Traceless, non-invasive and spatiotemporal control of protein activity in cells

Protein characterisation is an experimental process, where scientists build a profile of the physical, chemical and biological properties of the protein of interest. Information obtained from this protein characterisation has great importance in scientific research, particularly in areas related to health, ageing and drug development. Often, the physical and chemical properties of a protein can be characterised in a test tube isolated from its natural partners in a cell. However, these studies cannot provide a full biological characterisation because the molecular environment within a cell is much more complex and contains other protein molecules, genetic material such as DNA, in addition to carbohydrates and small molecules. It would therefore be desirable to analyse target proteins in their natural environment in a cell rather than in the test tube. However, at current there are no good methods available to introduce proteins into cells and induce their activity within a cell without modifying them chemically; such modifications often alter the properties of a protein.

We now wish to create a specialised tool that allows us to introduce a protein of interest into cells and subsequently activate it with infrared light, which will not damage living cells. In the past such activation has relied on ultra-violet light which is toxic to cells. Our tool will allow researchers to introduce any protein into any cell type at any time point in an unmodified form. Moreover, integrity of the biological experiment will not be affected by the use of this tool, as IR activation is harmeless and traceless.

To this end we will engineer proteins to install a specialised chemical group into proteins for activation by infrared light. Highly positively-charged molecular tags will be appended to proteins, so that their solubility in water and ability to enter cell can be enhanced. Upon light activation the tag and the chemical auxiliary will be removed and the protein will be present within the cell in its natural unmodified form.

We will apply our technology to activate a specific muscle-related protein, Myf-5, which upon activation with light will induce undifferentiated cells to change their morphology and turn into muscle cells. Overall our technology has the potential to activate externally supplied biologically active proteins in diseased cells and will therefore offer opportunities for future protein therapies. It therefore has huge potential for wide ranging biomedical research and commercial applications.

Here we propose to develop a novel gain-of-function tool to investigate the function of proteins in their natural environment. In inducible Delivered Protein (iDEP), a multifunctional intein is embedded into the protein of interest. The intein contains a cell-penetrating peptide for protein transduction and an infrared-responsive photocage added to a functionally important cysteine. Upon IR irradiation, intein splicing is activated and the cell-penetrating peptide is lost, releasing the protein of interest in its natural state.

We will evolve orthogonal aminoacyl-tRNA synthetases to incorporate IR-responsive cysteine analogues. This will enable us to activate the cysteine residue without causing cell damage. We will develop cell-penetrating peptides for efficient cellular uptake and maximal solubility. The wide-ranging implications of this work will be demonstrated by using iDEP for light-triggered phenotype switching. Myf-5 is a transcription factor responsible for the transformation of fibroblast to myogenic cells. iDEP constructs of Myf-5 will be prepared and transduced into 10T1/2 fibroblasts. Spatiotemporal activation by IR irradiation will consequently lead to the activation of muscle specific genes and differentiation of the fibroblast to myoblast.

Since no modification is needed for the protein of interest, iDEP has unrivalled versatility and offers unique advantages over existing gain of function methods. This tool will find use in fundamental biology research and biomedical science and be applicable in a wide range of areas of technology development such as pharmaceutical, biomedical and veterinary industries.

This programme will develop an unprecedented chemical biology tool to spatiotemporally activate a protein of interest in cells. This tool will allow researchers to fully decipher protein function in vivo, which is often dictated by its location, subcellular concentration and post-translational modification status. Hence, this work has direct consequences for protein scientists, including those in the fields of molecular biology, synthetic biology, drug delivery and protein therapy. Researchers on the project will gain excellent training, providing scientific knowledge, skills and transferable skills suitable for future employment in a range of sectors. They will also benefit from immersion in a high quality interdisciplinary research environment and the dissemination of results will greatly benefit their prospects of a career in science either in academia or industry.

iDEP and the associated light induced phenotype switch experiment form the basis of a fundamentally new strategy for protein therapy, a promising option for treatments of many human disorders. Malfunctioning proteins are associated with different human disorders, including MECP2 in Rett syndrome, p53 in Li-Fraumeni syndrome and STAT in breast cancer. By introducing normally functioning protein into cells, normal biological function can be restored. The development of our approach for medical applications is outside the scope of this proposal, but the light induced phenotype switch experiment experiment serves as a proof of concept to attract future collaboration with the pharmaceutical and veterinary industry for drug development. It will also positively impact the UK, enriching our country economically and enhancing the reputation of our science.

We will periodically discuss our progress and findings with the Research and Innovation Services (RIS) at Cardiff University to assess any outcomes that should be protected as intellectual property. RIS is well equipped to protect intellectual property, set up license arrangements and handle all aspects of commercial exploitation in support of this project.

In order that the results can be fully exploited by the wider scientific community and the society, communication is vital. The work will be published in open access form in internationally leading, peer-reviewed journals with minimal delay. Full experimental details containing data of experimental replicas will be included in the publications, when appropriate as free electronic supplementary information. In addition, DOIs will be provided in the publications to retrieve metadata related to the project. Plasmids produced will be deposited in the Addgene library to make them available to other workers. Results will be presented at national and international conferences, public lectures, meetings and workshops to foster exchange of opinions with other professions. This will be extended to the popular press when appropriate. Allemann and Jones regularly engages with media organisations.

Also, we will actively disseminate our results to the general public through the web and a project specific theatre production, which will not only increase the scientific knowledge of the general public in an entertaining manner, but also increase their interest in fundamental research. Jones has a very strong background in public engagement that will be exploited here in the form of training for researchers and delivery at events.