On May 19, 2022, Yu Zhilin’s team from the School of Chemistry of Nankai University was in the middle of the J. Am. Chem. Soc. published a new study titled “In Situ Self-Sorting Peptide Assemblies in Living Cells for Simultaneous Organelle-Targeting.”
For the first time, the paper reports the construction strategy of intracellular self-classification assembly system to achieve collaborative regulation of different biological processes, and provides a new strategy for the development of in situ bioactive materials. The corresponding author of the paper is Yu Zhilin, and the first author is Liu Xin and Li Mingming.
Self-classification assembly refers to the process of different assembly primitives constructing an ordered structure formed by specific primitive components through selective non-covalent interaction, which is the most common way to construct complex and ordered biological structures in living systems. For example, microtubules and microfilaments in the cytoskeleton are fiber structures formed by the aggregation of motorized proteins and tubulins through self-identification, respectively. Polypeptide molecules have the same composition as proteins, and its in-situ assembly and construction of ordered nanostructures in cells is an effective means of developing biomedical functional materials, and has a wide range of application prospects in disease diagnosis and drug delivery. Inspired by the phenomenon of protein self-classification and assembly, it is of great significance to construct a polypeptide self-classification assembly system in situ in intracellular area to achieve the coordinated regulation of different biological processes. However, due to the structural similarity (peptide bonds) of the peptide backbone, it is extremely challenging to establish selective interactions between different peptide radicals, so there have been no reports since the classification and assembly of intracellular peptides.
Recently, Yu Zhilin’s team of Nankai University proposed a glutathione (GSH) stimulation response polypeptide in situ self-classification assembly system based on different assembly modes, which has the function of targeting both the organelle endoplasmic reticulum and The Golgi apparatus, and realizes the effective killing of tumor cells by coordinating the regulation of organelle dysfunction.
Figure 1: Schematic diagram of the in situ self-classification assembly process of intracellular peptides. Image credit: J. Am. Chem. Soc.
On the basis of the preliminary construction of a polypeptide controllable assembly strategy and biomedical functional materials (J. Am. Chem. Soc. 2022, 144, 6907；J. Am. Chem. Soc. 2021, 143, 13854；Nano Lett. 2021, 21, 5730；Nano Today 2021, 38, 101198；Chem. Sci. 2020, 11, 1383；Nano Lett. 2019, 19, 7965；J. Am. Chem. Soc. In 2017, 139, 7823), Yu Zhilin’s team designed and synthesized two polypeptide motifs, E3C16E6 and EVMSeO. Among them, the E3C16E6 molecule forms the E3C16SH molecule under the condition of GSH reduction of disulfide bonds, and further assembles the superhel fibers by interspersing and assembling in a bilayer stacking mode similar to the phospholipid molecule, while EVMSeO forms a dumbbell-type amphiphilic polypeptide primitive containing selenium ether in GSH, and assembles to form a nanoscale structure. The differences in these two peptide assembly matrices, especially the stacking patterns, are expected to enable an in situ self-classification assembly process of the GSH response. The self-assembly behavior of the peptide matrix E3C16 and EVMSe was fully verified by atomic force microscopy (AFM) and transmission electron microscopy (TEM) experiments. Using structured light illumination confocal microscopy (SIM), the authors directly observed the independent formation of nanostructures of different fluorescent molecule-labeled peptides.
Figure 2: Schematic diagram of peptide self-assembly and self-classification assembly characterization and self-classification assembly mechanism. Image credit: J. Am. Chem. Soc.
The polypeptide nanofibers formed by E3C16SH contain thiol groups that can be combined with the golgi body rich in thiol proteins by forming disulfide bonds, which in turn has the property of targeting the Golgi body. The endoplasmic reticulum-targeted peptide Ts-EVMSe is introduced in the EVMSe reduced assembly to co-assemble the co-assembly ER-EVMSe with endoplasmic reticulum targeting function. The self-sorting assembly process with GSH-responsive E3C16E6 and ER-EVMSeO primitives in solution and intracellular was validated by super-resolved fluorescence microscopy SIM experiments. Experimental results show that the two polypeptide motifs can independently form nanostructures with different fluorescent labels. Laser confocal experiments demonstrate the targeting effect of different peptide nanostructures on Golgi bodies and endoplasmic reticulum.
Figure 3: In-solution and intracellular GSH response polypeptides are self-classified in situ. Image credit: J. Am. Chem. Soc.
Finally, through cytotoxicity experiments, western blotting and protein immunofluorescence experiments, the authors confirmed that the peptide in situ self-classification assembly system coordinated the regulation of organelle dysfunction through the dual targeting of Golgi and the endoplasmic reticulum, and finally achieved the effect of effectively killing tumor cells. The successful construction of this system provides a new strategy for the functionalization of peptides to regulate intracellular structure and signaling pathways, and provides new ideas for disease diagnosis and drug targeted delivery. (Source: Science Network)
Related paper information:https://doi.org/10.1021/jacs.2c01025