Changing an oxygen atom of the phosphoester bond in phosphopeptides by a sulfur atom enables instantly targeting Golgi apparatus (GA) and selectively killing cancer cells by enzymatic self-assembly. Specifically, conjugating cysteamine S-phosphate to the C-terminal of a self-assembling peptide generates a thiophosphopeptide. Being a substrate of alkaline phosphatase (ALP), the thiophosphopeptide undergoes rapid ALP-catalyzed dephosphorylation to form a thiopeptide that self-assembles. The thiophosphopeptide enters cells via caveolin-mediated endocytosis and macropinocytosis and instantly accumulates in GA because of dephosphorylation and formation of disulfide bonds in Golgi by themselves and with Golgi proteins. Moreover, the thiophosphopeptide potently and selectively inhibits cancer cells (HeLa) with the IC 50 (about 3 mM), which is an order of magnitude more potent than that of the parent phosphopeptide.

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Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of α-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an α-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes.

Herein, we show intranuclear nanoribbons formed upon dephosphorylation of leucine-rich L-or Dphosphopeptide catalyzed by alkaline phosphatase (ALP) to selectively kill osteosarcoma cells. Being dephosphorylated by ALP, the peptides are first transformed into micelles and then converted into nanoribbons. The peptides/assemblies first aggregate on cell membranes, then enter cells via endocytosis, and finally accumulate in nuclei (mainly in nucleoli). Proteomics analysis suggests that the assemblies interact with histone proteins. The peptides kill osteosarcoma cells rapidly and are nontoxic to normal cells. Moreover, the repeated stimulation of the osteosarcoma cells by the peptides sensitizes the cancer cells rather than inducing resistance. This work not only illustrates a novel mechanism for nucleus targeting, but may also pave a new way for selectively killing osteosarcoma cells and minimizing drug resistance.

The Golgi apparatus (GA) is the hub of intracellular trafficking, but selectively targeting GA remains a challenge. We show an unconventional types of peptide thioesters, consisting of an aminoethyl thioester and acting as substrates of thioesterases, for instantly targeting the GA of cells. The peptide thioesters, above or below their critical micelle concentrations, enter cells mainly via caveolin-mediated endocytosis or macropinocytosis, respectively. After being hydrolyzed by GA-associated thioesterases, the resulting thiopeptides form dimers and accumulate in the GA. After saturating the GA, the thiopeptides are enriched in the endoplasmic reticulum (ER). Their buildup in ER and GA disrupts protein trafficking, thus leading to cell death via multiple pathways. The peptide thioesters target the GA of a wide variety of cells, including human, murine, and Drosophila cells. Changing d-diphenylalanine to l-diphenylalanine in the peptide maintains the GA-targeting ability. In addition, targeting GA redirects protein (e.g., NRAS) distribution. This work illustrates a thioesterase-responsive and redox-active molecular platform for targeting the GA and controlling cell fates.

Induced resistance against SBPH via microbial pesticides is considered as an eco-friendly and promising management approach. In this study, the induced resistance against SBPH in rice seedling by a new potential microbial pesticide, decoyinine (DCY), a secondary metabolite produced by Streptomyces hygroscopicus, was evaluated to investigate the effects of DCY on SBPH’s biological and population parameters along with defense-related physiological and biochemical indices in rice against SBPH feeding. We found that DCY has potential to improve rice resistance and significantly reduced the fecundity of SBPH. Laboratory results revealed that DCY treated rice significantly changed SBPH’s fecundity and population life table parameters. The concentrations of hydrogen peroxide (H2O2), soluble sugars and malondialdehyde (MDA) were significantly lower in DCY treated rice plants against SBPH infestation at 24, 48 and 96 hours post infestation (hpi), respectively. The concentrations of antioxidant enzymes, catalase (CAT) was significantly higher at 72 hpi, while super oxidase dismutase (SOD) and peroxidase (POD) concentrations were recorded higher at 96 hpi. The concentrations of synthases enzymes, phenyl alanine ammonia-lyase (PAL) was higher at 48 hpi, whereas polyphenol oxidase (PPO) concentration was maximum at 72 hpi against SBPH infestation. The results imply that DCY has unique properties to enhance rice resistance against SBPH by stimulating plant defensive responses. Microbial pesticides may be developed as an alternative to chemical pest control.

Plant architecture is an important factor for crop production. Plant height, tiller pattern, and panicle morphology are decisive factors for high grain yield in rice. Here, we isolated and characterized a T-DNA insertion rice mutant Osdmt1 (Oryza sativa dwarf and multi-tillering1) that exhibited a severe dwarf phenotype and multi-tillering. Molecular cloning revealed that DMT1 encodes a plasma membrane protein that was identified as a putative Ca2+ permeable mechanosensitive channel. The transcript expression level was significantly higher in the dmt1 mutant compared to wild type (WT). Additionally, the dmt1 homozygous mutant displayed a stronger phenotype than that of the WT and heterozygous seedlings after gibberellic acid (GA) treatment. RNA-seq and iTRAQ-based proteome analyses were performed between the dmt1 mutant and WT. The transcriptome profile revealed that several genes involved in GA and strigolactone (SL) biosyntheses were altered in the dmt1 mutant. Ca2+ and other ion concentrations were significantly enhanced in the dmt1 mutant, suggesting that DMT1 contributes to the accumulation of several ions in rice. Moreover, several EF-hand Ca2+ sensors, including CMLs (CaM-like proteins) and CDPKs (calcium-dependent protein kinases), displayed markedly altered transcript expression and protein levels in the dmt1 mutant. Overall, these findings aid in the elucidation of the multiply regulatory roles of OsDMT1/OsMCA1 in rice.

Despite the great progress in the field of drug delivery systems for cancer treatment over the last decade, many challenges still lie ahead, such as low drug loading, deep penetration of tumors, side effects, and the development of drug resistance. A class of cationic membrane lytic peptides has shown potential as an anticancer agent by inducing cancer cell death via membrane disruption; meanwhile, their intrinsic selectivity renders them as having low cytotoxicity towards noncancerous cells. Here, we report the use of a cationic peptide amphiphile (PA), named PAH6, to load doxorubicin (Dox) that is intercalated in an ATP-binding aptamer-incorporated DNA scaffold. The PA contains a cationic lytic sequence, (KLAKLAK)2, a polyhistidine segment for the “proton sponge” effect, and a hydrophobic alkyl tail to drive the self-assembly. Dox-loaded DNA was found to form a spherical nanocomplex (NC) with PAH6 with particle sizes below 100 nm at various ratios. Since the carrier PAH6 is also a therapeutic agent, the drug loadings of the NC reached up to ~86% within the ratios we tested, and Dox was released from the NC in an ATP-rich environment. In vitro studies indicate that the presence of PAH6 could permeabilize cell membranes and kill cells through fast membrane disruption and depolarization of mitochondrial membranes. The cytotoxicity tests were conducted using A549 nonsmall cell lung cancer cells and NIH-3T3 fibroblast cells. PAH6 showed selectivity towards A549 cells. Significantly, the Dox-DNA/PAH6 NC exhibited a synergistic effect against A549 cells, with the IC50 decreased up to ~90% for Dox and ~69% for PAH6 when compared to the IC50 values of the two components, respectively. Furthermore, the selectivity of PAH6 conferred to the complex an improved therapeutic index between A549 and NIH-3T3 cells. A 3D-cultured A549 spheroid model was adopted to test the capability of Dox-DNA/PAH6 for tumor penetration. The PAH6 or Dox-DNA/PAH6 complex was found to break the spheroids into pieces, while Dox-treated spheroids maintained their shapes. In summary, this work provides a new strategy for constructing nanomedicines using therapeutic agents to meet the features required by anticancer treatment.