivable biosynthetic route is accepted. Together, these factors afford greater plasticity to the biosynthetic pathway by equalizing the processing of each cassette, enabling access to chemical diversity.
Assuntos
Peptídeos Cíclicos/biossíntese , Precursores de Proteínas/metabolismo , Alquilação , Sequência de Aminoácidos , Ciclização , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Domínios Proteicos , Precursores de Proteínas/química , Processamento de Proteína Pós-Traducional , Processos Estocásticos , Especificidade por SubstratoRESUMO
Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules.
Assuntos
Geraniltranstransferase/metabolismo , Peptídeos Cíclicos/biossíntese , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Cianobactérias/química , Cianobactérias/metabolismo , Geraniltranstransferase/isolamento & purificação , Peptídeos Cíclicos/químicaRESUMO
Recent innovations in peptide natural product biosynthesis reveal a surprising wealth of previously uncharacterized biochemical reactions that have potential applications in synthetic biology. Among these, the cyanobactins are noteworthy because these peptides are protected at their N- and C-termini by macrocyclization. Here, we use a novel bifunctional enzyme AgeMTPT to protect linear peptides by attaching prenyl and methyl groups at their free N- and C-termini. Using this peptide protectase in combination with other modular biosynthetic enzymes, we describe the total synthesis of the natural product aeruginosamide B and the biosynthesis of linear cyanobactin natural products. Our studies help to define the enzymatic mechanism of macrocyclization, providing evidence against the water exclusion hypothesis of transpeptidation and favoring the kinetic lability hypothesis.
Assuntos
Produtos Biológicos/metabolismo , Metiltransferases/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Transferases/metabolismo , Produtos Biológicos/química , Metiltransferases/química , Conformação Molecular , Peptídeo Hidrolases/química , Peptídeos/química , Transferases/químicaRESUMO
Neuronal activity drives global alterations in gene expression within neurons, yet how it directs transcriptional and epigenomic changes in neighboring astrocytes in functioning circuits is unknown. Here we show that neuronal activity induces widespread transcriptional upregulation and downregulation in astrocytes, highlighted by the identification of a neuromodulator transporter Slc22a3 as an activity-inducible astrocyte gene regulating sensory processing in the olfactory bulb. Loss of astrocytic Slc22a3 reduces serotonin levels in astrocytes, leading to alterations in histone serotonylation. Inhibition of histone serotonylation in astrocytes reduces expression of GABA biosynthetic genes and GABA release, culminating in olfactory deficits. Our study reveals that neuronal activity orchestrates transcriptional and epigenomic responses in astrocytes, while illustrating new mechanisms for how astrocytes process neuromodulatory input to gate neurotransmitter release for sensory processing.
RESUMO
Neuronal activity drives alterations in gene expression within neurons, yet how it directs transcriptional and epigenomic changes in neighboring astrocytes in functioning circuits is unknown. We found that neuronal activity induces widespread transcriptional up-regulation and down-regulation in astrocytes, highlighted by the identification of Slc22a3 as an activity-inducible astrocyte gene that encodes neuromodulator transporter Slc22a3 and regulates sensory processing in the mouse olfactory bulb. Loss of astrocytic Slc22a3 reduced serotonin levels in astrocytes, leading to alterations in histone serotonylation. Inhibition of histone serotonylation in astrocytes reduced the expression of γ-aminobutyric acid (GABA) biosynthetic genes and GABA release, culminating in olfactory deficits. Our study reveals that neuronal activity orchestrates transcriptional and epigenomic responses in astrocytes while illustrating new mechanisms for how astrocytes process neuromodulatory input to gate neurotransmitter release for sensory processing.
Assuntos
Astrócitos , Histonas , Bulbo Olfatório , Percepção Olfatória , Proteínas de Transporte de Cátions Orgânicos , Serotonina , Transmissão Sináptica , Animais , Camundongos , Astrócitos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Histonas/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Serotonina/metabolismo , Bulbo Olfatório/metabolismo , Epigênese Genética , Percepção Olfatória/genética , Percepção Olfatória/fisiologiaRESUMO
In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems.
Assuntos
Proteínas do Capsídeo/síntese química , Capsídeo/química , Complexos Multienzimáticos/síntese química , Montagem de Vírus , Proteínas do Capsídeo/genética , Eletroforese em Gel de Ágar , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/genéticaRESUMO
Astrocyte reactivity can directly modulate nervous system function and immune responses during disease and injury. However, the consequence of human astrocyte reactivity in response to specific contexts and within neural networks is obscure. Here, we devised a straightforward bioengineered neural organoid culture approach entailing transcription factor-driven direct differentiation of neurons and astrocytes from human pluripotent stem cells combined with genetically encoded tools for dual cell-selective activation. This strategy revealed that Gq-GPCR activation via chemogenetics in astrocytes promotes a rise in intracellular calcium followed by induction of immediate early genes and thrombospondin 1. However, astrocytes also undergo NF-κB nuclear translocation and secretion of inflammatory proteins, correlating with a decreased evoked firing rate of cocultured optogenetic neurons in suboptimal conditions, without overt neurotoxicity. Altogether, this study clarifies the intrinsic reactivity of human astrocytes in response to targeting GPCRs and delivers a bioengineered approach for organoid-based disease modeling and preclinical drug testing.
Assuntos
Astrócitos/metabolismo , Bioengenharia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Organoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Trifosfato de Adenosina/farmacologia , Astrócitos/patologia , Cálcio/metabolismo , Linhagem Celular , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Inflamação/patologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Reprodutibilidade dos Testes , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Sinaptofisina/metabolismoRESUMO
Oligodendrocyte precursor cell differentiation into myelinating oligodendrocytes is critical for remyelination in the central nervous system after injury. In this issue of Neuron, Niu et al. (2021) detail a novel role for ring finger protein Rnf43, which is expressed in response to injury and is essential to promote remyelination in vivo.
Assuntos
Células Precursoras de Oligodendrócitos , Remielinização , Diferenciação Celular , Sistema Nervoso Central , OligodendrogliaRESUMO
The role of transcription factors during astrocyte development and their subsequent effects on neuronal development has been well studied. Less is known about astrocytes contributions towards circuits and behavior in the adult brain. Astrocytes play important roles in synaptic development and modulation, however their contributions towards neuronal sensory function and maintenance of neuronal circuit architecture remain unclear. Here, we show that loss of the transcription factor Sox9 results in both anatomical and functional changes in adult mouse olfactory bulb (OB) astrocytes, affecting sensory processing. Indeed, astrocyte-specific deletion of Sox9 in the OB results in decreased odor detection thresholds and discrimination and it is associated with aberrant neuronal sensory response maps. At functional level, loss of astrocytic Sox9 impairs the electrophysiological properties of mitral and tufted neurons. RNA-sequencing analysis reveals widespread changes in the gene expression profiles of OB astrocytes. In particular, we observe reduced GLT-1 expression and consequential alterations in glutamate transport. Our findings reveal that astrocytes are required for physiological sensory processing and we identify astrocytic Sox9 as an essential transcriptional regulator of mature astrocyte function in the mouse OB.
Assuntos
Astrócitos/metabolismo , Bulbo Olfatório/fisiologia , Fatores de Transcrição SOX9/metabolismo , Sensação/fisiologia , Animais , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Fatores de Transcrição SOX9/genética , Fatores de TranscriçãoRESUMO
Astrocytes are the most abundant type of glial cell in the central nervous system and perform a myriad of vital functions, however, the nature of their diversity remains a longstanding question in neuroscience. Using transcription factor motif discovery analysis on region-specific gene signatures from astrocytes we uncovered universal and region-specific transcription factor expression profiles. This analysis revealed that motifs for Nuclear Factor-I (NFI) are present in genes enriched in astrocytes from all regions, with NFIB and NFIX exhibiting pan-astrocyte expression in the olfactory bulb, hippocampus, cortex, and brainstem. Further analysis into region-specific motif patterns, identified Nkx3-1, Stat4, Pgr, and Nkx6-1 as prospective region-specific transcription factors. Validation studies revealed that Nkx6-1 is exclusively expressed in astrocytes in the brainstem and associates with the promoters of several brainstem specific target genes. These studies illustrate the presence of multiple transcriptional layers in astrocytes across diverse brain regions and provide a new entry point for examining how astrocyte diversity is specified and maintained.
RESUMO
Astrocytes play essential roles in brain function by supporting synaptic connectivity and associated circuits. How these roles are regulated by transcription factors is unknown. Moreover, there is emerging evidence that astrocytes exhibit regional heterogeneity, and the mechanisms controlling this diversity remain nascent. Here, we show that conditional deletion of the transcription factor nuclear factor I-A (NFIA) in astrocytes in the adult brain results in region-specific alterations in morphology and physiology that are mediated by selective DNA binding. Disruptions in astrocyte function following loss of NFIA are most pronounced in the hippocampus, manifested by impaired interactions with neurons, coupled with diminution of learning and memory behaviors. These changes in hippocampal astrocytes did not affect basal neuronal properties but specifically inhibited synaptic plasticity, which is regulated by NFIA in astrocytes through calcium-dependent mechanisms. Together, our studies reveal region-specific transcriptional dependencies for astrocytes and identify astrocytic NFIA as a key transcriptional regulator of hippocampal circuits.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Cálcio/metabolismo , Regulação da Expressão Gênica , Aprendizagem/fisiologia , Fatores de Transcrição NFI/genética , Animais , Astrócitos/fisiologia , Encéfalo/citologia , Encéfalo/fisiopatologia , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiopatologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/fisiopatologia , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Camundongos , Camundongos Knockout , Vias Neurais , Plasticidade Neuronal , Neurônios , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Bulbo Olfatório/fisiopatologia , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Memória Espacial/fisiologiaRESUMO
Reactive astrocytes are associated with every form of neurological injury. Despite their ubiquity, the molecular mechanisms controlling their production and diverse functions remain poorly defined. Because many features of astrocyte development are recapitulated in reactive astrocytes, we investigated the role of nuclear factor I-A (NFIA), a key transcriptional regulator of astrocyte development whose contributions to reactive astrocytes remain undefined. Here, we show that NFIA is highly expressed in reactive astrocytes in human neurological injury and identify unique roles across distinct injury states and regions of the CNS. In the spinal cord, after white matter injury (WMI), NFIA-deficient astrocytes exhibit defects in blood-brain barrier remodeling, which are correlated with the suppression of timely remyelination. In the cortex, after ischemic stroke, NFIA is required for the production of reactive astrocytes from the subventricular zone (SVZ). Mechanistically, NFIA directly regulates the expression of thrombospondin 4 (Thbs4) in the SVZ, revealing a key transcriptional node regulating reactive astrogenesis. Together, these studies uncover critical roles for NFIA in reactive astrocytes and illustrate how region- and injury-specific factors dictate the spectrum of reactive astrocyte responses.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Sistema Nervoso Central/lesões , Sistema Nervoso Central/metabolismo , Fatores de Transcrição NFI/metabolismo , Adulto , Animais , Barreira Hematoencefálica , Diferenciação Celular , Sistema Nervoso Central/patologia , Humanos , Camundongos , Camundongos Knockout , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Fatores de Transcrição NFI/deficiência , Fatores de Transcrição NFI/genética , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Remielinização , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Trombospondinas/genética , Trombospondinas/metabolismoRESUMO
Ribosomally synthesized natural products are found in all forms of life. Their biosynthesis uses simple ribosomally synthesized peptides as starting materials that are transformed into complex structures via posttranslational modifications, enriched with elaborate chemical scaffolds that make them desirable as pharmacological tools. In addition, these natural products often exhibit combinatorial biosynthesis, making them attractive targets for engineering. An increasing knowledge of their biosynthetic machinery has provided key insights into their fascinating chemistry. Marine organisms have been a rich source of this class of natural products and here we review the lessons learned from marine life that enables exploitation of their potential for combinatorial engineering, opening up new routes for peptide-based drug discovery.
Assuntos
Biologia Marinha , Biossíntese Peptídica , Biblioteca de Peptídeos , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016]. METHODS: A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. RESULTS: When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. CONCLUSION: It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. SIGNIFICANCE: The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems.
Assuntos
Ascomicetos/enzimologia , Sistema Enzimático do Citocromo P-450/química , Proteínas Fúngicas/química , Peroxidase/química , Catálise , OxirreduçãoRESUMO
Macrocyclases and other posttranslational enzymes afford derived peptides with improved properties for pharmaceutical and biotechnological applications. Here, we asked whether multiple posttranslational modifications could be simultaneously controlled and matched to rationally generate new peptide derivatives. We reconstituted the cyanobactin peptide natural products in vitro with up to five different posttranslational enzymes in a single tube. By manipulating the order of addition and identity of enzymes and exploiting their broad-substrate tolerance, we engineered the production of highly unnatural derivatives, including an N-C peptide macrocycle of 22 amino acids in length. In addition to engineering, this work better defines the macrocyclization mechanism, provides the first biochemical demonstration of Ser/Thr posttranslational prenylation, and is the first example of reconstitution of a native, multistep RiPP pathway with multiple enzymes in one pot. Overall, this work demonstrates how the modularity of posttranslational modification enzymes can be used to design and synthesize desirable peptide motifs.
Assuntos
Cianobactérias/enzimologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/síntese química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways pat and tru have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit "substrate evolution", where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme-substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.