5 research outputs found
The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation
Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAFMEK- ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS
Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells
Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium
Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells
Cholera toxin (CT) enters host intestinal
epithelia cells, and
its retrograde transport to the cytosol results in the massive loss
of fluids and electrolytes associated with severe dehydration. To
initiate this intoxication process, the B subunit of CT (CTB) first
binds to a cell surface receptor displayed on the apical surface of
the intestinal epithelia. While the monosialoganglioside GM1 is widely
accepted to be the sole receptor for CT, intestinal epithelial cell
lines also utilize fucosylated glycan epitopes on glycoproteins to
facilitate cell surface binding and endocytic uptake of the toxin.
Further, l-fucose can competively inhibit CTB binding to
intestinal epithelia cells. Here, we use competition binding assays
with l-fucose analogs to decipher the molecular determinants
for l-fucose inhibition of cholera toxin subunit B (CTB)
binding. Additionally, we find that mono- and difucosylated oligosaccharides
are more potent inhibitors than l-fucose alone, with the
LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding
to two colonic epithelial cell lines (T84 and Colo205). Finally, a
non-natural fucose-containing polymer inhibits CTB binding two orders
of magnitude more potently than the LeY glycan when tested against
Colo205 cells. This same polymer also inhibits CTB binding to T84
cells and primary human jejunal epithelial cells in a dose-dependent
manner. These findings suggest the possibility that polymeric display
of fucose might be exploited as a prophylactic or therapeutic approach
to block the action of CT toward the human intestinal epithelium