Engineering Intracellular Protein Sensors in Mammalian Cells
Summary
Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.
Abstract
Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.
Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington’s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.
Introduction
The study and modulations of cellular responses via controllable engineered gene circuits are major goals in synthetic biology1,2,3 for the development of prospective tools with relevant biological or medical applications in cancer4, infections5, metabolic diseases6, and immunology7.
Reprogramming cell functions in response to specific signals requires the design of smart interfaces that link sensing of extracellular or intracellular dynamic changes (input) to downstream processing, triggering specific output either for diagnostic purposes (i.e., reporter genes) or to rewire cell response (therapeutics). The inputs detected by the sensing module can be small analytes8, proteins9,10,11 or microRNAs11,12,13, specific for the onset or progression of a disease. Moreover, complex circuit regulation can be achieved by multiple input information processing, increasing the tight control over transgene expression in response to the defined conditions14,15,16. For example, microRNA-based sensors can identify specific cell types, such as cancer cells, inducing their clearance with the expression of an apoptotic gene13. Since microRNAs are easily implementable in synthetic circuits in a modular manner, they represent a widely used input for genetically encoded biosensors12,13,17. Proteins are also a valid biomarker for genetic mutations, cancer and infections, and indeed a number of extracellular protein-sensing devices have been reported18,19.
Many of the circuits that detect extracellular proteins rely on the use of engineered receptors, which tether a transcription factor (TF) to the membrane, fused to a TEVp-responsive cleavage site (TCS). A major advantage of the TEVp is the specificity of the cleavage and lack of interference with endogenous protein processing. In these systems, TEVp is fused to a second peptide that interacts with the engineered receptor upon binding of the extracellular molecules. Thus, the external inputs induce TEVp-mediated cleavage and TF release. Systems that function with this mechanism are Tango/TEVp18, light-induced20 and Modular Extracellular Sensor Architecture (MESA)19. Despite progress in detecting extracellular proteins, the technology for sensing intracellular proteins in a modular fashion was never realized before, with the limitation of going through many build-and-test-iterations for single devices responsive to a specific protein.
Our system is the first platform for intracellular protein sensing21. The modularity is guaranteed by the use of intrabodies that define the specificity to the target, whereas the cell-reprogramming is TEVp-mediated. Specifically, one intrabody is membrane bound and fused to the C-terminal to a fluorescent protein mKate, a TCS and a TF (fusion protein 1); the second intrabody is fused to the TEVp and located in the cytosol (fusion protein 2) (Figure 1).
Thus, the interaction between two intrabodies and the target protein occurs in the cytoplasm and leads to TCS cleavage by TEVp, resulting in TF translocation into the nucleus to activate functional output. The sensing—actuating device was successfully tested for four intracellular disease-specific proteins: NS3 serine protease expressed by the HCV virus22, Tat and Nef proteins from HIV infection23,24, and mutated huntingtin (HTT) of the Huntington’s disease25. Output expression includes fluorescent reporters, apoptotic gene (hBax)26 and immunomodulators (XCL-1)27. We demonstrate that the system can also impair pathological functionality of its targets. For instance, the Nef-responsive device interferes with the spreading of viral infection by sequestering the target protein and reverting the downmodulation of HLA-I receptor on infected T cells24. The described sensing-actuating platform is the first of this kind for the detection of intracellular proteins and can be potentially implemented to sense abnormal protein expression, post-translational or epigenetic modifications, for diagnostic and therapeutic purposes20.
Protocol
Representative Results
Discussion
Until recently, interrogating cells based on intracellular environment was performed with systems developed de novo for specific targets. The present protocol describes an example of the most recent, cell engineering approach for protein sensing and actuating in one device, that can be rapidly adapted to new desired biomarkers.
This pioneering system sense intracellular proteins and provide a specific output to detect or neutralize the disease. The advantage of this class of genetic circuits i…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Istituto Italiano di Tecnologia.
Materials
| AlexaFluor 647 mouse anti-human HLA-A, B, C antibody clone W6/32 | Biolegend | 311414 | Antibodies |
| Annexin V | LifeTechnologies | A35122 | Apoptosis marker |
| Attractene | Qiagen | 301005 | Transfection reagent |
| BD Falcon Round-Bottom Tubes | BD Biosciences | 352053 | FACS tubes |
| Doxycycline | Clonetech | – | Cell Culture: Drugs |
| Dulbecco's modified Eagle medium | Cellgro | 10-013-CM | Cell Culture: Medium |
| Evos Cell Imaging System | Life Technology | EVOS M5000 | Imaging systems; Infectious molecular clones |
| FACSDiva8 software | BD Biosciences | 659523 | FACS software |
| Fast SYBR Green Master Mix | ThermoFisher Scientific | 4385612 | qPCR reaction |
| FBS (Fetal Bovin Serum) | Atlanta BIO | S11050 | Cell Culture: Medium |
| Gateway System | Life Technologies | – | Plasmid Construction |
| Golden Gate System | in-house | – | Plasmid Construction |
| HEK 293FT | Invitrogen | R70007 | Cell Culture: Cells |
| Infusion Cloning System | Clonetech | 638920 | Plasmid Construction |
| JetPRIME reagent | Polyplus transfection | 114-15 | Transcfection reagent |
| Jurkat Cells | ATCC | TIB-152 | Cell Culture: Cells |
| L-Glutamine | Sigma-Aldrich | G7513-100ML | Cell Culture: Medium |
| Lipofectamine LTX with Plus Reagent | Thermo Fisher Scientific | 15338030 | Transfection reagent |
| LSR Fortessa flow cytometer (405, 488, and 561 nm lasers) | BD Biosciences | 649225 | Flow cytometer |
| MicroAmp Fast Optical 96-Well Reaction Plate (0.1 mL) | ThermoFisher Scientific | 4346907 | qPCR reaction |
| Neon Transfection System | Life Technologies | MPK10025 | Transfection reagent |
| Non-essential amino acids | HyClone | SH3023801 | Cell Culture: Medium |
| Opti-MEM I reduced serum medium | Life Technologies | 31985070 | Transfection medium |
| Penicillin/Streptomycin | Sigma-Aldrich | P4458-100ML | Cell Culture: Medium |
| QuantiTect Reverse Transcription Kit | Qiagen | 205313 | Rev Transcriptase kit |
| RNeasy Mini Kit | Qiagen | 74106 | RNA extraction kit |
| RPMI-1640 | ATCC | ATCC 302001 | Cell Culture: Medium |
| Shield | Clonetech | 632189 | Cell Culture: Drugs |
| SpheroTech RCP-30-5-A beads | Spherotech | RCP-30- 5A-2 | Compensation set up |
| StepOnePlus 7500 Fast machine | Applied Biosystems | 4351106 | qPCR reaction |
Referências
- Bernardo, D., Marucci, L., Menolascina, F., Siciliano, V. Predicting Synthetic Gene Networks. Synthetic Gene Networks: Methods and Protocols. 813, 57-81 (2012).
- Krams, R., et al. Mammalian synthetic biology: emerging medical applications. J. R. Soc. Interface. 12, (2015).
- MacDonald, J. T., Siciliano, V. Computational Sequence Design with R2oDNA Designer. Methods in molecular biology. 1651, 249-262 (2017).
- Zah, E., Lin, M. -. Y., Silva-Benedict, A., Jensen, M. C., Chen, Y. Y. T cells expressing CD19/CD20 bi-specific chimeric antigen receptors prevent antigen escape by malignant B cells. Cancer Immunology Research. 4, 498-508 (2016).
- Wu, F., Bethke, J. H., Wang, M., You, L. Quantitative and synthetic biology approaches to combat bacterial pathogens. Current Opinion in Biomedical Engineering. 4, 116-126 (2017).
- Ye, H., et al. Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome. Proceedings of the National Academy of Sciences of the United States of America. 110, 141-146 (2013).
- Caliendo, F., Dukhinova, M., Siciliano, V. Engineered Cell-Based Therapeutics: Synthetic Biology Meets Immunology. Frontiers in Bioengineering and Biotechnology. 7, (2019).
- Thaker, M. N., Wright, G. D. Opportunities for synthetic biology in antibiotics: Expanding glycopeptide chemical diversity. ACS Synthetic Biology. 4, 195-206 (2015).
- Culler, S. J., Hoff, K. G., Smolke, C. D. Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins. Science. 330, 1251-1255 (2010).
- Ausländer, S., et al. A general design strategy for protein-responsive riboswitches in mammalian cells. Nature Methods. 11, 1154-1160 (2014).
- Cella, F., Siciliano, V. Protein-based parts and devices that respond to intracellular and extracellular signals in mammalian cells. Current Opinion in Chemical Biology. 52, 47-53 (2019).
- Miki, K., et al. Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches. Cell Stem Cell. 16, 699-711 (2015).
- Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R., Benenson, Y. Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science. , 1307-1311 (2011).
- Sun, J., et al. Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions. Nature Communications. 8, 477 (2017).
- Shigeto, H., et al. Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic β cells. Analyst. 144, 3765-3772 (2019).
- Cella, F., Wroblewska, L., Weiss, R., Siciliano, V. Engineering protein-protein devices for multilayered regulation of mRNA translation using orthogonal proteases in mammalian cells. Nature Communications. 9, (2018).
- Siciliano, V., et al. MiRNAs confer phenotypic robustness to gene networks by suppressing biological noise. Nature communications. 4, 2364 (2013).
- Barnea, G., et al. The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences of the United States of America. 105, 64-69 (2008).
- Schwarz, K. A., Daringer, N. M., Dolberg, T. B., Leonard, J. N. Rewiring human cellular input–output using modular extracellular sensors. Nature Chemical Biology. 13, 202-209 (2016).
- Wieland, M., Fussenegger, M. Engineering molecular circuits using synthetic biology in mammalian cells. Annual review of chemical and biomolecular engineering. 3, 209-234 (2012).
- Siciliano, V., et al. Engineering modular intracellular protein sensor-actuator devices. Nature Communications. 9, 1881 (2018).
- Lin, C. . HCV NS3-4A Serine Protease. , (2006).
- Romani, B., Engelbrecht, S., Glashoff, R. H. Functions of Tat: the versatile protein of human immunodeficiency virus type 1. Journal of General Virology. 91, 1-12 (2010).
- Basmaciogullari, S., Pizzato, M. The activity of Nef on HIV-1 infectivity. Frontiers in microbiology. 5, 232 (2014).
- Ross, C. A., Tabrizi, S. J. Huntington’s disease: from molecular pathogenesis to clinical treatment. Lancet neurology. 10, 83-98 (2011).
- Pfeffer, C. M., Singh, A. T. K. Apoptosis: A Target for Anticancer Therapy. International journal of molecular sciences. 19, 448 (2018).
- Guzzo, C., et al. The CD8-derived chemokine XCL1/lymphotactin is a conformation-dependent, broad-spectrum inhibitor of HIV-1. PLoS pathogens. 9, 1003852 (2013).
- Marasco, W. A., LaVecchio, J., Winkler, A. Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS. Journal of Immunological Methods. 231, 223-238 (1999).
- Gal-Tanamy, M., et al. HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen. Journal of molecular biology. 347, 991-1003 (2005).
- Southwell, A. L., et al. Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity. The Journal of neuroscience the official journal of the Society for Neuroscience. 28, 9013-9020 (2008).
- Bouchet, J., et al. Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody. Blood. 117, 3559-3568 (2011).
- Arold, S., et al. The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling. Structure. 5, 1361-1372 (1997).
- Beal, J. Signal-to-Noise Ratio Measures Efficacy of Biological Computing Devices and Circuits. Frontiers in Bioengineering and Biotechnology. 3, 93 (2015).
- Arold, S., et al. The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling. Structure. 5, 1361-1372 (1997).
- Tyshchuk, O., et al. Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein. mAbs. 9, 94-103 (2017).
.