Assay of Protein Turnover Using a Bioluminescent Reporter Essay

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Assay of Protein Turnover Using a Bioluminescent Reporter Essay

Assay of protein turnover using a bioluminescent reporter Affiliation Results and Discussion Answer to Question The results were relatively different for the corresponding LUCWTs and LUCPs. For the sample LUCWT, light emitted in 10 seconds per ul (RLU/ul) at time zero was 1236.8 RLU/ul. The average protein concentration at time zero was 0.53 ug/ul while the Relative Luciferase Activity at time zero was 2333.58. For the sample LUCP, light emitted in 10 seconds per ul (RLU/ul) at time zero was 1205.5 RLU/ul. The average protein concentration at time zero was 0.53 ug/ul while the Relative Luciferase Activity at time zero was 2274.47. The difference could have been caused by various factors including pipetting errors (Gould & Subramani, 1988).  Assay of Protein Turnover Using a Bioluminescent Reporter Essay.
2. Answer to Question 2
An estimate of a half-life for the LUCP enzyme is (How to Calculate Half Life, 2015):
Half-life = t*In 2
10seconds = t*0.301
t=10seconds*0.301 =30.1 seconds.
3. Answer to Question 3
It is important for some proteins to have a short half-life but no others like clusterin because they have to control excessive growth of cells. Clusterin in this case controls prostate cancer cells (Rizzi, Caccamo, Belloni, & Bettuzzi, 2009).
4. Answer to Question 4
In designing an experiment using MetLUC to assay the efficacy of three compounds with respect to BF A, the first thing to consider is that the activity of BF A is known. The BF A would thus be used as the control variable (Schultz, Cegielski, & Hastings, 2005). Analysis of each of the three novel compounds would be done. This would be based on their respective signal sequence, in which each of them would be expressed in tissue culture cess. Comparison of how the MetLUC protein for each compound would be translocated to the ER and the resulting secretion into the medium in which the cells are growing would be done. The results of each would be compared to the known facts about the same experiment in BF A.

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References
Gould, S., & Subramani, S. (1988). Firefly luciferase as a tool in molecular and cell biology”. . Anal. Biochem. 175 (1), 5–13.
How to Calculate Half Life. (2015). Retrieved from wikihow: http://www.wikihow.com/Calculate-Half-Life
Rizzi, F., Caccamo, A., Belloni, L., & Bettuzzi, S. (2009). Clusterin is a short half-life, poly-ubiquitinated protein, which controls the fate of prostate cancer cells. Journal of Cell Physioly, 19(2), 14-23.
Schultz, L. L., Cegielski, M., & Hastings, J. (2005). Crystal structure of a pH-regulated luciferase catalyzing the bioluminescent oxidation of an open tetrapyrrole .  Proc. Natl. Acad. Sci. U.S.A., 102 (5), 1378–83. Assay of Protein Turnover Using a Bioluminescent Reporter Essay.

Protein–protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein–protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein–channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders. Assay of Protein Turnover Using a Bioluminescent Reporter Essay.

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Introduction

Rapid progress in the complementary fields of molecular genetics and proteomics has led to the appreciation of protein–protein interactions within macromolecular complexes as key determinants of ion channel functional modulation.1,2 These macromolecular complexes play a critical role in regulating biophysical properties, surface expression, and membrane localization of channels through highly specific contact surfaces.2,3 The specificity of these protein–channel interactions usually resides in a few critical amino acid residues at the interface, referred to as “hot spots.” An emerging concept in the field of ion channel research is to leverage these hot spots as new targets for drug development.4,5 Yet, the ever growing number of protein–protein interactions poses a challenge in target selection. We propose that functional significance of the target and availability of structural information on the protein–channel complex are likely to provide the fundaments for a successful drug discovery campaign, facilitating hit-to-lead transition (structural information) and preclinical testing (functional significance).

Voltage-gated Na+ (Nav) channels are heteromeric transmembrane proteins consisting of a pore-forming α-subunit (Nav1.1–Nav1.9) and accessory β-subunits (β1 to β4); these channels are activated by membrane depolarization giving rise to action potentials and providing the basis for excitability in neurons and cardiomyocytes.6 Recent discoveries indicate that intracellular fibroblast growth factor 14 (FGF14) is a biologically relevant component of the Nav channel macromolecular complex. FGF14 is a member of the intracellular FGFs (iFGFs; FGF11–13), a group of molecules that remain intracellular, are not secreted, and exhibit selective tissue localization.7 Through a high affinity monomeric interaction with the intracellular C-terminal tail of Nav channel α subunits (Nav1.1–Nav1.9), FGF14 acts as a multivalent molecule that controls neuronal excitability promoting gating, stability, and targeting of native Nav channels to the action potential initiation site.8–13 Co-expression of neuronal FGF14 (FGF14-1b isoform) with different Nav channel isoforms results in modulation of Nav current amplitude and of voltage dependence of channel activation and inactivation of a magnitude and direction that depend upon the channel isoform and are distinct compared with any reported effects of other iFGFs on Nav channel function.8–19 Furthermore, genetic deletion of fgf14 in rodents impairs neuroplasticity and cognitive function, and single missense mutations of FGF14 in humans results in neurodegeneration, highlighting the functional relevance of FGF14 as a critical component of the Nav channel macromolecular complex.10,12,20–23 Although the structure of the FGF14:Nav channel complex, or of any other iFGF:Nav channel complexes, has not been resolved yet, information on critical residues of the iFGFs:Nav channel interface has been inferred from the FGF13 dimer crystal structure. The analysis of crystal packing contacts of the FGF13 dimer combined with mutagenesis experiments has demonstrated the existence of a conserved iFGF monomer interface that is proposed to mediate both homodimerization and Nav channel binding.9 Point mutations of presumptive hot spots at this interface impair FGF13 regulation of Nav currents and disrupt subcellular targeting and co-localization of FGF14 with native Nav channels at the axonal initial segment (AIS).9 Overall, the functional significance of FGF14 and the availability of structural information on the iFGFs:Nav channel complex makes the FGF14:Nav channel complex a potential target for proteomics-based discoveries and drug development directed toward regulation of Nav channel function and, ultimately, for treatment of disorders associated with dysregulation and/or mutations of Nav channels (epilepsy, neurodegeneration, pain, or other channelopathies). Assay of Protein Turnover Using a Bioluminescent Reporter Essay.

As a first step in an FGF14-based medication development, we identified the need for simple and rapid methods for the detection and functional evaluation of protein–channel complexes that could rapidly translate into drug development campaigns targeting ion channels.

Traditional biochemical methods used for protein–protein interaction studies include enzyme-linked immunosorbent assays, surface plasmon resonance, and fluorescence polarization, while the functional effect of protein binding to ion channels has been studied using manual and/or automated patch-clamp electrophysiogy,24 fluorescence-based methods,25 or ion flux assays.26 However, these assays are either relatively low throughput, not optimized for protein–channel complexes (electrophysiology), or costly and time demanding because they require the use of antibodies, high yield of purified proteins, or chemical derivation of the interacting protein pair. Conversely, split-protein reporters have emerged as a powerful methodology for the detection of biomolecular interactions in intact systems.27 The concept behind this approach relies on the complementation of two separated halves of a monomeric enzyme driven by the assembly of two interacting partners. First utilized with ubiquitin,28 the approach has been extended to dihydrofolate reductase,29 β-lactamase,30 GFP,31,32 and various luciferase species, such as Renilla luciferase,33 Gaussia luciferase,34 and Photinus firefly luciferase.35 The use of bioluminescence-based assays has become progressively more prominent in recent years.36 High signal-to-noise ratio, favorable dynamic range, and reversibility of luminescence-based signals have revealed the split-luciferase complementation as a very sensitive assay to detect protein–protein interactions, protein localization, intracellular protein dynamics, and protein activity in real time and in living cells and animals.35,37

In the present study, we sought to assess the utility of the Photinus firefly split-luciferase complementation assay (LCA) for rapid evaluation of the FGF14:Nav1.6 channel C-tail complex in living cells. Assay of Protein Turnover Using a Bioluminescent Reporter Essay. Toward this goal, we adapted and optimized the LCA to detect the FGF14:Nav1.6 channel C-tail complex assembly, and further employed the assay to identify critical amino acid residues responsible for the protein–channel interaction and to screen for upstream modulatory elements that alter complex formation. The data presented here support the use of the LCA as an innovative platform for rapid screening of protein–protein interactions within ion channel complexes.

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Materials and Methods

Chemicals

D-luciferin was purchased from Gold Biotechnology (St. Louis, MO) and prepared as a 30 mg/mL stock solution in phosphate-buffered saline (PBS); SP600125 (1,9-pyrazoloanthrone) was purchased from EMD Chemicals (San Diego, CA); PD169316 (4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole) was purchased from Sigma-Aldrich (St. Louis, MO); and BAY 11-7082 ((E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile) was purchased from Cayman Chemical (Ann Arbor, MI). The compounds were dissolved in dimethyl sulfoxide (DMSO).

DNA constructs

Mammalian expression vectors coding for N-terminal (pcDNA3.1-V5_HIS TOPO; rapamycin-binding domain [FRB]-N-terminal luciferase fragment [FRB-NLuc]) and C-terminal (pEF6-V5_HIS TOPO; C-terminal luciferase fragment [CLuc-FKBP]) fragments of firefly (Photinus pyralis) luciferase were a gift of Dr. Piwnica-Worms (Washington University, St. Louis, MO). To generate the CLuc-FGF14 construct, FKBP was replaced with neuronal FGF14 (1b isoform) in the CLuc-FKBP fusion vector. CLuc-FGF14 was engineered by polymerase chain reaction (PCR) amplification of the FGF14 open reading frame (nt 1–855) using a 5′ primer containing a BsiWI site up to a linker region and a 3′ primer containing a NotI site and ligated into the CLuc vector. To generate the CD4-Nav1.6-NLuc construct, a chimera carrying the C-terminal fragment of Nav1.6 (amino acids 1763–1976) fused with CD4ΔCtail (amino acids 1–395; gift of Dr. Benedict Dargent, INSERM, France) was similarly replaced with FRB in the FRB-NLuc construct using PCR amplification and ligation into BamHI at the 5′ end and BsiWI at the 3′ end. The choice of using the CD4 chimera fused to Nav1.6 C-tail was based on previous validations of this and other similar constructs in primary hippocampal neurons.38–40 Because the N-terminus of the Nav channels is located intracellularly, the fusion of the NLuc fragment to the Nav1.6 C-tail resulted in intracellular reconstitution of the two halves of luciferase. The following primers were used for PCR amplification: Assay of Protein Turnover Using a Bioluminescent Reporter Essay.

  • CLuc-FGF14:

  • Sense: 5′-CTCGTACGCGTCCCGGGGCGTAAAACCGGTGCCCCTCTTC-3′;

  • Antisense: 5′-GTTTAGCGGCCGCCTATGTTGTCTTACTCTTGTTGACTGG-3′.

  • CD4-Nav1.6-C-tail-NLuc:

  • Sense: 5′-CGGGGTACCCAAGCCCAGAGCCCTGCCATTTCTGTGGGCTCAGGT3′;

  • Antisense: 5′-CGCGTACGAGATCTGGCACTTGGACTCCCTGACCTCTTTTTGCCT-3′.

The FGF14Y153N/V155N mutant was engineered similarly to CLuc-FGF14 using pQBI-FGF14Y158N/V160N–GFP as a template in the PCR reaction.9,11 Note that the FGF14Y153N/V155N mutant presented in this study corresponds to the FGF14Y158N/V160N mutant described in previous studies.9 All constructs were verified by DNA sequencing. The plasmid pGL3 expressing full length firefly luciferase, used for counter screenings of kinase inhibitors, was a gift of Dr. Sarkar (University of Texas Medical Branch [UTMB], Department of Neurology).

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Cell culture and transient transfections

HEK293 cells were incubated at 37°C with 5% CO2 in medium composed of equal volumes of Dulbecco modified essential medium (DMEM) and F12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. For transfection cells were seeded in 24-well CELLSTAR®tissue culture plates (Greiner Bio-One, Monroe, NC) at 4.5×105 cells per well and incubated overnight to give monolayers at 90%–100% confluency. The cells were then transiently cotransfected with pairs of plasmids or single plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Assay of Protein Turnover Using a Bioluminescent Reporter Essay/ Each plasmid used per transfection per well was 1 μg, unless otherwise indicated. Cells co-transfected with the CLuc-FGF14 and CD4-Nav1.6-C-tail-NLuc constructs (1 μg of each construct per transfection) were used as a positive control; cells co-transfected with CLuc-FGF14 and pcDNA3.1 empty vector (1 μg of each construct per transfection; this pair is referred to in the text as CLuc-FGF14 alone) were used as background luminescence. The same ratio and plasmid DNA amounts were used for the experiments involving the CLuc-FGF14Y153N/V155N and CD4-Nav1.6-C-tail-NLuc complex.

Bioluminescence assays

Forty-eight hours post-transfection, cells were trypsinized (0.25%) for 10 min at 37°C, triturated in a medium, and seeded in white, clear-bottom CELLSTAR μClear® 96-well tissue culture plates (Greiner Bio-One) at ∼105 cells per well in 200 μL of medium. The cells were incubated for 24 h and then the growth medium was replaced with 100 μL of serum-free, phenol red–free DMEM/F12 medium (Invitrogen). In experiments involving protein kinase inhibitors compounds dissolved in DMSO (stock solution=10 mM; intermediate dilutions=0.2 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM in DMSO) were added to the final concentration of 1–50 μM in the culture medium; the final concentration of DMSO was maintained at 0.5% and positive control wells were also treated with 0.5% DMSO (treated positive control). Luminescence measurements were performed 1 h after the application of compounds. The reporter reaction was initiated by injection of 100 μL of substrate solution containing 1.5 mg/mL of D-luciferin (final concentration=0.75 mg/mL) dissolved in serum-free, phenol red–free DMEM/F12 medium. Dispensing of the substrate was performed by the Synergy™ H4 Multi-Mode Microplate Reader (BioTek, Winooski, VT). Luminescence readings were initiated after 3 s of mild plate shaking and performed at 2-min intervals for 20 min, integration time 0.5 s. The cells were maintained at 37°C throughout the measurements. Signal intensity for each well was calculated as a mean value of peak luminescence and luminescence measured at two adjacent time points; the calculated values were expressed as percentage of mean signal intensity in the control samples from the same experimental plate. Assay of Protein Turnover Using a Bioluminescent Reporter Essay.