The Design and Application of Inhibiting Peptide for Rapid In vitro Downregulation of Post-Translational Modifications Levels at a Specific Site

Summary

Here, we present a protocol that outlines the design specifications, quality control standards, and functional verification system of TAT-PIP, a post-translational modification-inhibiting peptide.

Abstract

Proteins are the primary executors of life activities and are regulated at transcriptional, translational, and post-translational modification (PTM) levels. Moreover, PTM represents a more complicated mechanism for regulating protein activities as a protein generally has various types of PTMs and multiple sites for a specific PTM. Plasmid transfection or lentivirus and adenovirus could be used in initial screening of the functionally important PTM site among all identified sites; however, these methods always face challenges such as low-efficiency in cell and tissue entry, time-consuming and high costs, potential immune reaction, etc. To address this, we recently developed and successfully employed a type of recombined peptide called TAT-PIP, TAT-conjugated PTM inhibitory peptide. TAT-PIP consists of a TAT module that facilitates cell and tissue entry and a PIP module that specifically downregulates the PTM at targeted sites through competitive binding. Here, we present a protocol that outlines the design specifications, quality control standards, and functional verification system of TAT-PIP. In the design section, we describe the consistency, the optional length, specificity, and conservation of TAT-PIP. Next, we introduce the quality testing requirements of TAT-PIP, which guarantee its efficacy and safety. In the application of the TAT-PIP part, we introduced the concentration gradient test of TAT-PIP, the incubation process of the tested cells, and the subsequent phenotypic detection. In summary, we describe an effective method for screening PTM sites by selectively knocking down a specific site and observing the resulting phenotype to infer its function. Due to its low synthetic cost and high efficiency, this method overcomes the limitations of existing technologies, such as plasmid transfection.

Introduction

Proteins are the primary executors of life activities, and their biological regulatory processes involve two core aspects of gene expression regulation-gene transcription and mRNA translation. However, during the past decades, post-translational modification (PTM) has become another indispensable mechanism for regulating cellular functions¹. Phosphorylation, the most well-known PTM to researchers, plays a particularly important role in physiological functions. For example, after extracellular signal-regulated kinase (ERK) is phosphorylated by MEK kinase at the Thr202 and Tyr204 sites, its conformation changes, exposing the active site, which activates downstream target proteins, such as transcription factors². In contrast, when the N-terminal Ser9 of GSK-3β is phosphorylated by Akt/PKB kinase, its substrate-binding pocket is blocked, leading to a loss of kinase activity³. Ubiquitination is another important PTM that regulates various cellular processes through the covalent attachment of ubiquitin molecules to substrate proteins. Ubiquitin contains 7 lysine residues (K6, K11, K27, K29, K33, K48, K63) and an N-terminal methionine (M1). The linkage of ubiquitin chains at different sites imparts different destinies to substrate proteins. The primary function of K48-linked ubiquitination is to mark substrate proteins for degradation by the proteasome⁴. Conversely, the primary function of K63-linked ubiquitination is to engage in non-degradative signal transduction, such as DNA damage repair, inflammatory responses, and endocytosis⁵. In recent years, with the rapid advancement of proteomics technologies, an increasing number of novel PTMs have been discovered. Beyond classical acetylation, novel acylations such as propionylation, crotonylation, and glutarylation have been identified⁶. In the field of histone modifications, histone lactosylation interferes with RNA m⁶A modification and the homeostasis of the immune microenvironment⁷. In the field of non-histone modifications, the reduction of ATP5O crotonylation is a major detrimental factor in the downregulation of phospholipid metabolism⁸.

Conducting research on PTM requires systematically advancing the analysis of the PTM proteome⁹. This field encompasses two major research directions: the analysis of PTM profiles at the whole proteome level and the comprehensive identification of the PTM sites of a specific protein. Although modern mass spectrometry can effectively identify various types of PTMs and the specific sites, the screening and validation of key functional sites remains a core scientific challenge that restricts the research progress. Establishing efficient site screening strategies is the first step towards resolving the function of key regulatory sites. Traditional methods involve constructing mutant plasmids for specific sites (inactivating mutations, activating mutations), introducing them into target cells through in vitro transfection techniques, and systematically evaluating changes in phenotypic indicators such as cell proliferation, apoptosis, and oxidative stress. However, this strategy faces significant technical obstacles in transcriptionally silent cell models like oocytes and primary tissue culture systems. Although the lentiviral or adenovirus transduction system can partially overcome the issue of transfection efficiency, its application faces dual challenges: high economic costs and low penetration efficiency of viral particles in vitro tissue models. While the strategy of constructing mutant knock-in mice is extremely long-lasting and costly. Overall, it is urgent that economic and efficient strategies are developed for the initial screening of functionally important PTM sites.

In recent years, our team has successfully developed and employed a rapid PTM downregulation technique called TAT-PIP, TAT-conjugated PTM inhibitory peptide, based on polypeptide competitive inhibition. Its core advantage is its universality and high efficiency for conducting preliminary screening of functionally important PTM sites, which provides a crucial foundation for in-depth analyses of the molecular mechanisms of key PTM sites. This study systematically elucidates the principal framework and implementation process of TAT-PIP, and demonstrates its effectiveness in regulating the PTM level at a specific site through representative experimental data.

Protocol

1. Designing the TAT-PIP sequence

NOTE: TAT-PIP consists of two parts of peptides (Figure 1): The N-terminal part is TAT, CYGRKKRRQRRR, which remains unchanged for every TAT-PIP and can help efficiently enter in vitro cultured cells or in vivo tissues; the C-terminal part is a polypeptide sequence around the specific PTM site (Figure 1, usually lysine in symbol "K", red highlighted) of an object protein. The length of PIP is about 10-15 AA residues (if longer, the cost increases; if shorter, the specificity significantly decreases).

1. Optimize the sequence of PIP through Blast analysis (Figure 2).
   1. Log in to https://blast.ncbi.nlm.nih.gov/, click on the blastp search for protein sequences.
   2. Copy the target protein sequence into the enter the query sequence window.
   3. In choose search, select the Standard databases option. Select the blastp (protein-protein BLAST) option in the program selection. Finally, click the BLAST button (Figure 2B).
   4. Select peptide segments with top specificity as candidate PIPs.
2. Conduct a homology comparison between humans and mice for the final PIP.
   1. Log in to https://blast.ncbi.nlm.nih.gov/, click on the blastp search for protein sequences.
   2. Copy the sequences of the protein from both human and mouse species and paste them into the input sequence box.
   3. Click the submit button to get the results.
   4. Select a PIP with 100% homology between human and mouse (Figure 2C).
3. Determine the composition of TAT-PIP, which consists of the N-terminal TAT and C-terminal PIP.

2. Synthesis and quality verification of TAT-PIP

1. Select a company with extensive project experience to ensure peptide purity of no less than 98% with mass spectrometry, avoiding impurities that could impact subsequent experiments.
   NOTE: The endotoxin level of the polypeptide should be ≤10, and it should be pathogen-free.

3. The application of TAT-PIP

1. Use pathogen-free ultra-pure water to dissolve TAT-PIP to achieve a stock concentration of 10 mg/mL. Dispense the stock into small tubes to avoid repeated freeze-thaw cycles, which will cause partial denaturation.
   NOTE: Adding 1% dimethyl sulfoxide (DMSO) will help protect the structure of TAT-PIP.
2. Treat cells with TAT-PIP.
   1. Culture human embryonic kidney 293T cells in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under standard culture conditions (37 °C, 5% CO₂).
      NOTE: After seeding, the cells were cultured for about 12 h, and TAT-PIP treatment was performed when the confluency reached approximately 70%.
   2. Dilute the dissolved TAT to 1 mg/mL using PBS, then directly added to the cell culture medium at the appropriate dosage (recommended final concentration of the culture medium: 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL) for 24 h.
   3. Wash the cells with the original culture medium 3 times.
   4. Perform western blot analysis to verify the extent of downregulation of the PTM.
      NOTE: Based on experience and subsequent experimental results, the optimum final concentration of TAT-PIP is 0.1 mg/ mL.

4. Western blot

1. Prepare cell protein samples.
   1. Wash the cells with phosphate buffered saline (PBS) 3 times, add 200 µL of strong radioimmunoprecipitation assay (RIPA) lysis buffer (with phenylmethylsulfonyl fluoride [PMSF]-protease inhibitor added) into the cell pellet, and incubate for 5 min for preliminary lysis, scrape them off with a cell scraper.
   2. Sonicate the mixture using an ultrasonic crusher at 12% energy intensity for 5 s.
   3. Chill the cell lysate on ice for 30 min and centrifuge at 13400 g at 4 °C for 10 min.
   4. Transfer the supernatant containing proteins to a pre-cooled 1.5 mL centrifuge tube. Add 1/4 volume of 5x sample buffer to the supernatant (final concentration 1x).
   5. Vortex the mixture, heat it at 95 °C for 5 min in a dry thermostat, and chill on ice for 2 min.
   6. Measure the total protein concentration using a Bradford assay on a Nanodrop.
2. Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
   1. Use a 12% resolving gel and a 4% stacking gel for the SDS-PAGE.
   2. Pipette the protein samples into the wells according to the specified volume.
   3. Turn on the electrophoresis apparatus and first run at 80 V, 300 mA for 30 min to align the bands at the interface between the stacking and resolving gels. Then, run at 120 V, 300 mA for 45 min to resolve the proteins.
   4. Transfer the proteins from the gel to the polyvinylidene fluoride (PVDF) membrane using a fast transfer apparatus.
3. Perform blocking, antibody incubation, and visualization.
   1. Block the PVDF membrane in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubate at room temperature (RT) for 1 h.
   2. Incubate the membrane with the primary antibody diluted 1:1000 in 5% skim milk/PBST overnight at 4 °C on a rotator.
   3. Wash the membrane three times with TBST (10 min each).
   4. Incubate the membrane with the secondary antibody diluted 1:1000 in 5% skim milk/TBST at RT for 1 h.
   5. Wash the membrane three times with TBST (10 min each).
   6. Expose the membrane in a time series (5 s, 10 s, 20 s, 30 s, 60 s, 120 s) to obtain the optimal image under an electrogenerated chemiluminescence (ECL) device.

5. Functional validation of TAT-PIP in in vitro cultured cells

1. Detect cellular reactive oxygen species (ROS) levels.
   1. Plate 293T cells at an optimal density of 1 x 10⁵ cells/well in a 24-well culture plate.
   2. Treat cells with TAT-conjugated Histone3 lysine 116 Crotonylation inhibitory peptide (THCIP) (0.1 mg/mL) for 24 h, then wash the cells with PBS.
   3. Incubate the cells with 10 µM dichloro-dihydro-fluorescein diacetate (DCFH-DA) in serum-free DMEM at 37 °C in the dark for 20 min.
   4. Capture images using an inverted fluorescence microscope, and measure fluorescence intensity using the Measure function in ImageJ.
      NOTE: The FITC filter set was used, with an emission filter of 465-495 nm, a dichroic mirror 505A, and a second emission filter of 512-550 nm.
2. Cell proliferation assay
   1. Seed 293T cells at an optimal density of 1 x 10⁵ cells per well in a 24-well culture plate.
   2. Add 10 µL of CCK-8 reagent to each well.
   3. Incubate the cells at 37 °C for 2 h.
   4. Record the absorbance at 450 nm using a microplate reader and subtract the background signal from the blank wells (medium + CCK-8).
   5. Normalize the data to calculate cell viability.
3. Detection of cellular ATP levels
   1. Lyse the cells with a lysis buffer. Centrifuge the lysate at 12,000 × g for 5 min at 4 °C. Collect the supernatant for subsequent analysis.
   2. Dilute the ATP standard solution with the lysis buffer to create a concentration gradient of 0.5 µM, 1 µM, 2 µM, 3 µM, 4 µM, and 5 µM.
   3. Add 20 µL of ATP detection solution to each well containing either the cell supernatant or ATP standard.
   4. Immediately measure the luminescent signal using a microplate reader.
      1. Click Plate Layout, select the corresponding plate template in Change Plate Template to set up samples (standards and samples).
      2. Then click Protocol on the left, choose the Chemiluminescence button, then click Start to initiate detection.
   5. Generate a standard curve from the luminescent signals of the ATP standards and use it to quantify the ATP concentration in the cell supernatant.

Results

This study describes a strategy that uses TAT-PIP to downregulate the PTM level of a target protein at a specific site based on competitive inhibition. In a typical example, the TAT-PIP here is renamed specifically as THCIP. It comprises two functional modules: (1), the N-terminal- cell-penetrating peptide TAT (CYGRKKRRQRRR), which allows THCIP to penetrate cells; (2) the C-terminal sequence around K116 of H3, AIHAKRVTIMPKD, which specifically competes with the endogenous H3 to be crotonylated at K116 (

Discussion

In this protocol, the following steps are critical. First, the TAT-PIP design addresses the specificity of the peptide. Second, optimize the application and final concentration; the optimized concentration must be carefully tested to achieve efficient and rapid knockdown. Third, to verify the effect of the TAT-PIP, multiple experiments are recommended rather than relying on a single experiment to confirm its additional effects.

Comparing the existing methods, this method mainly demonstrates th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automatic multifunctional imaging system	Tanon	4600	The exposure meter identifies and visualizes the target protein bands by detecting the fluorescence or chemiluminescence signals on the membrane after the development treatment.
Cell counting kit-8	GLPBIO	GK10001	Based on the correlation between cell metabolic viability and cell number, this method evaluates the proliferative capacity of cells by measuring the reduction ability of cells to CCK-8 reagents
Dulbecco’s Modified Eagle’s Medium (DMEM)	Gibco	11995065	Dulbecco's modified Eagle's medium (DMEM) is a widely used basal medium to support the growth of a wide range of mammalian cells.
ECL chemiluminescent substrate	Biosharp	BL520B	ECL substrates generate light signals through chemiluminescence reactions that enable detection of specific proteins or nucleic acids.
Electrophoresis apparatus	Tanon	EPS600	The main role of Electrophoresis apparatus is to separate and analyze biological macromolecules ‌
Enhanced ATP assay kit	Beyotime	S0027	The kit can effectively detect the ATP level of samples
Fetal bovineserum (FBS)	Gibco	16000-044	Fetal bovine serum (FBS) provides essential nutrients and growth factors for cell maintenance and growth.
GenScript eBlotL1	GenScript	L00686	The device is highly efficient and it is able to rapidly achieve protein transfer from polyacrylamide gel to PVDF membrane within 15 minutes
Inverted fluorescence microscope	Olympus Corporation	IX73	Fluorescence microscopy uses ultraviolet light as a light source to irradiate the examined object to make it emit fluorescence, and then observe the shape and location of the object under the microscope.
NanoDrop2000 microvolume UV-Vis spectrophotometer	Thermo Scientific	2000	NanoDrop was used to determine the concentration of DNA or RNA
PVDF membrane	BioRAD	1620177	This type of PVDF membrane has excellent chemical resistance and high protein binding ability
ROS assay kit	Beyotime	S0033S	The kit can accurately detect the level of reactive oxygen species in cells
TAT-conjugater H3K16 Crotonylation inhibitory peptide	Nanjing Taopu Biotechnology Co. LTD	CN218107533U	It can specifically compete with the endogenous H3 to be crotonylated at K16
TBST	Biosharp	BL602A	TBST can clean irrelevant substances on the membrane, reduce experimental errors, and ensure the accuracy of experimental results ‌
Ultrasonic crusher	LICHEN	LC-AUD-150P	The main functions of the apparatus include cell fragmentation, particle dispersion and emulsification
Varioskan LUX	Thermo Fisher	VL0000D0	Microplate readers can achieve high-throughput quantitative analysis of biological samples by detecting the optical signals (such as absorbance, fluorescence or luminescence) of samples in microplates