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 ac....

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).

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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