Dataset Information

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

ABSTRACT: Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.

SUBMITTER: Lee SC

PROVIDER: S-EPMC4106332 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

Lee Shao-Chen SC Lin Chien-Chu CC Wang Chia-Hui CH Wu Po-Long PL Huang Hsuan-Wei HW Chang Chung-I CI Wu Wen-guey WG

The Journal of biological chemistry 20140604 29

Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese ...[more]

PMID: 24898246

Similar Datasets

Project description:Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-?) and interferon gamma (IFN-?). While TNF-?/IFN-? treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.

Project description:In this project, we combine two areas of research, experimental characterization and molecular docking studies of the interaction of positively charged oligopeptides with crucial blood plasma proteins. The investigated peptides are rich in NH2 groups of amino acid side chains from Dap, Orn, Lys, and Arg residues, which are relevant in protein interaction. The peptides are 9- and 11-mer with the following sequences: (Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt), (Lys-Dab-Ala-Gly-Orn-Pro-His-Lys-Arg), and (Lys-Dab-Dab-Gly-Orn-Pro-Phe(2-F)-Lys-Arg). The net charge of the compound strongly depends on the pH environment and it is an important aspect of protein binding. The studied oligopeptides exhibit therapeutic properties: anti-inflammatory activity and the capacity to diminish reactive oxygen species (ROS). Therefore, the mechanism of potential binding with blood plasma components is the next challenge. The binding interaction has been investigated under pseudo-physiological conditions with the main blood plasma proteins: albumin (BSA), α1-acid glycoprotein (AAG), and γ-globulin fraction (GGF). The biomolecular quenching constant (kq) and binding constant (Kb) were obtained by fluorescence spectroscopy at various temperatures. Simultaneously, the changes in the secondary structure of proteins were monitored by circular dichroism (CD) and infrared spectroscopy (IR) by quantity analysis. Moreover, molecular docking studies were conducted to estimate the binding affinity, the binding domain, and the chemical nature of these interactions. The results show that the investigated oligopeptides could be mainly transported by albumin, and the binding domain I is the most favored cavity. The BSA and GGF are able to form stable complexes with the studied compounds as opposed to AAG. The binding reactions are spontaneous processes. The highest binding constants were determined for Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt peptide, in which the values of the binding constants Kb to BSA and GGF were 10.1 × 104 dm3mol-1 and 3.39 × 103 dm3mol-1, respectively. The positively charged surface of peptides participated in salt bridge interaction with proteins; however, hydrogen bonds were also formed. The secondary structure of BSA and GGF after contact with peptides was changed. A reduction in the α-helix structure was observed with an increase in the β-sheet and β-turn and random coil structures.

Dataset Information

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

Publications

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets