RESUMEN
Algae encode up to five different types of cryptochrome photoreceptors. So far, relatively little is known about the biological functions of the DASH (Drosophila, Arabidopsis, Synechocystis and Homo)-type cryptochromes. The green alga Chlamydomonas reinhardtii encodes two of them. CRY-DASH1 also called DCRY1 has its maximal absorption peak in the UV-A range. It is localized in the chloroplast and plays an important role in balancing the photosynthetic machinery. Here, we performed a comparative analysis of chloroplast proteins from wild type and a knockout mutant of CRY-DASH1 named cry-dash1mut, using label-free quantitative proteomics as well as immunoblotting. Our results show upregulation of enzymes involved in specific pathways in the mutant including key enzymes of chlorophyll and carotenoid biosynthesis consistent with increased levels of photosynthetic pigments in cry-dash1mut. There is also an increase in certain redox as well as photosystem I and II proteins, including D1. Strikingly, CRY-DASH1 is coregulated in a D1 deletion mutant, where its amount is increased. In contrast, key proteins of the central carbon metabolism, including glycolysis/gluconeogenesis, dark fermentation and the oxidative pentose phosphate pathway are downregulated in cry-dash1mut. Similarly, enzymes of histidine biosynthesis are downregulated in cry-dash1mut leading to a reduction in the amount of free histidine. Yet, transcripts encoding for several of these proteins are at a similar level in the wild type and cry-dash1mut or even opposite. We show that CRY-DASH1 can bind to RNA, taking the psbA RNA encoding D1 as target. These data suggest that CRY-DASH1 regulates plastidial metabolic pathways at the posttranscriptional level.
Asunto(s)
Chlamydomonas reinhardtii , Proteínas de Cloroplastos , Criptocromos , Fotosíntesis , Plastidios , Vías Biosintéticas , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Regulación hacia Abajo , Histidina/biosíntesis , Histidina/genética , Plastidios/genética , Plastidios/metabolismo , Rayos Ultravioleta , Eliminación de Gen , Transcripción GenéticaRESUMEN
l-Histidine is an essential proteinogenic amino acid in food with extensive applications in the pharmaceutical field. Herein, we constructed a Corynebacterium glutamicum recombinant strain for efficient biosynthesis of l-histidine. First, to alleviate the l-histidine feedback inhibition, the ATP phosphoribosyltransferase mutant HisGT235P-Y56M was constructed based on molecular docking and high-throughput screening, resulting in the accumulation of 0.83 g/L of l-histidine. Next, we overexpressed rate-limiting enzymes including HisGT235P-Y56M and PRPP synthetase and knocked out the pgi gene in the competing pathway, which increased the l-histidine production to 1.21 g/L. Furthermore, the energy status was optimized by decreasing the reactive oxygen species level and enhancing the supply of adenosine triphosphate, reaching a titer of 3.10 g/L in a shake flask. The final recombinant strain produced 5.07 g/L of l-histidine in a 3 L bioreactor, without the addition of antibiotics and chemical inducers. Overall, this study developed an efficient cell factory for l-histidine biosynthesis by combinatorial protein engineering and metabolic engineering.
Asunto(s)
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ingeniería de Proteínas/métodos , Ingeniería Metabólica/métodos , Histidina/biosíntesis , Simulación por Computador , Biocatálisis , Mutación , Reactores BiológicosRESUMEN
Selenium is an essential micronutrient in diverse organisms. Two routes are known for its insertion into proteins and nucleic acids, via selenocysteine and 2-selenouridine, respectively1. However, despite its importance, pathways for specific incorporation of selenium into small molecules have remained elusive. Here we use a genome-mining strategy in various microorganisms to uncover a widespread three-gene cluster that encodes a dedicated pathway for producing selenoneine, the selenium analogue of the multifunctional molecule ergothioneine2,3. We elucidate the reactions of all three proteins and uncover two novel selenium-carbon bond-forming enzymes and the biosynthetic pathway for production of a selenosugar, which is an unexpected intermediate en route to the final product. Our findings expand the scope of biological selenium utilization, suggest that the selenometabolome is more diverse than previously thought, and set the stage for the discovery of other selenium-containing natural products.
Asunto(s)
Vías Biosintéticas , Genes Microbianos , Histidina/análogos & derivados , Compuestos de Organoselenio , Selenio , Productos Biológicos/química , Productos Biológicos/metabolismo , Vías Biosintéticas/genética , Carbono/metabolismo , Enzimas , Ergotioneína , Genes Microbianos/genética , Histidina/biosíntesis , Metaboloma/genética , Micronutrientes/biosíntesis , Familia de Multigenes/genética , Proteínas , Selenio/metabolismoRESUMEN
The [4Fe-4S]-dependent radical S-adenosylmethionine (SAM) proteins is one of large families of redox enzymes that are able to carry a panoply of challenging transformations. Despite the extensive studies of structure-function relationships of radical SAM (RS) enzymes, the electronic state-dependent reactivity of the [4Fe-4S] cluster in these enzymes remains elusive. Using combined MD simulations and QM/MM calculations, we deciphered the electronic state-dependent reactivity of the [4Fe-4S] cluster in Dph2, a key enzyme involved in the biosynthesis of diphthamide. Our calculations show that the reductive cleavage of the S-C(γ) bond is highly dependent on the electronic structure of [4Fe-4S]. Interestingly, the six electronic states can be classified into a low-energy and a high-energy groups, which are correlated with the net spin of Fe4 atom ligated to SAM. Due to the driving force of Fe4-C(γ) bonding, the net spin on the Fe4 moiety dictate the shift of the opposite spin electron from the Fe1-Fe2-Fe3 block to SAM. Such spin-regulated electron transfer results in the exchange-enhanced reactivity in the lower-energy group compared with those in the higher-energy group. This reactivity principle provides fundamental mechanistic insights into reactivities of [4Fe-4S] cluster in RS enzymes.
Asunto(s)
Histidina/análogos & derivados , Proteínas Hierro-Azufre/metabolismo , Proteínas/metabolismo , Transporte de Electrón , Histidina/biosíntesis , Histidina/química , Humanos , Proteínas Hierro-Azufre/química , Estructura Molecular , Oxidación-Reducción , Proteínas/químicaRESUMEN
All radical S-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1-Dph2, require at least one [4Fe-4S](Cys)3 cluster for activity. It is well-known in the radical-SAM enzyme community that the [4Fe-4S](Cys)3 cluster is extremely air-sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1-Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe-4S] cluster is easily degraded into a [3Fe-4S] cluster. Remarkably, the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. This mechanism to maintain radical-SAM enzyme activity in aerobic environments is likely general, and Dph3-like proteins may exist to keep other radical-SAM enzymes functional in aerobic environments.
Asunto(s)
Histidina/análogos & derivados , Proteínas Hierro-Azufre/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ditionita/metabolismo , Histidina/biosíntesis , Hierro/química , Proteínas Hierro-Azufre/química , Factor 2 de Elongación Peptídica/metabolismo , Proteínas Represoras/química , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Biobased processes to minimize environmental pollutants have attracted much attention. l-Carnosine has been produced by chemical synthesis, and as an alternative to this method, we newly developed engineered Corynebacterium glutamicum synthesizing l-carnosine. To develop the strain, the pentose phosphate pathway (PPP) was enhanced by attenuating flux to nonoxidative PPP. Enhanced PPP strengthened the histidine pathway and produced 5.0 g/L l-histidine and 3.9 mg/L l-carnosine. Then, the histidine synthetic pathway was reinforced by overexpressing HisG and Rel. This pathway reduced feedback inhibition by l-histidine and strengthened the flux of the histidine pathway; thus, it produced 552.20 mg/g DCW l-histidine. As a result, enhancement of the PPP accumulates more l-histidine than the histidine pathway; thus, the PPP was further enhanced by pgi gene alteration. For sufficient ß-alanine products, PanD was overexpressed and produced 99.17 mg/L l-carnosine. The final strain, Car15, which consolidated all three pathways, produced 323.26 mg/L l-carnosine via fed-batch fermentation. Finally, we confirmed the antioxidant and antiglycation effects of biologically synthesized l-carnosine, and the biologically synthesized l-carnosine showed inhibitory activity similar to that of commercial l-carnosine. Consequently, this study suggested a new biosynthetic process for l-carnosine and showed potential as a treatment for metabolic disorders through the assessment of its functions.
Asunto(s)
Antioxidantes/metabolismo , Proteínas Bacterianas/biosíntesis , Carnosina/biosíntesis , Corynebacterium glutamicum/metabolismo , Ingeniería Metabólica/métodos , Vía de Pentosa Fosfato/genética , Técnicas de Cultivo Celular por Lotes/métodos , Carboxiliasas/metabolismo , Corynebacterium glutamicum/genética , Fermentación , Histidina/biosíntesisRESUMEN
The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry.
Asunto(s)
Aminohidrolasas/metabolismo , Histidina/biosíntesis , Pirofosfatasas/metabolismo , Adenosina Monofosfato/metabolismo , Aminohidrolasas/química , Aminohidrolasas/genética , Catálisis , Dominio Catalítico , Medicago truncatula/enzimología , Medicago truncatula/metabolismo , Redes y Vías Metabólicas , Filogenia , Estructura Terciaria de Proteína , Pirofosfatasas/química , Pirofosfatasas/genética , Alineación de SecuenciaRESUMEN
Genetic engineering of the filamentous fungus Aspergillus oryzae still requires more suitable selection markers for fungal transformation. Our previous work has shown that Agrobacterium tumefaciens-mediated transformation (ATMT) based on the uridine/uracil auxotrophic mechanism with pyrG as the selection marker is very efficient for gene transfer in A. oryzae. In the present study, we delete the hisB gene, which is essential for histidine biosynthesis, in A. oryzae via homologous recombination and demonstrate that hisB is a reliable selection marker for genetic transformation of this fungus. Under optimal conditions, the ATMT efficiency of the histidine auxotrophic A. oryzae reached 515 transformants per 106 spores. Especially, we have succeeded in constructing a new ATMT system based on dual auxotrophic A. oryzae mutants with two different selection markers including hisB and pyrG. This dual auxotrophic ATMT system displayed a transformation efficiency of 232 transformants per 106 spores for the hisB marker and 318 transformants per 106 spores for the pyrG marker. By using these selectable markers, the co-expression of the DsRed and GFP fluorescent reporter genes was implemented in a single fungal strain. Furthermore, we could perform both the deletion and complementation of the laeA regulatory gene in the same strain of A. oryzae to examine its function. Conclusively, the ATMT system constructed in our work represents a promising genetic tool for studies on recombinant expression and gene function in the industrially important fungus A. oryzae.
Asunto(s)
Agrobacterium tumefaciens/fisiología , Aspergillus oryzae/crecimiento & desarrollo , Proteínas Fúngicas/genética , Ingeniería Genética/métodos , Aspergillus oryzae/genética , Eliminación de Gen , Genes Reporteros , Histidina/biosíntesis , Transformación Genética , Uracilo/biosíntesisRESUMEN
Intestinal flora structure and function change with age and have been associated with a variety of aging-related diseases. Until now, how age affects the functions of gut bacteria has not been fully understood. We used 16S-rRNA-sequencing technology and PICRUSt2 analysis to predict the functions encoded by intestinal flora in male Wistar rats across lifespan. We found that the abundance of gut microbiota genes encoding the L-tryptophan, L-histidine, L-leucine, inositol and catechol degradation pathways as well as L-arginine, ectoine, flavin and ubiquinol synthesis pathways increased with age. Differential analysis of the associated genera revealed that Rhodococcus spp. were significantly abundant during middle-old aged stage. This genus contributed greatly to the L-tryptophan, catechol and inositol degradation pathways as well as ectoine and L-arginine biosynthesis pathways. We concluded that gut bacteria-encoded functions such as amino acid metabolism, B vitamin metabolism, aromatic compound metabolism and energy metabolism varied in an age-dependent manner, and Rhodococcus spp. were the most associated functional bacteria in middle-old aged rats. These may be closely associated with the physiological phenotype of the aging process, which offers new insights for evaluating the relationship between intestinal flora and aging.
Asunto(s)
Envejecimiento/fisiología , Bacterias/aislamiento & purificación , Microbioma Gastrointestinal , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , ADN Bacteriano/genética , Heces/microbiología , Histidina/biosíntesis , Humanos , Masculino , ARN Ribosómico 16S/genética , Ratas , Ratas Wistar , Triptófano/biosíntesisRESUMEN
Intracellular pathogens including Mycobacterium tuberculosis (Mtb) have evolved with strategies to uptake amino acids from host cells to fulfil their metabolic requirements. However, Mtb also possesses de novo biosynthesis pathways for all the amino acids. This raises a pertinent question- how does Mtb meet its histidine requirements within an in vivo infection setting? Here, we present a mechanism in which the host, by up-regulating its histidine catabolizing enzymes through interferon gamma (IFN-γ) mediated signalling, exerts an immune response directed at starving the bacillus of intracellular free histidine. However, the wild-type Mtb evades this host immune response by biosynthesizing histidine de novo, whereas a histidine auxotroph fails to multiply. Notably, in an IFN-γ-/- mouse model, the auxotroph exhibits a similar extent of virulence as that of the wild-type. The results augment the current understanding of host-Mtb interactions and highlight the essentiality of Mtb histidine biosynthesis for its pathogenesis.
Asunto(s)
Histidina/biosíntesis , Interacciones Huésped-Patógeno , Interferón gamma/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Animales , Femenino , Histidina/deficiencia , Humanos , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7 , Conejos , Transducción de Señal , Células THP-1 , VirulenciaRESUMEN
Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.
Asunto(s)
Quinasa del Factor 2 de Elongación/genética , Histidina/análogos & derivados , Factores de Transcripción de Tipo Kruppel/genética , Antígenos de Histocompatibilidad Menor/genética , Proteínas Supresoras de Tumor/genética , Animales , Dominio BTB-POZ/genética , Sistemas CRISPR-Cas/genética , Regulación de la Expresión Génica/genética , Histidina/biosíntesis , Histidina/genética , Humanos , Metiltransferasas , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Procesamiento Proteico-Postraduccional/genética , Células RAW 264.7 , Factores de Transcripción/genéticaRESUMEN
The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.
Asunto(s)
Ascomicetos/genética , Ascomicetos/metabolismo , Sistemas CRISPR-Cas , Proteínas del Sistema Complemento/genética , Eliminación de Gen , Edición Génica/métodos , Plásmidos/genética , Farmacorresistencia Bacteriana/genética , Ingeniería Genética , Histidina/biosíntesis , Orotato Fosforribosiltransferasa/metabolismo , Triptófano/biosíntesis , Uracilo/biosíntesisRESUMEN
Histidine biosynthesis is an ancestral pathway that was assembled before the appearance of the Last Universal Common Ancestor; afterwards, it remained unaltered in all the extant histidine-synthesizing (micro)organisms. It is a metabolic cross-road interconnecting histidine biosynthesis to nitrogen metabolism and the de novo synthesis of purines. This interconnection is due to the reaction catalyzed by the products of hisH and hisF genes. The latter gene is an excellent model to study which trajectories have been followed by primordial cells to build the first metabolic routes, since its evolution is the result of different molecular rearrangement events, i.e. gene duplication, gene fusion, gene elongation, and domain shuffling. Additionally, this review summarizes data concerning the involvement of hisF and its product in other different cellular processes, revealing that HisF very likely plays a role also in cell division control and involvement in virulence and nodule development in different bacteria. From the metabolic viewpoint, these results suggest that HisF plays a central role in cellular metabolism, highlighting the interconnections of different metabolic pathways.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Genes Bacterianos/genética , Histidina/biosíntesis , Histidina/genética , Aminohidrolasas , Evolución Molecular , Duplicación de Gen , Fusión Génica , Redes y Vías Metabólicas/genética , VirulenciaRESUMEN
l-Histidine is a functional amino acid with numerous therapeutic and ergogenic properties. It is one of the few amino acids that is not produced on a large scale by microbial fermentation due to the lack of an efficient microbial cell factory. In this study, we demonstrated the engineering of wild-type Escherichia coli to overproduce histidine from glucose. First, removal of transcription attenuation and histidine-mediated feedback inhibition resulted in 0.8 g/L histidine accumulation. Second, chromosome-based optimization of the expression levels of histidine biosynthesis genes led to a 4.75-fold increase in histidine titer. Third, strengthening phosphoribosyl pyrophosphate supply and rerouting the purine nucleotide biosynthetic pathway improved the histidine production to 8.2 g/L. Fourth, introduction of the NADH-dependent glutamate dehydrogenase from Bacillus subtilis and the lysine exporter from Corynebacterium glutamicum enabled the final strain HW6-3 to produce 11.8 g/L histidine. Finally, 66.5 g/L histidine was produced under fed-batch fermentation, with a yield of 0.23 g/g glucose and a productivity of 1.5 g/L/h. This is the highest titer and productivity of histidine ever reported from an engineered strain. Additionally, the metabolic strategies utilized here can be applied to engineering other microorganisms for the industrial production of histidine and related bioproducts.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/metabolismo , Histidina/biosíntesis , Ingeniería Metabólica/métodos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , Corynebacterium glutamicum/metabolismo , Fermentación , Glutamato Deshidrogenasa/metabolismo , Lisina/metabolismo , Fosforribosil Pirofosfato/metabolismo , Nucleótidos de Purina/biosíntesisRESUMEN
Protein therapeutics, also known as biologics, are currently manufactured at centralized facilities according to rigorous protocols. The manufacturing process takes months and the delivery of the biological products needs a cold chain. This makes it less responsive to rapid changes in demand. Here, we report on technology application for on-demand biologics manufacturing (Bio-MOD) that can produce safe and effective biologics from cell-free systems at the point of care without the current challenges of long-term storage and cold-chain delivery. The objective of the current study is to establish proof-of-concept safety and efficacy of Bio-MOD-manufactured granulocyte colony-stimulating factor (G-CSF) in a mouse model of total body irradiation at a dose estimated to induce 30% lethality within the first 30 days postexposure. To illustrate on-demand Bio-MOD production feasibility, histidine-tagged G-CSF was manufactured daily under good manufacturing practice-like conditions prior to administration over a 16-day period. Bio-MOD-manufactured G-CSF improved 30-day survival when compared with saline alone (p = .073). In addition to accelerating recovery from neutropenia, the platelet and hemoglobin nadirs were significantly higher in G-CSF-treated animals compared with saline-treated animals (p < .05). The results of this study demonstrate the feasibility of consistently manufacturing safe and effective on-demand biologics suitable for real-time release.
Asunto(s)
Productos Biológicos/farmacología , Almacenaje de Medicamentos , Factor Estimulante de Colonias de Granulocitos/farmacología , Neutropenia/tratamiento farmacológico , Animales , Plaquetas/efectos de los fármacos , Sistema Libre de Células , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Hemoglobinas/efectos de los fármacos , Histidina/biosíntesis , Histidina/química , Humanos , Ratones , Neutropenia/sangre , Neutropenia/etiología , Neutropenia/patología , Irradiación Corporal Total/efectos adversosRESUMEN
Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2, or DPH3, which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that Drosophila nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.
Asunto(s)
Adhesión Celular/genética , Adhesión Celular/fisiología , Histidina/análogos & derivados , Riñón/metabolismo , Podocitos/metabolismo , Animales , Línea Celular , Drosophila , Fibronectinas/metabolismo , Proteínas del Choque Térmico HSP40/genética , Ensayos Analíticos de Alto Rendimiento , Histidina/biosíntesis , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Antígenos de Histocompatibilidad Menor/genética , Proteínas/genética , ARN Interferente Pequeño , Proteínas Supresoras de Tumor/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation elongation factor 2 (EF2). In the first step of diphthamide biosynthesis, a [4Fe-4S] cluster-containing radical SAM enzyme, Dph1-Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2. It was demonstrated previously that for the archaeal Dph2 homodimer, only one [4Fe-4S] cluster is necessary for the in vitro activity. Here, we demonstrate that for the eukaryotic Dph1-Dph2 heterodimer, the [4Fe-4S] cluster-binding cysteine residues in each subunit are required for diphthamide biosynthesis to occur in vivo. Furthermore, our in vitro reconstitution experiments with Dph1-Dph2 mutants suggested that the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. Our results reveal the asymmetric functional roles of the Dph1-Dph2 heterodimer and may help to understand how the Fe-S clusters in radical SAM enzymes are reduced in biology.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Histidina/análogos & derivados , Proteínas Hierro-Azufre/metabolismo , Secuencia de Aminoácidos , Histidina/biosíntesis , Histidina/química , Proteínas Hierro-Azufre/química , Mutación , Multimerización de Proteína , Pyrococcus horikoshii/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Kmmig1 as a disrupted mutant of MIG1 encoding a regulator for glucose repression in Kluyveromyces marxianus exhibits a histidine-auxotrophic phenotype. Genome-wide expression analysis revealed that only HIS4 in seven HIS genes for histidine biosynthesis was down-regulated in Kmmig1. Consistently, introduction of HIS4 into Kmmig1 suppressed the requirement of histidine. Considering the fact that His4 catalyzes four of ten steps in histidine biosynthesis, K. marxianus has evolved a novel and effective regulation mechanism via Mig1 for the control of histidine biosynthesis. Moreover, RNA-Seq analysis revealed that there were more than 1,000 differentially expressed genes in Kmmig1, suggesting that Mig1 is directly or indirectly involved in the regulation of their expression as a global regulator.
Asunto(s)
Vías Biosintéticas , Regulación Fúngica de la Expresión Génica , Histidina/biosíntesis , Kluyveromyces/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termotolerancia , Kluyveromyces/crecimiento & desarrollo , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Histidine biosynthesis, which is absent in animals, was shown to be highly conserved among gram-negative bacteria, thus making it an attractive target for antibiotic design. There are many fusion forms of enzymes in the histidine biosynthetic pathway and people still have limited knowledge about their domain organizations and catalytic mechanisms, due to the lack of structural information. Here we report the first crystal structure of Shigella flexneri bi-functional enzyme HisIE (SfHisIE) that functions in the 2nd and 3rd steps in the histidine biosynthetic pathway. This structure shows that HisIE exists as dimers with two loops (fusion loop) connecting the individual dimer of HisE and HisI in its N-terminus and C-terminus respectively. Our mutagenesis study shows mutations in this fusion loop are lethal for bacteria indicating the advantage of gene fusion in Histidine biosynthesis. Structural analysis revealed several highly conserved residues in the putative ligand binding grooves of HisE and HisI, showing an evolutionarily conserved catalytic mechanism shared among gram negative-bacteria.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Histidina/biosíntesis , Shigella flexneri/enzimología , Secuencia de Aminoácidos , Biocatálisis , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5 mg of DHHC3 and 1.0 mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1 mg/mL by SEC.