Synthetic Mitochondria-Targeting Peptides Incorporating α-Aminoisobutyric Acid with a Stable Amphiphilic Helix Conformation in Plant Cells

Abstract

In the genetic modification of plant cells, the mitochondrion is an important target in addition to the nucleus and plastid. However, gene delivery into the mitochondria of plant cells has yet to be established by conventional methods, such as particle bombardment, because of the small size and high mobility of mitochondria. To develop an efficient mitochondria-targeting signal (MTS) that functions in plant cells, we designed the artificial peptide (LURL)3 and its analogues, which periodically feature hydrophobic α-aminoisobutyric acid (Aib, U) and cationic arginine (R), considering the consensus motif recognized by the mitochondrial import receptor Tom20. Circular dichroism measurements and molecular dynamics simulation studies revealed that (LURL)3 had a propensity to form a stable α-helix in 0.1 M phosphate buffer solution containing 1.0 wt % sodium dodecyl sulfate. After internalization into plant cells via particle bombardment, (LURL)3 revealed highly selective accumulation in the mitochondria, whereas its analogue (LARL)3 was predominantly located in the vacuoles in addition to mitochondria. The high selectivity of (LURL)3 can be attributed to the incorporation of Aib, which promotes the hydrophobic interaction between the MTS and Tom20 by increasing the hydrophobicity and helicity of (LURL)3. The present study provided a prospective mitochondrial targeting system using the simple design of artificial peptides.

An Interplay between Mitochondrial and ER Targeting of a Bacterial Signal Peptide in Plants

“…Many mitochondrial pre-sequences possess a hydrophobic motif with the consensus ϕxβϕϕ (where ϕ is a hydrophobic residue, x is any residue, and β is a basic residue) within a helical structure, which mediates the binding to Tom20 on the outer mitochondria membrane [ 43 , 44 ]. Analysis using MitoFates [ 45 ] did not predict the presence of a mitochondrial pre-sequence in sp-GFP; however, it detected two sequences within the signal peptide which conform to the hydrophobic motif structure (LERMA and VGKLL).…”

Synthesis and structure‐activity relationship of mitochondria‐targeting peptoids with varying hydrophobicity and cationic charge

“…To assess the cellular toxicity of peptoids, we performed an MTS assay using MRC-5 and HeLa cells (Table 3). For minimization of spectroscopic interference, the MTS assay was conducted with unlabeled peptoids (7)(8)(9)(10)(11)(12). Hexameric peptoids (…”

“…Nascent proteins expressed in the cytoplasm contain mitochondria-targeting signals (MTSs) that form cationic amphiphilic helices, which readily interact with the mitochondrial membrane surface, facilitating protein import through the mitochondrial transporter. [10][11][12] While MTS promote mitochondrial delivery of cytosolic proteins, their sequences and sizes vary widely, and transport Soyoung Kim and Ji-Yu Lee contributed equally to this work. of extracellular substances is inefficient, highlighting the requirement for reliable and readily accessible approaches for targeted mitochondrial delivery.…”

Solubility Behavior of α-Aminoisobutyric Acid in 13 Individual Solvents at Temperatures Ranging from 283.15 to 323.15 K

“…α-Aminoisobutyric acid and its derivatives are also fruit preservation agents, which can prevent plant fungal infection. 2 Solubility, as a significant characteristic of active pharmaceutical ingredients (APIs), is closely related to bioavailability, crystal size distribution, and cocrystal formation. 5 Optimizing the crystallization process based on solubility data can improve the quality of the product, which includes purity, particle size, final yield, and so on.…”