Transduction of biologically active motifs of the small heat shock‐related protein, HSP20, leads to relaxation of vascular smooth muscle

Abstract

Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.

Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

“…HSP20 phosphorylation at serine 16 can be catalyzed by the cAMP-dependent kinase, PKA (13). Phosphopeptide analogs of HSP20 induce the relaxation of several different smooth muscle tissues and cell types (46,49,76,103,137). In vitro, phosphorylated HSP20 interacts directly with 14-3-3, the chaperone protein that sequesters cofilin (26).…”

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

“…Peptide mimetics are advantageous in determining full-length protein activity as they have fewer domains available for binding/interaction, are usually easier to synthesize in large quantities, and less susceptible to proteolytic activity. A synthesized HSP20 analog, PTD-pHSP20 composed of an optimized protein transduction domain, fused to sequences encoding the active portion (phosphorylation site, serine 16 of HSP20) [6] has been shown to relax a wide array of smooth muscles including bovine carotid artery, porcine coronary artery, human saphenous vein and human umbilical artery [8,10,12,13]. To compare the magnitude of the physiological responses of the entire recombinant molecule to the peptide mimetics, rings of rat aorta contracted with 5-HT (0.5 μM) displayed steady contractions that were sustained for long periods of time (> 20 min), whereas treatment of aorta rings with increasing amounts of PTD-pHSP20 (100 μM, 500 μM and 1 mM respectively) led to a dosedependent decrease in contraction that was maximal at a PTD-pHSP20 concentration of 1 mM (data not shown).…”

“…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].…”