RESUMO
Rare earth elements (REEs) are critical for our modern lifestyles and the transition to a low-carbon economy. Recent advances in our understanding of the role of REEs in biology, particularly methylotrophy, have provided opportunities to explore biotechnological innovations to improve REE mining and recycling. In addition to bacterial accumulation and concentration of REEs, biological REE binders, including proteins (lanmodulin, lanpepsy) and small molecules (metallophores and cofactors) have been identified that enable REE concentration and separation. REE-binding proteins have also been used in several mechanistically distinct REE biosensors, which have potential application in mining and medicine. Notably, the role of REEs in biology has only been known for a decade, suggesting their considerable scope for developing new understanding and novel applications.
Assuntos
Bactérias , Metais Terras Raras , Metais Terras Raras/metabolismo , Metais Terras Raras/química , Bactérias/metabolismo , Bactérias/genética , Bactérias/química , Biotecnologia/métodosRESUMO
Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.
Assuntos
Técnicas Biossensoriais , RNA Mensageiro , Técnicas Biossensoriais/métodos , RNA Mensageiro/genética , RNA Mensageiro/análise , Humanos , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismoRESUMO
Eukaryotic cell-free protein expression systems enable rapid production of recombinant multidomain proteins in their functional form. A cell-free system based on the rapidly growing protozoan Leishmania tarentolae (LTE) has been extensively used for protein engineering and analysis of protein interaction networks. However, like other eukaryotic cell-free systems, LTE deteriorates at ambient temperatures and requires deep freezing for transport and storage. In this study, we report the development of a lyophilized version of LTE. Use of lyoprotectants such as poly(ethylene glycol) and trehalose during the drying process allows retention of 76% of protein expression activity versus nonlyophilized controls. Lyophilized LTE is capable of withstanding storage at room temperature for over 2 weeks. We demonstrated that upon reconstitution the lyophilized LTE could be used for in vitro expression of active enzymes, analysis of protein-protein interactions by AlphaLISA assay, and functional analysis of protein biosensors. Development of lyophilized LTE lowers the barriers to its distribution and opens the door to its application in remote areas.
Assuntos
Leishmania , Leishmania/metabolismo , Sistema Livre de Células/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , ProteômicaRESUMO
The emergence of viral threats such as Ebola, ZIKA, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires a rapid and efficient approach for elucidating mechanisms of pathogenesis and development of therapeutics. In this context, cell-free protein synthesis (CFPS) holds a promise to resolve the bottlenecks of multiplexed protein production and interaction analysis among host and pathogen proteins. Here, we applied a eukaryotic CFPS system based on Leishmania tarentolae extract (LTE) protein expression in combination with AlphaLISA proximity-based protein interaction technology to identify intraviral and viral-human protein interactions of SARS-CoV-2 virus that can potentially be targeted by the existing or novel antiviral therapeutics. We produced and tested 54 putative human-viral protein pairs in vitro and identified 45 direct binary protein interactions. As a casing example of the assay's suitability for drug development applications, we analyzed the effect of a putative biologic on the human angiotensin-converting enzyme 2/receptor-binding domain (hACE2/RBD) interaction. This suggests that the presented pathogen characterization platform can facilitate the development of new therapeutic agents.
RESUMO
The construction and assembly of artificial allosteric protein switches into information and energy processing networks connected to both biological and non-biological systems is a central goal of synthetic biology and bionanotechnology. However, designing protein switches with the desired input, output and performance parameters is challenging. Here we use a range of reporter proteins to demonstrate that their chimeras with duplicated receptor domains produce YES gate protein switches with large (up to 9,000-fold) dynamic ranges and fast (minutes) response rates. In such switches, the epistatic interactions between largely independent synthetic allosteric sites result in an OFF state with minimal background noise. We used YES gate protein switches based on ß-lactamase to develop quantitative biosensors of therapeutic drugs and protein biomarkers. Furthermore, we demonstrated the reconfiguration of YES gate switches into AND gate switches controlled by two different inputs, and their assembly into signalling networks regulated at multiple nodes.
RESUMO
Protein biosensors hold a promise to transform the way we collect physiological data by enabling quantification of biomarkers outside of specialized laboratory environment. However, achieving high specificity and sensitivity in homogeneous assay format remains challenging. Here we report construction of fluorescent biosensor arrays based on artificial allosteric α-amylase-activated PQQ-dependent glucose dehydrogenase (Amy-GDH). Amy-GDH was covalently immobilized on silica nanoparticles that were then arrayed on fiberglass sheets. The activity of the biosensor was monitored using a smartphone camera via emergence of bright fluorescence (λex 365 nm) originating from reduced phenazine methosulfate upon glucose oxidation by Amy-GDH. We show that such biosensor arrays demonstrate an apparent Kd of 115 pM for α-amylase with a detection limit of 2 pM. Using the developed biosensor arrays, we were able to specifically and accurately quantify the concentration of α-amylase in biological fluids such as serum and saliva. We propose that the presented approach can enable construction of ultrasensitive point-of-care diagnostic arrays.
Assuntos
Técnicas Biossensoriais , alfa-Amilases , Glucose , Saliva , OxirreduçãoRESUMO
The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.
Assuntos
Anti-Infecciosos , Triagem , Espécies Reativas de Oxigênio/metabolismo , NADP/metabolismo , Macrófagos/metabolismo , Anti-Infecciosos/metabolismo , Via de Pentose Fosfato/fisiologiaRESUMO
Biological homeostasis is a dynamic and elastic equilibrium of countless interlinked biochemical reactions. A key goal of life sciences is to understand these dynamics; bioengineers seek to reconfigure such networks. Both goals require the ability to monitor the concentration of individual intracellular metabolites with sufficient spatiotemporal resolution. To achieve this, a range of protein or protein/DNA signalling circuits with optical readouts have been constructed. Protein biosensors can provide quantitative information at subsecond temporal and suborganelle spatial resolution. However, their construction is fraught with difficulties related to integrating the affinity- and selectivity-endowing components with the signal reporters. We argue that development of efficient approaches for construction of chemically induced dimerisation systems and reporter domains with large dynamic ranges will solve these problems.
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Técnicas Biossensoriais , Proteínas , Proteínas/metabolismoRESUMO
Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.
Assuntos
Anti-Inflamatórios , Produtos Biológicos , Colite , Proteínas de Helminto , Doenças Inflamatórias Intestinais , Animais , Anti-Inflamatórios/farmacologia , Produtos Biológicos/farmacologia , Colite/tratamento farmacológico , Proteínas de Helminto/genética , Proteínas de Helminto/farmacologia , Helmintos , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/parasitologia , CamundongosRESUMO
Protein biosensors play increasingly important roles in cell and neurobiology and have the potential to revolutionise the way clinical and industrial analytics are performed. The gradual transition from multicomponent biosensors to fully integrated single chain allosteric biosensors has brought the field closer to commercial applications. We evaluate various approaches for converting constitutively active protein reporter domains into analyte operated switches. We discuss the paucity of the natural receptors that undergo conformational changes sufficiently large to control the activity of allosteric reporter domains. This problem can be overcome by constructing artificial versions of such receptors. The design path to such receptors involves the construction of Chemically Induced Dimerisation systems (CIDs) that can be configured to operate single and two-component biosensors.
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Técnicas Biossensoriais , Engenharia de Proteínas , Domínios Proteicos , ProteínasRESUMO
Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera's activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen-deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.
Assuntos
Regulação Alostérica , Calmodulina , Glucose Desidrogenase , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão , Calmodulina/química , Calmodulina/genética , Glucose Desidrogenase/química , Glucose Desidrogenase/genética , Ligantes , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , TermodinâmicaRESUMO
Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method's suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing.
Assuntos
Técnicas Biossensoriais , Tacrolimo , Técnicas Biossensoriais/métodos , Ciclosporina , Glucose 1-Desidrogenase/metabolismo , Humanos , Proteínas Recombinantes de FusãoRESUMO
Innate immune signaling by Toll-like receptors (TLRs) involves receptor phosphorylation, which helps to shape and drive key inflammatory outputs, yet our understanding of the kinases and mechanisms that mediate TLR phosphorylation is incomplete. Spleen tyrosine kinase (Syk) is a nonreceptor protein tyrosine kinase, which is known to relay adaptive and innate immune signaling, including from TLRs. However, TLRs do not contain the conserved dual immunoreceptor tyrosine-based activation motifs that typically recruit Syk to many other receptors. One possibility is that the Syk-TLR association is indirect, relying on an intermediary scaffolding protein. We previously identified a role for the palmitoylated transmembrane adapter protein SCIMP in scaffolding the Src tyrosine kinase Lyn, for TLR phosphorylation, but the role of SCIMP in mediating the interaction between Syk and TLRs has not yet been investigated. Here, we show that SCIMP recruits Syk in response to lipopolysaccharide-mediated TLR4 activation. We also show that Syk contributes to the phosphorylation of SCIMP and TLR4 to enhance their binding. Further evidence pinpoints two specific phosphorylation sites in SCIMP critical for its interaction with Syk-SH2 domains in the absence of immunoreceptor tyrosine-based activation motifs. Finally, using inhibitors and primary macrophages from SCIMP-/- mice, we confirm a functional role for SCIMP-mediated Syk interaction in modulating TLR4 phosphorylation, signaling, and cytokine outputs. In conclusion, we identify SCIMP as a novel, immune-specific Syk scaffold, which can contribute to inflammation through selective TLR-driven inflammatory responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Membrana , Quinase Syk , Receptor 4 Toll-Like , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Macrófagos/enzimologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Quinase Syk/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks. We hypothesize that circular permutation of proteins increases the probability of functional coupling of new N- and C- termini with the protein's active center through increased local structural disorder. To test this we construct a synthetically allosteric version of circular permutated NanoLuc luciferase that can be activated through ligand-induced intramolecular non-covalent cyclisation. This switch module is tolerant of the structure of binding domains and their ligands, and can be used to create biosensors of proteins and small molecules. The developed biosensors covers a range of emission wavelengths and displays sensitivity as low as 50pM and dynamic range as high as 16-fold and could quantify their cognate ligand in human fluids. We apply hydrogen exchange kinetic mass spectroscopy to analyze time resolved structural changes in the developed biosensors and observe ligand-mediated folding of newly created termini.
Assuntos
Regulação Alostérica , Luciferases/genética , Luciferases/metabolismo , Engenharia Metabólica , Regulação Alostérica/genética , Regulação da Expressão Gênica , Humanos , Ligantes , Luciferases/química , Modelos MolecularesRESUMO
Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli-based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment.
Assuntos
Sistema Livre de Células/efeitos dos fármacos , Medicamentos Genéricos/química , Medicamentos Genéricos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Animais , Anticorpos , Análise Custo-Benefício , Interpretação Estatística de Dados , Dissulfetos , Drosophila melanogaster , Escherichia coli , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leishmania , Peptídeos/genética , Agregados Proteicos , Domínios Proteicos , RNA Ribossômico 16S , Biologia Sintética , TermodinâmicaRESUMO
Protein biosensors play an increasingly important role as reporters for research and clinical applications. Here we present an approach for the construction of fully integrated but modular electrochemical biosensors based on the principal component of glucose monitors PQQ-glucose dehydrogenase (PQQ-GDH). We designed allosterically regulated circular permutated variants of PQQ-GDH that show large (>10-fold) changes in enzymatic activity following intramolecular scaffolding of the newly generated N- and C termini by ligand binding domain/ligand complexes. The developed biosensors demonstrated sub-nanomolar affinities for small molecules and proteins in colorimetric and electrochemical assays. For instance, the concentration of Cyclosporineâ A could be measured in 1â µL of undiluted blood with the same accuracy as the leading diagnostic technique that uses 50 times more sample. We further used this biosensor to construct highly porous gold bioelectrodes capable of robustly detecting concentrations of Cyclosporineâ A as low as 20â pM and retained functionality in samples containing at least 60 % human serum.
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Técnicas Biossensoriais , Ciclosporina/sangue , Técnicas Eletroquímicas , Glucose Desidrogenase/química , Glucose Desidrogenase/metabolismo , HumanosRESUMO
Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.
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Técnicas Biossensoriais/instrumentação , Dimerização , Eletrônica , Metotrexato/química , Eletroquímica , Humanos , Ligantes , Metotrexato/sangue , Peptídeos/química , Polímeros/química , Proteínas/metabolismoRESUMO
Rapid techniques for producing high-quality recombinant proteins are essential for fast protein functional analysis, as well as various screening applications. Cell-free protein expression is an enabling tool in protein research capable of producing high-quality proteins within a few hours. In this chapter, we describe the use of a Leishmania tarentolae-based cell-free expression system to produce antibody fragments coupled to the analysis of their interaction with their ligands. Interaction analysis is performed using the scalable and sensitive AlphaLISA bead proximity assay. The method presented in this chapter offers a rapid and inexpensive approach for production of putative interacting protein pairs, as well as a multiplexable approach for their rapid interaction analysis.