Proteomics of appetite-regulating system influenced by menstrual cycle and intensive exercise in female athletes: a pilot study.

Female athletes who endure intense training are at risk of developing the 'female athlete triad,' making energy intake management crucial. However, the fluctuations in estradiol and progesterone levels throughout the menstrual cycle present a challenge in maintaining consistent energy intake. This study aimed to uncover the underlying factors associated with appetite regulation linked to menstrual phases and exercise using proteomic approach. Five female athletes engaged in 60 min of bicycle exercise, followed by 90 min of rest, during both the follicular and luteal phases. Serum samples were collected before, during, and after exercise, and the serum proteome was analyzed using 2D-gel electrophoresis. A total of 511 spots were detected in the subjects' serum profiles, with significant decreases observed in haptoglobin during the luteal phase and complement component 3 during bicycle training. Unsupervised learning with a generalized estimating equation analysis showed that serum peptide YY (PYY), an appetite suppressor, significantly influenced the fluctuations of serum proteins induced by exercise (p < 0.05). Regression analysis demonstrated a positive correlation between PYY and serum IgM (R = 0.87), implying that the intestinal environment and the immune response in female athletes may contribute to appetite regulation.

E3MPH16: An efficient endosomolytic peptide for intracellular protein delivery.

To facilitate the introduction of proteins, such as antibodies, into cells, a variety of delivery peptides have been engineered. These peptides are typically highly cationic and somewhat hydrophobic, enabling cytosolic protein delivery at the cost of causing cell damage by rupturing membranes. This balance between delivery effectiveness and cytotoxicity presents obstacles for their real-world use. To tackle this problem, we designed a new endosome-disruptive cytosolic delivery peptide, E3MPH16, inspired by mastoparan X (MP). E3MPH16 was engineered to incorporate three Glu (E3) and 16 His (H16) residues at the N- and C-termini of MP, respectively. The negative charges of E3 substantially mitigate the cell-surface damage induced by MP. The H16 segment is known to enhance cell-surface adsorption and endocytic uptake of the associated molecules. With these modifications, E3MPH16 was successfully trapped within endosomes. The acidification of endosomes is expected to protonate the side chains of E3 and H16, enabling E3MPH16 to rupture endosomal membranes. As a result, nearly 100% of cells achieved cytosolic delivery of a model biomacromolecule, Alexa Fluor 488-labeled dextran (10 kDa), via endosomal escape by co-incubation with E3MPH16. The delivery process also suggested the involvement of macropinocytosis and caveolae-mediated endocytosis. With the assistance of E3MPH16, Cre recombinase and anti-Ras-IgG delivered into HEK293 cells and HT1080 cells enabled gene recombination and inhibited cell proliferation, respectively. The potential for in vivo application of this intracellular delivery method was further validated by topically injecting the green fluorescent protein fused with a nuclear localization signal (NLS-GFP) along with E3MPH16 into Colon-26 tumor xenografts in mice.

Structural Dissection of Epsin-1 N-Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature.

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.

Split luciferase-based estimation of cytosolic cargo concentration delivered intracellularly via attenuated cationic amphiphilic lytic peptides.

Intracellular delivery of biomacromolecules is challenging as these molecules are taken up by cells and encapsulated into vesicular compartments called endosomes, and the fraction of molecules that are translocated to the cytosol are particularly important to obtain desired biological responses. This study aimed to estimate the cytosolic concentrations of intracellularly delivered peptides and proteins to aid the design of novel and effective biopharmaceutical delivery systems. To this end, we employed the split NanoLuc luciferase system, using the 11-residue HiBiT peptide segment as a probe for the delivered molecules in cells expressing the complementary LgBiT protein segment. The efficacy in cytosolic HiBiT delivery was determined by measuring the resultant luciferase activity when the HiBiT segment delivered into the cytosol forms a complex with LgBiT. Mean cytosolic HiBiT concentration was calculated using cell number and cell volume analysis. L17E and HAad peptides, developed in our laboratory for intracellular protein delivery, yielded approximately 6-fold cellular HiBiT concentrations than that obtained in their absence.

Stearylated Macropinocytosis-Inducing Peptides Facilitating the Cellular Uptake of Small Extracellular Vesicles.

Macropinocytosis is a form of endocytosis that allows massive uptake of extracellular materials and is a promising route for intracellular delivery of biofunctional macromolecules and nanoparticles. Our laboratory developed a potent macropinocytosis-inducing peptide named P4A. However, the ability of this peptide is not apparent in the presence of serum. This study aims to endow P4A and related peptides with the ability to induce macropinocytosis in the presence of serum by N-terminal acylation with long-chain fatty acids (i.e., decanoic, myristic, and stearic acids). Stearylated P4A (stearyl-P4A) had the highest effect on stimulating macropinocytotic uptake. Moreover, the intramolecularly disulfide-bridged analogue, stearyl-oxP4A, showed an even higher ability. The effect of stearyl-oxP4A to facilitate the intracellular delivery of small extracellular vesicles (sEVs) was evaluated in terms of (i) cellular uptake using sEVs labeled with an enhanced green fluorescent protein (EGFP) and (ii) cytosolic liberation and expression of sEV-encapsulated luciferase mRNA in recipient cells. The two- to threefold uptake of both sEVs in the presence of stearyl-oxP4A suggests the potential of the peptide for sEV delivery in the presence of serum.

Modular Redesign of a Cationic Lytic Peptide To Promote the Endosomal Escape of Biomacromolecules.

Endocytosis is an important route for the intracellular delivery of biomacromolecules, wherein their inefficient endosomal escape into the cytosol remains a major barrier. Based on the understanding that endosomal membranes are negatively charged, we focused on the potential of cationic lytic peptides for developing endosomolysis agents to release such entrapped molecules. As such, a venom peptide, Mastoparan X, was employed and redesigned to serve as a delivery tool. Appending a tri-glutamate unit to the N-terminus attenuates the cytotoxicity of Mastoparan X by about 40 fold, while introduction of a NiII -dipicolylamine complex enhances cellular uptake of the peptide by about 17 fold. Using the optimized peptide, various fluorescently labeled macromolecules were successfully delivered to the cytosol, enabling live-cell imaging of acetylated histones.