概要

Engineering Intracellular Protein Sensors in Mammalian Cells

Published: April 28, 2020
doi:

概要

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

1. Design principles for construction and test the sensor-actuator device Select a protein of interest. NOTE: We designed a system for proteins located in the cytoplasm or shuttling between the cytoplasm and other compartments. Select two intrabodies binding different epitopes of the target protein. In our study we selected proteins for which the intrabodies were already developed and tested28,29,30<…

Representative Results

An architecture for modular intracellular protein detection As shown in Figure 1, the device is composed of: 1) intrabody 1 connected to the membrane-tethered fluorescent marker (mKate) and TEVp cleavage site (TCS), followed by a transcription activator GAL4VP16 (TF); 2) intrabody 2 fused to TEV protease (TEVp), free in the cytosol; 3) a synthetic promoter responsive to GAL4VP16, driving the expression of a reporter gene. The modularity is guaranteed by intrabodies tha…

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…

開示

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

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記事を引用
Duhkinova, M., Crina, C., Weiss, R., Siciliano, V. Engineering Intracellular Protein Sensors in Mammalian Cells. J. Vis. Exp. (158), e60878, doi:10.3791/60878 (2020).

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