In cellulo phosphorylation induces pharmacological reprogramming of maurocalcin, a cell-penetrating venom peptide

Abstract

The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr 26 belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr 26 phosphorylation leads to charge neutralization of Arg 24 , a residue crucial for MCa agonist activity. The functional effect of Thr 26 phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs.maurocalcin | phosphorylation | pharmacology | toxin | ryanodine receptor

Maurocalcin phosphorylated at threonin 26 maintains its activity on ryanodine receptor-mediated Ca 2+ release in intact muscle fibers

“…Altogether, we believe that the above results are an important complement to the data presented by Ronjat et al (1) and there are reasons worth discussing as to why MCaThr 26 -Phospho becomes inefficient in in vitro assays but remains active in intact muscle fibers. One possibility could be that high phosphatase activity inside the muscle fibers so completely dephosphorylates intracellular MCaThr 26 -Phospho as to release active MCa.…”

“…Ronjat et al (1) report that a phosphorylated form of the scorpion venom toxin maurocalcin (MCa) loses its ability to activate its preferred target, the type 1 ryanodine receptor (RYR1). RYR1 is the sarcoplasmic reticulum (SR) Ca 2+ release channel responsible for the rise in cytosolic Ca 2+ , which activates contraction during excitation-contraction coupling of skeletal muscle.…”

Integration of phylogenomics and molecular modeling reveals lineage-specific diversification of toxins in scorpions

“…Calcins rapidly activate ryanodine receptors (RyRs) in cardiac or skeletal muscle cells in mammals with high affinity and specificity (reviewed in Xiao et al (2016) ). These peptides bind to cell surface glycosaminoglycans and membrane lipids ( Mabrouk et al, 2007 ; Ram et al, 2008 ), translocating also into cells, and undergo posttranslational modifications by target cell enzymes ( Ronjat et al, 2016 ). To date, calcins have only been discovered in 16 species from eight families (but not Buthidae).…”

Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

“…For example, the plant‐derived cyclotides kalata B1 (kB1) and Momordica cochinchinensis trypsin inhibitors I and II (MCoTI‐I and MCoTI‐II), as well as their analogs, have been shown to enter mammalian cells and have been proposed to be promising scaffolds not only for stabilizing bioactive peptides but also for drug delivery . Examples of venom‐derived CPPs include crotamine, from the South American Rattlesnake, which shows nuclear localization after being internalized by cells, and maurocalcine, from the venom of the scorpion Scorpio maurus palmatus, which binds to the intracellular ryanodine receptor and triggers internal Ca 2+ release seconds after being applied extracellularly …”