The SGYS motif of TAF15 prion-like domain is critical to amyloid fibril formation.

Misfolding of TATA-box binding protein-associated factor 15 (TAF15) may cause neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Some mutations of prion-like domain (PrLD) have been detected in patients with sporadic ALS, suggesting the importance of TAF15-PrLD in ALS pathogenesis. Herein, combining experiments and molecular dynamics (MD) simulations, we investigated the influences of several TAF15-PrLD mutations on the amyloid fibril formation of TAF15-PrLD-extracted peptide segments, and identified an essential ß-amyloid-forming segment from TAF15-PrLD. A pathogenic mutation T2 E71G resulted in significantly enhanced aggregation of the TAF15-PrLD segment T2 (Y56GQSQSGYSQSYGGYENQ73). In addition, the peptide T2 with a strong ß-amyloid-forming tendency was able to induce the liquid to solid phase transition of TAF15-PrLD protein. Further study identified the SGYS motif as a critical segment that promoted the formation of amyloid fibrils, which maintained a stable ß-sheet structure through intermolecular hydrogen bonds and π-π stacking interaction. This work provides a clue to elucidate the molecular pathogenic mechanism of TAF15-associated neurodegenerative diseases, and will direct drug development targeting TAF15.

Natural Wood-Derived Macroporous Cellulose for Highly Efficient and Ultrafast Elimination of Double-Stranded RNA from In Vitro-Transcribed mRNA.

Double-stranded RNA (dsRNA) is a major impurity that can induce innate immune responses and cause adverse drug reactions. Removing dsRNA is an essential and non-trivial process in manufacturing mRNA. Current methods for dsRNA elimination use either high-performance liquid chromatography or microcrystalline cellulose, rendering the process complex, expensive, toxic, and/or time-consuming. This study introduces a highly efficient and ultrafast method for dsRNA elimination using natural wood-derived macroporous cellulose (WMC). With a naturally formed large total pore area and low tortuosity, WMC removes up to 98% dsRNA within 5 min. This significantly shortens the time for mRNA purification and improves purification efficiency. WMC can also be filled into chromatographic columns of different sizes and integrates with fast-protein liquid chromatography for large-scale mRNA purification to meet the requirements of mRNA manufacture. This study further shows that WMC purification improves the enhanced green fluorescent protein mRNA expression efficiency by over 28% and significantly reduces cytokine secretion and innate immune responses in the cells. Successfully applying WMC provides an ultrafast and efficient platform for mRNA purification, enabling large-scale production with significant cost reduction.

Novel branched amphiphilic peptides for nucleic acids delivery.

Highly efficient and safe non-viral vectors for nucleic acids delivery have attracted much attention due to their potential applications in gene therapy, gene editing and vaccination against infectious diseases, and various materials have been investigated and designed as delivery vectors. Herein, we designed a series of branched amphiphilic peptides (BAPs) and tested their applications as pDNA/mRNA delivery vectors. The BAP structure was inspired by the phospholipids, in which lysine oligomers were used as the "polar head", segments containing phenylalanine, histidine and leucine were used as the "hydrophobic tails", and a lysine residue was used as the branching point. By comparing the gel retardation, particle sizes and zeta potentials of the BAP/pDNA complexes of the short-branch BAPs (BAP-V1 âˆ¼ BAP-V4), we determined the optimal lysine oligomer was K6. However, their cell transfection efficiencies were not satisfactory, and thus three long-branch BAPs (BAP-V5 âˆ¼ BAP-V7) were further designed. In these long-branch BAPs, more hydrophobic residues were added and the overall amphiphilicity increased accordingly. The results showed that these three BAPs could effectively compact the nucleic acids, including both pDNA and mRNA, and all could transfect nucleic acids into HEK 293 cells, with low cytotoxicity. Among the three long-branch BAPs, BAP-V7 (bis(FFLFFHHH)-K-K6) showed the best transfection efficiency at N/P = 10, which was better than the commercial transfection reagent PEI-25 K. These results indicate that increased amphiphilicity would also benefit for BAP mediated nucleic acid delivery. The designed BAPs provide more documents of such novel type of nucleic acids delivery vectors, which is worth of further investigation as a new gene theranostic platforms.

Self-assembling branched amphiphilic peptides for targeted delivery of small molecule anticancer drugs.

Water insolubility poses a significant challenge in the clinical applications of many small molecule drugs. To improve the drug delivery efficiency, two branched amphiphilic peptides (BAPs) were designed in a computer-aided manner, for drug-loading through peptide self-assembling. The structures of the two BAPs, bis(LVFFA)-K-RGD (PepV-1) and bis(FHF)-K-RGD (PepV-2), were inspired by phospholipids, containing the RGD sequence as the hydrophilic head and two hydrophobic sequences as the hydrophobic tails. PepV-1 could self-assemble into nano-fibrils with a hydrophobic core and the RGD moiety on the surface. Its drug-loading efficiency (DE%) of three small molecule anticancer drugs (doxorubicin, camptothecin and curcumin) ranged from 9.90% to 11.74%, and entrapment efficiency (EE%) ranged from 37.30% to 43.00%. Pep-V2 could self-assemble into bilayer delimited nano-vesicles. The DE% of PepV-2 for these drugs ranged from 15.87% to 18.55%, and the EE% ranged from 60.45% to 73.23%. Both BAP carriers could prolong the release of the small molecule drugs, and the PepV-2 vesicles also showed pH-triggered increase of drug release due to the histidine residues. Bothe BAP carriers could increase the cytotoxicity against cancer cells, which might be due to the targeting on the cancer overexpressed integrins. The designed BAP carriers represent promising functional drug carriers for targeted drug delivery, and will be useful for improving the clinical use of small molecule drugs, especially for those with poor water solubility.

Self-assembly of chimeric peptides toward molecularly defined hexamers with controlled multivalent ligand presentation.

This work demonstrates a self-assembling peptide strategy to form finite, molecularly defined trigonal bipyramidal-like hexamers which offer control over multivalent ligand display for enhanced tumor targeting.

How charge distribution influences the function of membrane-active peptides: Lytic or cell-penetrating?

Lytic and cell-penetrating peptides (CPPs) are both membrane-active peptides sharing similar physicochemical properties. Although their respective functions have been intensively investigated, the difference of intrinsic properties between these two types of peptides is rarely discussed. In this study, we designed a series of analogs of a recently discovered CPP ZXR-1 (FKIGGFIKKLWRSKLA) by varying the charge distributions both on the helical wheel projection and along the sequence. These peptides showed different functions on cell membranes, including membrane lytic (peptide Z1), cell-penetrating (peptide ZXR-1, Z2 and Z3), and inactive (peptide Z4) peptides. The three groups of peptides displayed different interactions with model lipid monolayer, and found that peptide insertion might be an important dynamic step to distinguish lytic and cell penetrating functions. Based on the analysis of charge distribution patterns, it was proposed that the charge distributions on the helical wheel and along the sequence are both able to influence the functions of the membrane-active peptides. This finding provides a further understanding about the effect of charge distribution on the functions of membrane-active peptides, and will be helpful for the design of functional peptides.