Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle

Abstract

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

“…pACYC Duet PKG was constructed by ligating the BglII-XhoI insert of pET29a PKG and the backbone of BglII-XhoIdigested pACYC Duet (Stratagene). Construction of pET14b TAT-HSP20 has been described previously [13].…”

“…One and two dimensional SDS-PAGE, immunoblot analyses and quantification were performed as described previously [13].…”

“…Like other members of the heat shock protein family, HSP20 requires phosphorylation at a serine residue (S16) to become functionally active [5,6]. HSP20 is a specific substrate protein of PKA and PKG and accumulating evidence suggests HSP20 has several physiological and biochemical roles including controlling muscle tone, regulating cell motility, modulating actin filament dynamics and protection against intimal hyperplasia and ischemia/reperfusion injury [7][8][9][10][11][12][13][14]. Although the molecular mechanisms of action for HSP20 have not been determined fully, it is an actinassociated protein and we have shown that HSP20 peptide treatment leads to loss of actin stress fibers and disruption of focal adhesion complexes [9][10][11].…”

“…We transduced phosphoproteins and peptides into tissues using protein transduction domains (PTDs) and in doing so prevented smooth muscle contraction [12,13]. The use of PTDs as tools for intracellular delivery is a relatively new approach that is receiving increased attention as new methods for its implementation are developed and evidence for its efficacy is established.…”

“…PTDs, first discovered independently by the Green and Frankel laboratories, are positively charged amino acid sequences facilitating the entry of proteins into cells through macropinocytotic mechanisms [15][16][17]. Our laboratory has successfully implemented the use of these sequences to transduce different proteins and peptides into numerous tissues and cells [8][9][10][11][12][13][14].…”