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 biology^1,2,3 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, proteins^9,10,11 or microRNAs^11,12,13, 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 conditions^14,15,16. 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 biosensors^12,13,17. Proteins are also a valid biomarker for genetic mutations, cancer and infections, and indeed a number of extracellular protein-sensing devices have been reported^18,19.

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 infection^23,24, 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

1. Select a protein of interest.
   NOTE: We designed a system for proteins located in the cytoplasm or shuttling between the cytoplasm and other compartments.
2. Select two intrabodies binding different epitopes of the target protein. In our study we selected proteins for which the intrabodies were already developed and tested^28,29,30,31. When intrabodies are not available, they should be newly developed.
   NOTE: An alternative to intrabodies can be other molecules that interact with the protein of interest. For example, the Nef sensing device include one intrabody (sdAb19) and the SH3 domain of a p59fyn protein tyrosine kinase³².
3. Design in silico the plasmids with DNA sequences encoding the fusion proteins.
   NOTE: Software for plasmid design are available for free or can be purchased.
   1. Generate sequences that include the following modules: membrane tag, fluorescent marker (e.g., mKate), intrabody, TCS and TF for fusion protein 1, and intrabody and TEVp for fusion protein 2.
      NOTE: Building a new architecture based on the interaction of several components requires attention to parameters that may maximize the signal activation when the target protein is detected. Specifically, we focused on TEVp activity and flexibility of the chimeric proteins to facilitate protein-protein interaction (Table 1). All these features are specified in the following points.
   2. Insert a flexible glycine-serine linker domain (LD) between the intrabody–TCS and/or TEVp–intrabody. The LD may be of variable length (0, 10 or 15 amino acids-AA).
   3. Fusion protein 1: Design variants that include the high affinity TCS (S) or the low affinity TCS (L) to regulate TEVp cleavage activity. The expression of this construct is driven by a constitutive promoter. Include the GAL4VP16 transcription factor at the C-terminal of TCS to allow translocation to the nucleus upon TEVp cleavage.
      NOTE: We used the hEF1a promoter, but other constitutive promoters can be chosen.
   4. Fusion protein 2: Use a constitutive (hEF1a) or doxyclycine-inducible (pTET) promoter to tune TEVp transcription. Use a degradation domain (DD) regulated by the small molecule shield to modulate protein stability.
      NOTE: Other inducible systems that tune protein expression can potentially be chosen.
   5. Output: Place a reporter gene downstream UAS promoter that respond to GAL4VP16 transcription factor. Choose the output according to the application of the device (i.e., fluorescent protein, cytokine, etc.).
4. Perform cloning with any preferred strategy. Here, we built plasmids with golden gate or gateway technologies.
5. Test the various configuration of the device in the desired cell lines by transient transfection. Co-transfect the cells with the obtained plasmids for fusion proteins 1 and 2 and fluorescent reporter gene, such as UAS-EYFP. Fusion protein 1 include a red fluorescent protein (mkate) used as transfection marker.
   NOTE: Start with simple-to-transfect cell lines such as HEK293FT cells to test the devices.
   1. Transfection in HEK293FT cells. Seed 2 x 10⁵ cells into a 24-well plate with 500 µL of DMEM. Transfect cells with 300 ng of total DNA using transfection reagent, vortex the mix and incubate at RT for 20 minutes.
      1. Place cells in a 37 °C/5% CO₂ incubator. Add fresh culture medium 24 h after transfection.
   2. Transfection in Jurkat cells. Use 3 x 10⁵ cells in 500 µL of RPMI cell culture medium for 24-well plate. Perform electroporation following manufacturer’s instruction. Use 2-4 µg of total DNA for each sample. Add 500 µL of fresh cell culture media 24 h after transfection.
      1. During electroporation, place cells and DNA mix on ice. Then, place the cells in the 37 °C/5% CO₂ incubator.
         NOTE: Different cell lines and transfection methods may be chosen according to the specific application of the device.
6. Acquire microscope images to obtain a qualitative understanding of the functionality of the device. Confirm membrane localization of the fusion protein 1 by detecting the fluorescent signal (mkate) in proximity to the cell membrane. Visualize the fluorescent output (EYFP) in the presence or absence (negative control-NC) of the target protein.
   1. Use the cell imaging system equipped with Tx Red and GFP light cubes to detect mKate and EYFP fluorescent proteins. Use the 10x or 20x objective.
      NOTE: Use non-transfected cells to ensure that there is no autofluorescence.
7. Analyze the transfection with flow cytometry (FACS).
   NOTE: See Protocol 2 for sample preparation.
   1. Evaluate the devices in the presence of the target protein. Create an experimental matrix that combine variants of fusion protein 1 and 2, modulating TEVp with (i) constitutive hEf1a promoter (ii) doxycycline-inducible TET promoter and (iii) shield for protein stability. Add doxycycline and shield at concentrations (0-1000 nM).
   2. Perform FACS analysis to compare the fold induction of EYFP between samples with or without protein of interest.
      NOTE: The highest fold induction corresponds to the greatest input sensitivity in ON (presence of the target protein) as compared to the OFF (absence of the target protein) mode.
8. Test the functionality of the device with respect to the desired applications.
   1. Run FACS analysis to measure the levels of output proteins or apoptosis, or other relevant assays.
   2. Perform RT PCR analysis if the output part of the device regulates expression levels of target genes. Calculate 2^-ddCT in respect to the house-keeping gene (GAPDH or equivalent) and compare the fold change of output expression in ON and OFF modes.

2. Sample preparation for flow cytometry analysis

1. Perform quantitative analysis of protein devices with flow cytometer (FACS) 48 h after transfection.
   1. Aspirate the DMEM media and wash HEK293FT cells with 300 µL of PBS. Add 100 µL of trypsin and leave in the 37 °C/5% CO₂ incubator for 2-4 minutes.
      NOTE: Jurkat cells do not require trypsin.
   2. Add 400 µL of DMEM media without phenol red and resuspend the cells to avoid clumps formation. Transfer in FACS tubes.
   3. Use a flow cytometer equipped with 405, 488, and 561 nm lasers.
   4. Collect 30,000 – 100,000 events selected from live cell population using the following cytometer settings: 488 nm laser and 530/30 nm bandpass filter for EYFP/EGFP, 561 nm laser and 610/20 nm filter for mKate, and 405 nm laser, 450/50 filter for EBFP.
      NOTE: To set up the flow cytometer, include non-transfected cells and cells transfected with single fluorescent proteins.
   5. Perform analysis of collected samples using preferred software (e.g., DIVA software, Flowjo, or TASBE³³ method).

3. Characterization of the HCV sensor-actuator device

1. Select intrabodies to detect NS3 protein. scFv35 and scFv162 bind to two different epitopes and were thus chosen.
2. Design and test variants of the NS3-responsive device following the rational described in step 1.3 (e.g., Fusion protein 1: membrane tag, mKate, single chain fragment intrabody scFv35, LD0, TCS-L and TF GAL4VP16; Fusion protein 2: single chain fragment intrabody scFv162, LD0 and TEVp; Output: UAS-EYFP or UAS-hBax (apoptosis); NS3 (see Table 2)).
3. Test the constructs in HEK293FT cells.
   NOTE: For each experiment, set a negative control that is the delivery of the device and output quantification in the absence of the protein of interest.
   1. Test the devices for TEVp under a constitutive promoter in the presence or absence of NS3. Perform transfection of fusion protein 1 (step 1.3.3), fusion protein 2 (step 1.3.4) and EYFP output step (1.3.5) in HEK293FT cells as described in step 1.5.1. Analyze the samples 48 h post-transfection by measuring the fluorescent reporter (EYFP) by flow cytometry (Protocol 2).
   2. Assess the interaction intrabody-NS3 by mKate and BFP colocalization using the BFP-NS3 fusion protein. Transfect cells with fusion protein 1 that includes mKate, and with a BFP-NS3. 48 h post-transfection, observe cells at confocal microscope (63x objective) to test for co-localization of fluorescent proteins.
      NOTE: Use a BFP (not fused to NS3) as negative control. In this condition BFP should show diffuse cellular localization and should not co-localize with the membrane-bound mKate.
   3. Test the devices for TEVp under the pTET promoter in the presence or absence of NS3. Compare EYFP in absence and presence of NS3. Co-transfect cells with fusion protein 1 (step 1.3.3), fusion protein 2 (step 1.3.4) and EYFP output (step 1.3.5) using the protocol described in step 1.5.1. Transfections are performed in the presence or absence of NS3. Analyze the samples 48 h post-transfection by measuring the fluorescent reporter (EYFP) by flow cytometry (Protocol 2).
4. Test cell killing driven by NS3 device.
   1. Replace EYFP output with the apoptotic gene hBax.
   2. Repeat step 3.3.3. Collect the cells 48 h after transfection and wash with PBS before starting the assay.
   3. Perform apoptosis assay with Annexin V conjugated to Pacific Blue. Stain cells with apoptotic marker Annexin V (conjugated with 2.5 µL of Pacific Blue) diluted in binding buffer (1:20 volume ratio) for 10 min at room temperature.
   4. Analyze the cells by flow cytometry. Calculate the apoptosis level within population as number of Pacific-Blue (Annexin V) positive cells.

4. Characterization of Nef-responsive HIV sensor-actuator device

1. Select intrabodies that bind to Nef. Single domain intrabody sdAb19 and SH3 were chosen as they bind to different epitopes.
   NOTE: SH3 is not an intrabody but a domain of p59^fyn protein tyrosine kinase that physiologically interacts with Nef.
2. Design and test variants of Nef-responsive device following the rational described in step 1.3 (e.g., Fusion protein 1: membrane tag, mKate, sdAb19, LD0, TCS-L and GAL4-VP16; Fusion protein 2: SH3, LD0 and TEVp; Output: UAS-EYFP; Nef (see Table 3)).
3. Test the devices in HEK293FT and Jurkat T cells.
   NOTE: For each experiment we set a negative control that is delivery of the device and output quantification in the absence of the protein of interest
   1. Transfection of HEK293FT: Follow the procedure described in step 1.5.1 in presence or absence of Nef.
   2. Transfection of Jurkat T cells: Follow the procedure described in step 1.5.2 in presence or absence of Nef.
   3. 48h post-transfection, analyze the samples with flow cytometer as described in Protocol 2.
4. Infect Jurkat cells with HIV LAI strains for HIV detection.
   1. Prepare viral stocks at least 48 h before the experiment. Use virus obtained from transfection of HEK-293T cells with the infectious molecular clones (NIH AIDS reagents program) and commercial reagents using manufacturer instructions. Concentrate virus after 40 h by ultracentrifugation for 1 h, 64,074 x g, 4°C on 20% sucrose to avoid viral particle-free proteins. Titer viral stocks with HIV-1 p24 ELISA.
   2. Infect cells with HIV-1 strains with 500 ng of p24 inoculum. 40 h post-infection quantify the percentage of infected cells by detecting the viral protein p24 with KC57-FITC conjugated antibody and performing flow-cytometry analysis.
   3. Stain the cells with AlexaFluor 647 conjugated antibodies clone W6/32 against HLA-A, B, C. Fix the cells and analyze on a flow cytometer. Nef mediated downmodulation of HLA-I in Jurkat T cells in the presence or absence of the protein sensor device.

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 that ...

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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