10-Residue MyD88-Peptide Adopts β-Sheet Structure, Self-Assembles, Binds to Lipopolysaccharides, and Rescues Mice from Endotoxin-Mediated Lung-Infection and Death

Abstract

Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel β-sheet AMPs, we identified two short cationic amphipathic β-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine–valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with β-sheet structures and self-assembling properties.

Characterization of a Myeloid Differentiation Factor 2-Derived Peptide that Facilitates THP-1 Macrophage-Mediated Phagocytosis of Gram-Negative Bacteria

“…The cell line was maintained by usual protocol in an Innova CO 2 incubator. 13,14 Animals. Female Balb/c 4−6-week mice (National Laboratory Animal Center, CSIR-Central Drug Research Institute, Lucknow) were given a standard laboratory diet and water ad libitum and housed under controlled environmental conditions.…”

“…THP-1 (human monocytic cell line derived from an acute monocytic leukemia patient) was grown in suspension in RPMI 1640 medium, supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% antibiotics at 37 °C in a humidified 5% CO 2 (v/v) incubator. THP-1 cells at a density of 1 × 10 6 cells/mL were treated with 0.3 mg/mL PMA (Sigma-Aldrich) for 24 h, , inducing plastic-adherent cells that were further rested in complete RPMI 1640 medium for an additional 24 h before stimulation with various treatments. Cells were counted with the help of an Olympus inverted microscope for the experiments.…”

“…THP-1, a human embryonic kidney cell line, was obtained from a CSIR-Central Drug Research Institute, Lucknow, cell line repository. The cell line was maintained by usual protocol in an Innova CO 2 incubator. , …”

Short peptides based on the conserved regions of MIEN1 protein exhibit anticancer activity by targeting the MIEN1 signaling pathway

“…Anticancer peptides (ACPs) have been shown to selectively inhibit tumor cell migration and invasion, or suppress angiogenesis and thus are less likely to cause chemoresistance ( 4 , 5 ). Peptides derived from naturally occurring proteins are known to counteract the signaling pathways of their parental proteins, demonstrating potent biological activities ( 6 , 7 , 8 , 9 ).…”