Substrate plasticity of a fungal peptide α-N-methyltransferase

This work was financially supported by the Commission for Technology and Innovation (CTI/Innosuisse Grant No. CTI 25951.2), the Swiss National Science Foundation (Grant No. 31003A_173097), Wellcome Trust (Grant No. 094476/Z/10/ Z), and BBSRC (Grant No. BB/R018189/1).The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.Publisher PDFPeer reviewe

Synthetic biology circuits: temperature and myocardial infarction sensors for precision therapeutics

Personalized medicine is the science for developing remedies tailored to the individual necessities of each and every patient. The regulatory approval of the first engineered mammalian cell therapies enhanced the anticipation that these are to become the next pillar of medical care for complex and chronic disease. Although such cells are biological entities, they are modified using engineering principles in what is now established as the field of synthetic biology, and could thus be better described as biocomputing units. The foundations of these systems lie in the integrated therapeutic circuitry, which may provide an open or closed-loop control over the beneficial cellular functions. Open-loops would allow for more flexibility as the input strength and processes can be varied, while closed-loops are rigid and closely emulate the natural physiology. A common feature of every cellular circuit are the input converters, which may also be transcription factors activated by specialized cues. In this work we engineered the first mammalian transcription factor directly sensing temperatures in the physiological range from 38 to 40 °C. This enabled its use as a fever sensor in pyrexia animal models as well as for precise thermoelectrical control over therapeutic implants of engineered cells for controlling glycemia in a type-1-diabetes model. Capitalizing on this work, we envisioned natural energy sources such as sunlight as facilitators for temperature-based cellular control. In particular, we engineered endocrine cells to accumulate diverse therapeutic proteins in their storage granules and release them within minutes upon thermal stimulation in physiological saline. This generated injection ready solutions in an emergency biopharmaceutical process for production of therapeutics in remote areas. Finally, we engineered a closed-loop circuit capable of sensing cardiac troponin I, a protein considered as a gold standard biomarker for myocardial infarction, and responding to it with the secretion of a half-life optimized cardioprotective peptide

Biomarker-driven feedback control of synthetic biology systems for next-generation personalized medicine

Many current clinical therapies for chronic diseases involve administration of drugs using dosage and bioavailability parameters estimated for a generalized population. This standard approach carries the risk of under dosing, which may result in ineffective treatment, or overdosing, which may cause undesirable side effects. Consequently, maintaining a drug concentration in the therapeutic window often requires frequent monitoring, adversely affecting the patient’s quality of life. In contrast, endogenous biosystems have evolved finely tuned feedback control loops that govern the physiological functions of the body based on multiple input parameters. To provide personalized treatment for chronic diseases, therefore, we require synthetic systems that can similarly generate a calibrated therapeutic response. Such engineered autonomous closed-loop devices should incorporate a sensor that actively tracks and evaluates the disease severity based on one or more biomarkers, as well as components that utilize these molecular inputs to bio compute and deliver the appropriate level of therapeutic output. Here, we review recent advances in applications of the closed-loop design principle in biomedical implants for treating severe and chronic diseases, highlighting translational studies of cellular therapies. We describe the engineering principles and components of closed-loop therapeutic devices, and discuss their potential to become a key pillar of personalized medicine.ISSN:2296-418

Sunlight-Controllable Biopharmaceutical Production for Remote Emergency Supply of Directly Injectable Therapeutic Proteins

Biopharmaceutical manufacturing requires specialized facilities and a long-range cold supply chain for the delivery of the therapeutics to patients. In order to produce biopharmaceuticals in locations lacking such infrastructure, a production process is designed that utilizes the trigger-inducible release of large quantities of a stored therapeutic protein from engineered endocrine cells within minutes to generate a directly injectable saline solution of the protein. To illustrate the versatility of this approach, it is shown that not only insulin, but also glucagon-like peptide 1 (GLP-1), nanoluciferase (NLuc), and the model biopharmaceutical erythropoietin (EPO) can be trigger-inducibly released, even when using biologically inactive insulin as a carrier. The facilitating beta cells are engineered with a controllable TRPV1-mediated Ca2+ influx that induces the fusion of cytoplasmic storage vesicles with the membrane, leading to the release of the stored protein. When required, the growth medium is exchanged for saline solution, and the system is stimulated with the small molecule capsaicin, with a hand-warming pack, or simply by using sunlight. Injection of insulin saline solution obtained in this way into a type-1 diabetes mouse model results in the regulation of blood glucose levels. It is believed that this system will be readily adaptable to deliver various biopharmaceutical proteins at remote locations.ISSN:1613-6810ISSN:1613-682

Engineering a Rapid Insulin Release System Controlled By Oral Drug Administration

Rapid insulin release plays an essential role in maintaining blood-glucose homeostasis in mammalians. Patients diagnosed with type-I diabetes mellitus experience chronic and remarkably high blood-sugar levels, and require lifelong insulin injection therapy, so there is a need for more convenient and less invasive insulin delivery systems to increase patients' compliance and also to enhance their quality of life. Here, an endoplasmic-reticulum-localized split sec-tobacco etch virus protease (TEVp)-based rapamycin-actuated protein-induction device (RAPID) is engineered, which is composed of the rapamycin-inducible dimerization domains FK506 binding protein (FKBP) and FKBP-rapamycin binding protein fused with modified split sec-TEVp components. Insulin accumulation inside the endoplasmic reticulum (ER) is achieved through tagging its C-terminus with KDEL, an ER-retention signal, spaced by a TEVp cleavage site. In the presence of rapamycin, the split sec-TEVp-based RAPID components dimerize, regain their proteolytic activity, and remove the KDEL retention signal from insulin. This leads to rapid secretion of accumulated insulin from cells within few minutes. Using liver hydrodynamic transfection methodology, it is shown that RAPID quickly restores glucose homeostasis in type-1-diabetic (T1DM) mice treated with an oral dose of clinically licensed rapamycin. This rapid-release technology may become the foundation for other cell-based therapies requiring instantaneous biopharmaceutical availability.ISSN:2198-384

Controlling therapeutic protein expression via inhalation of a butter flavor molecule

Precise control of the delivery of therapeutic proteins is critical for gene- and cell-based therapies, and expression should only be switched on in the presence of a specific trigger signal of appropriate magnitude. Focusing on the advantages of delivering the trigger by inhalation, we have developed a mammalian synthetic gene switch that enables regulation of transgene expression by exposure to the semi-volatile small molecule acetoin, a widely used, FDA-approved food flavor additive. The gene switch capitalizes on the bacterial regulatory protein AcoR fused to a mammalian transactivation domain, which binds to promoter regions with specific DNA sequences in the presence of acetoin and dose-dependently activates expression of downstream transgenes. Wild-type mice implanted with alginate-encapsulated cells transgenic for the acetoin gene switch showed a dose-dependent increase in blood levels of reporter protein in response to either administration of acetoin solution via oral gavage or longer exposure to acetoin aerosol generated by a commercial portable inhaler. Intake of typical acetoin-containing foods, such as butter, lychees and cheese, did not activate transgene expression. As a proof of concept, we show that blood glucose levels were normalized by acetoin aerosol inhalation in type-I diabetic mice implanted with acetoin-responsive insulin-producing cells. Delivery of trigger molecules using portable inhalers may facilitate regular administration of therapeutic proteins via next-generation cell-based therapies to treat chronic diseases for which frequent dosing is required.ISSN:1362-4962ISSN:0301-561

Genetically Encoded Protein Thermometer Enables Precise Electrothermal Control of Transgene Expression

Body temperature is maintained at around 37 degrees C in humans, but may rise to 40 degrees C or more during high-grade fever, which occurs in most adults who are seriously ill. However, endogenous temperature sensors, such as ion channels and heat-shock promoters, are fully activated only at noxious temperatures above this range, making them unsuitable for medical applications. Here, a genetically encoded protein thermometer (human enhanced gene activation thermometer; HEAT) is designed that can trigger transgene expression in the range of 37-40 degrees C by linking a mutant coiled-coil temperature-responsive protein sensor to a synthetic transcription factor. To validate the construct, a HEAT-transgenic monoclonal human cell line, FeverSense, is generated and it is confirmed that it works as a fever sensor that can temperature- and exposure-time-dependently trigger reporter gene expression in vitro and in vivo. For translational proof of concept, microencapsulated designer cells stably expressing a HEAT-controlled insulin production cassette in a mouse model of type-1 diabetes are subcutaneously implanted and topical heating patches are used to apply heat corresponding to a warm sensation in humans. Insulin release is induced, restoring normoglycemia. Thus, HEAT appears to be suitable for practical electrothermal control of cell-based therapy, and may also have potential for next-generation treatment of fever-associated medical conditions.ISSN:2198-384