RESUMO
The development of microbes for conducting bioprocessing via synthetic biology involves design-build-test-learn (DBTL) cycles. To aid the designing step, we developed a computational technique that suggests next genetic modifications on the basis of relatedness to the user's design history of genetic modifications accumulated through former DBTL cycles conducted by the user. This technique, which comprehensively retrieves well-known designs related to the history, involves searching text for previous literature and then mining genes that frequently co-occur in the literature with those modified genes. We further developed a domain-specific lexical model that weights literature that is more related to the domain of metabolic engineering to emphasize genes modified for bioprocessing. Our technique made a suggestion by using a history of creating a Corynebacterium glutamicum strain producing shikimic acid that had 18 genetic modifications. Inspired by the suggestion, eight genes were considered by biologists for further modification, and modifying four of these genes proved experimentally efficient in increasing the production of shikimic acid. These results indicated that our proposed technique successfully utilized the former cycles to suggest relevant designs that biologists considered worth testing. Comprehensive retrieval of well-tested designs will help less-experienced researchers overcome the entry barrier as well as inspire experienced researchers to formulate design concepts that have been overlooked or suspended. This technique will aid DBTL cycles by feeding histories back to the next genetic design, thereby complementing the designing step.
Assuntos
Corynebacterium glutamicum/genética , Biologia Sintética/métodos , Corynebacterium glutamicum/metabolismo , Glucose/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Família Multigênica , Projetos de Pesquisa , Ácido Chiquímico/metabolismoRESUMO
Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.
RESUMO
MOTIVATION: Exploring metabolic pathways is one of the key techniques for developing highly productive microbes for the bioproduction of chemical compounds. To explore feasible pathways, not only examining a combination of well-known enzymatic reactions but also finding potential enzymatic reactions that can catalyze the desired structural changes are necessary. To achieve this, most conventional techniques use manually predefined-reaction rules, however, they cannot sufficiently find potential reactions because the conventional rules cannot comprehensively express structural changes before and after enzymatic reactions. Evaluating the feasibility of the explored pathways is another challenge because there is no way to validate the reaction possibility of unknown enzymatic reactions by these rules. Therefore, a technique for comprehensively capturing the structural changes in enzymatic reactions and a technique for evaluating the pathway feasibility are still necessary to explore feasible metabolic pathways. RESULTS: We developed a feasible-pathway-exploration technique using chemical latent space obtained from a deep generative model for compound structures. With this technique, an enzymatic reaction is regarded as a difference vector between the main substrate and the main product in chemical latent space acquired from the generative model. Features of the enzymatic reaction are embedded into the fixed-dimensional vector, and it is possible to express structural changes of enzymatic reactions comprehensively. The technique also involves differential-evolution-based reaction selection to design feasible candidate pathways and pathway scoring using neural-network-based reaction-possibility prediction. The proposed technique was applied to the non-registered pathways relevant to the production of 2-butanone, and successfully explored feasible pathways that include such reactions.
Assuntos
Fenômenos Fisiológicos Celulares , Redes e Vias Metabólicas , CatáliseRESUMO
In the ascidian Ciona robusta (formerly C. intestinalis type A), the mechanism underlying sperm penetration through the egg investment remains unknown. We previously reported that proteins containing both an astacin metalloprotease domain and thrombospondin type 1 repeats are abundant in the sperm surface protein-enriched fraction of C. robusta. Here we investigated the involvement of those proteins in fertilisation. We refined the sequences of astacin metalloproteases, confirmed that five of them are present in the sperm, and labelled them as tunicate astacin and thrombospondin type 1 repeat-containing (Tast) proteins. Fertilisation of C. robusta eggs was potently inhibited by a metalloprotease inhibitor GM6001. The eggs cleaved normally when they were vitelline coat-free or the inhibitor was added after insemination. Furthermore, vitelline coat proteins were degraded after incubation with intact sperm. These results suggest that sperm metalloproteases are indispensable for fertilisation, probably owing to direct or indirect mediation of vitelline-coat digestion during sperm penetration. TALEN-mediated knockout of Tast genes and the presence of GM6001 impaired larval development at the metamorphic stage, suggesting that Tast gene products play a key role in late development.
Assuntos
Proteínas do Ovo/metabolismo , Metaloproteases/fisiologia , Espermatozoides/metabolismo , Membrana Vitelina/metabolismo , Animais , Ciona intestinalis , Feminino , Masculino , Interações Espermatozoide-ÓvuloRESUMO
Sea anemones belonging to the genera Adamsia and Stylobates have a remarkable symbiotic relationship with hermit crabs. These symbiotic sea anemones produce a shell-like structure, called a "carcinoecium," that covers and extends over the gastropod shell of the host hermit crab as hermit crabs grow. This structure has been described as "chitinous carcinoecium" or "chitinous coating." A previous study investigated carcinoecia of Stylobates aeneus, the results of which indicated that it contained at least 1.7% chitin, while the remaining components were unidentified. Moreover, the microscopic structure of a carcinoecium still remains to be detailed. We, therefore, conducted detailed observations using a stereoscopic microscope and scanning electron microscope (SEM) and the analyses of the chemical composition of carcinoecia produced by Stylobates sp. (apparently conspecific with Isadamsia sp. "J" reported in Uchida and Soyama, 2001) associated with a pagurid hermit crab Pagurodofleinia doederleini collected in the south of the Shima Peninsula, Mie, Honshu Island, Japan at a depth of 294-306 m. Our results indicate that carcinoecia of Stylobates sp. contain HCl-soluble components (13%), NaOH-soluble components (38%), chitin (11%) and unidentified remnants (39%). Additionally, our observations show that Stylobates sp. incorporates dark- and white-colored particles that could be sand and/or mud into the carcinoecium.
RESUMO
Peanut agglutinin (PNA) is an established marker of the mammalian acrosome. However, we observed that PNA specifically binds to a unique intracellular structure alongside the nucleus in ascidian sperm. Here, we characterize the PNA-binding structure in sperm of marine invertebrates. PNA bound to the region between the mitochondrion and nucleus in spermatozoa of ascidians, sea urchins, and an appendicularian. However, PNA-binding substances were not exposed by the calcium ionophore ionomycin in three ascidian species, indicating that it is a distinct structure from the acrosome. Instead, the ascidian PNA-binding region was shed with the mitochondrion from the sperm head via an ionomycin-induced sperm reaction. The ascidian PNA-binding substance appeared to be solubilized with SDS, but not Triton X-100, describing its detergent resistance. Lectins, PHA-L4 , SSA, and MAL-I were detected at an area similar to the PNA-binding region, suggesting that it contains a variety of glycans. The location and some of the components of the PNA-binding region were similar to known endoplasmic reticulum (ER)-derived structures, although the ER marker concanavalin A accumulated at an area adjacent to but not overlapping the PNA-binding region. Therefore, we conclude that ascidian sperm possess a non-acrosomal, Triton-resistant, glycan-rich intracellular structure that may play a general role in reproduction of tunicates and sea urchins given its presence across a wide taxonomic range.
Assuntos
Núcleo Celular/metabolismo , Ciona , Mitocôndrias/metabolismo , Aglutinina de Amendoim/química , Ouriços-do-Mar , Animais , Ciona/citologia , Ciona/metabolismo , Masculino , Camundongos , Ouriços-do-Mar/citologia , Ouriços-do-Mar/metabolismoRESUMO
Snake venom metalloproteases (SVMPs) are members of the a disintegrin and metalloprotease (ADAM) family of proteins, as they possess similar domains. SVMPs are known to elicit snake venom-induced haemorrhage; however, the target proteins and cleavage sites are not known. In this work, we identified a target protein of vascular apoptosis-inducing protein 1 (VAP1), an SVMP, relevant to its ability to induce haemorrhage. VAP1 disrupted cell-cell adhesions by relocating VE-cadherin and γ-catenin from the cell-cell junction to the cytosol, without inducing proteolysis of VE-cadherin. The Wnt receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are known to promote catenin relocation, and are rendered constitutively active in Wnt signalling by truncation. Thus, we examined whether VAP1 cleaves LRP5/6 to induce catenin relocation. Indeed, we found that VAP1 cleaved the extracellular region of LRP6 and LRP5. This cleavage removes four inhibitory ß-propeller structures, resulting in activation of LRP5/6. Recombinant human ADAM8 and ADAM12 also cleaved LRP6 at the same site. An antibody against a peptide including the LRP6-cleavage site inhibited VAP1-induced VE-cadherin relocation and disruption of cell-cell adhesions in cultured cells, and blocked haemorrhage in mice in vivo. Intriguingly, animals resistant to the effects of haemorrhagic snake venom express variants of LRP5/6 that lack the VAP1-cleavage site, or low-density lipoprotein receptor domain class A domains involved in formation of the constitutively active form. The results validate LRP5/6 as physiological targets of ADAMs. Furthermore, they indicate that SVMP-induced cleavage of LRP5/6 causes disruption of cell-cell adhesion and haemorrhage, potentially opening new avenues for the treatment of snake bites.
Assuntos
Proteínas ADAM/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Venenos de Crotalídeos/metabolismo , Hemorragia/induzido quimicamente , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Metaloendopeptidases/metabolismo , Proteínas ADAM/farmacologia , Proteína ADAM12/metabolismo , Proteína ADAM12/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Resistência a Medicamentos , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Domínios Proteicos , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Vertebrados/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologiaRESUMO
Sperm proteins mediating sperm-egg interaction should be exhibited on the sperm surface, or exposed or released when sperm approach an egg. In ascidians (protochordates), sperm undergo a sperm reaction, characterized by enhanced sperm motility and mitochondrial swelling and shedding on contact with the vitelline coat (VC) or by treatment with Ca(2+) ionophore. Here, proteomic analysis was conducted on sperm exudates and sperm surface proteins using ionomycin-induced sperm reaction and cell-impermeable labeling in Ciona intestinalis type A (C. robusta). In the exudate from sperm treated with ionomycin, membrane proteins including a possible VC receptor CiUrabin were abundant, indicating the release of membranous compartments during sperm reaction. Among the surface proteins XP_009859314.1 (uncharacterized protein exhibiting homology to HrTTSP-1) was most abundant before the sperm reaction, but XP_004227079.1 (unknown Ig superfamily protein) appears to be most abundantly exposed by the sperm reaction. Moreover, proteins containing a notable set of domains, astacin-like metalloprotease domain and thrombospondin type 1 repeat(s), were found in this fraction. Possible roles in fertilization as well as localizations and behaviors of these proteins are discussed.
Assuntos
Ciona intestinalis/metabolismo , Ionomicina/farmacologia , Proteômica , Espermatozoides/metabolismo , Animais , Ionóforos de Cálcio/farmacologia , Ciona intestinalis/efeitos dos fármacos , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Espermatozoides/efeitos dos fármacos , Ubiquitina/metabolismoRESUMO
Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing (15)N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a (15)N-enrichment ratio of approximately 80%. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.
Assuntos
Bombyx/genética , Glicoproteínas/química , Imunoglobulina G/química , Marcação por Isótopo/métodos , Animais , Baculoviridae , Cromatografia Líquida , Regulação da Expressão Gênica , Glicoproteínas/genética , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Larva , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas em TandemRESUMO
KEY MESSAGE: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.
Assuntos
Nicotiana/genética , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Adalimumab/genética , Sequência de Aminoácidos , Sequência de Carboidratos , Glicosilação , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Isótopos de Nitrogênio , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Nicotiana/metabolismoRESUMO
The recent patent expirations of erythropoietin (EPO) have promoted the development of biosimilars. Two and one biosimilar EPO products were approved in 2007 in Europe and in 2010 in Japan, respectively. Glycosylation heterogeneity of EPO is very complex, and its pattern has a large impact on its in vivo activity. In this study, glycoform profilings of biosimilar and innovator EPO products were performed using LC/ESI-MS. Glycoforms of EPO were detected within the range of m/z 1700-3600 at the 10(+)-16(+) charge states. The charge-deconvoluted spectra showed complex glycoform mass profiles at 28,000-32,000 Da, and most of the observed peaks were assigned to the peptide (18,236 Da)+glycans with the compositions of NeuAc10-14Hexn+3HexNAcnFuc3 (n=16-26) with or without some O-acetylations (+42 Da) and attachment of NeuGc for NeuAc or oxidation (+16 Da). Analysis of de-N-glycosylated EPO showed the distributions of O-glycans of NeuAc1-2Hex1HexNAc1 and site occupancy. Each EPO product showed a characteristic glycoform profile with respect to sialylation, glycan size, O-acetylation of sialic acids and O-glycosylation. Analysis of darbepoetin suggested that glycans of darbepoetin were highly sialylated and O-acetylated. LC/ESI-MS was shown to be useful to evaluate the similarity of the glycoform profiles of EPO.
Assuntos
Medicamentos Biossimilares/química , Cromatografia Líquida/métodos , Eritropoetina/análogos & derivados , Eritropoetina/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Acetilação , Darbepoetina alfa , Glicosilação , Oxirredução , Polissacarídeos/químicaRESUMO
Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino acids. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3-6 and B22-24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3-6, whereas they increased in B22-24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.
Assuntos
Medição da Troca de Deutério/métodos , Insulina Lispro/química , Insulina de Ação Curta/química , Insulina/análogos & derivados , Insulina/química , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Deutério/química , Humanos , Hidrogênio/química , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
Glycosylation of cells is known to alter with several biological events such as cell differentiations and proliferations as well as some diseases. "Glycomic approaches", comprehensive qualitative and quantitative glycan analyses of the cells, have become increasingly important as a means of discovering biomarkers that have the potential of being used as disease diagnostic markers and molecular markers for cell characterizations. In this paper, we introduce a method of quantitative glycan profiling by liquid chromatography/mass spectrometry with a combination of an isotope tagging method. In addition, we demonstrate the potential of glycan profiling as a tool for the identification of differentiated human bone marrow mesenchymal stem cell (hMSC) and non-differentiated hMSC.
Assuntos
Glicômica/métodos , Controle de Qualidade , Engenharia Tecidual , Animais , Biomarcadores , Células da Medula Óssea/citologia , Diferenciação Celular , Células/metabolismo , Cromatografia Líquida/métodos , Produtos Finais de Glicação Avançada , Humanos , Espectrometria de Massas/métodos , Células-Tronco Mesenquimais , Camundongos , Polissacarídeos/análiseRESUMO
Insulin analog products for subcutaneous injection are prepared as solutions in which insulin analog molecules exist in several oligomeric states. Oligomeric stability can affect their onset and duration of action and has been exploited in designing them. To investigate the oligomeric stability of insulin analog products having different pharmacokinetics, we performed hydrogen/deuterium exchange mass spectrometry (HDX/MS), which is a rapid method to analyze dynamic aspects of protein structures. Two rapid-acting analogs (lispro and glulisine) incorporated deuteriums more and faster than recombinant human insulin, whereas a long-acting analog (glargine) and two intermediate-acting preparations (protamine-containing formulations) incorporated them less and more slowly. Kinetic analysis revealed that the number of slowly exchanged hydrogens (D(s)) (k<0.01 min(-1)) accounted for the difference in HDX reactivity among analogs. Furthermore, we found correlations between HDX kinetics and pharmacokinetics reported previously. Their maximum serum concentration (C(max)) was linearly correlated with D(s) (r=0.88) and the number of maximum exchangeable hydrogens (D(∞)) (r=0.89). The maximum drug concentration time (t(max)) was also correlated with reciprocals of D(s) and D(∞) (r=0.86 and r=0.96, respectively). Here we demonstrate the ability of HDX/MS to evaluate oligomeric stability of insulin analog products.
Assuntos
Medição da Troca de Deutério/métodos , Insulina Lispro/química , Insulina de Ação Prolongada/química , Insulina/análogos & derivados , Insulina/análise , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Estabilidade de Medicamentos , Humanos , Injeções Subcutâneas , Insulina Glargina , Insulina Lispro/farmacocinética , Insulina de Ação Prolongada/farmacocinética , Cinética , Dados de Sequência Molecular , Análise de Componente PrincipalRESUMO
The sorting nexin (SNX) family proteins, which contain a Phox homology (PX) domain, play crucial roles in regulating the intracellular membrane trafficking of the endocytic pathway. The proper coordination of this pathway is important for axonal elongation; however, little is known about the expression and intracellular dynamics of the SNX members during the formation of the nervous system. Here the authors found that SNX18, which belongs to the Src-homology-3-PX-Bin/Amphiphysin/Rvs domain-containing SNX subfamily, was specifically expressed in differentiating motor neurons in the chick and mouse embryonic spinal cord. The expression of SNX18 in embryonic spinal motor neurons was transient and was downregulated as the neurons matured. The authors further demonstrated that the localization of EGFP-SNX18 in growth cones was dynamically regulated and accumulated especially at areas in contact with permissive substrates. These findings collectively suggest that SNX18 may play an active role in axonal elongation.