Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics.

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

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

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.

The small heat shock protein, HSPB6, in muscle function and disease.

The small heat shock protein, HSPB6, is a 17-kDa protein that belongs to the small heat shock protein family. HSPB6 was identified in the mid-1990s when it was recognized as a by-product of the purification of HSPB1 and HSPB5. HSPB6 is highly and constitutively expressed in smooth, cardiac, and skeletal muscle and plays a role in muscle function. This review will focus on the physiologic and biochemical properties of HSPB6 in smooth, cardiac, and skeletal muscle; the putative mechanisms of action; and therapeutic implications.

RGD-dependent binding of TP508 to integrin alphavbeta3 mediates cell adhesion and induction of nitric oxide.

TP508, a 23-amino acid RGD-containing synthetic peptide representing residues 508 to 530 of human prothrombin, mitigates the effects of endothelial dysfunction in ischaemic reperfusion injury. The objective of this study was to investigate whether TP508 binds to members of the integrin family of transmembrane receptors leading to nitric oxide synthesis. Immobilised TP508 supported adhesion of endothelial cells and alphavbeta3-expressing human embryonic kidney cells in a dose- and RGD-dependent manner. Soluble TP508 also inhibited cell adhesion to immobilised fibrinogen. The involvement of alphavbeta3 was verified with function-blocking antibodies and surface plasmon resonance studies. Adhesion of the cells to immobilised TP508 resulted in an induction of phosphorylated FAK and ERK1/2. In endothelial cells, TP508 treatment resulted in an induction of nitric oxide that could be inhibited by LM609, an alphavbeta3-specific, function-blocking monoclonal antibody. Finally, TP508 treatment of isolated rat aorta segments enhanced carbachol-induced vasorelaxation. These results suggest that TP508 elicits a potentially therapeutic effect through an RGD-dependent interaction with integrin alphavbeta3.

Transduction of peptide analogs of the small heat shock-related protein HSP20 inhibits intimal hyperplasia.

BACKGROUND: Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstructions. However, vasospasm can lead to early graft failure. The leading cause of delayed graft failure is intimal hyperplasia. OBJECTIVE: To develop a proteomic approach to prevent vein-graft spasm and intimal hyperplasia. METHODS: Biomimetic peptide analogs of the small heat shock-related protein HSP20, containing a protein transduction domain (PTD), a phosphorylated serine, and a sequence of HSP20 surrounding the phosphorylation site (PTD-pHSP20), or a scrambled sequence of the same amino acids surrounding the phosphorylation site (PTD-scHSP20) were synthesized. The peptides were used in muscle bath and organ culture experiments with human saphenous vein (HSV) segments. Cultured smooth muscle cell lines were used to determine the effect of the peptides on proliferation and migration. RESULTS: In HSV rings precontracted with norepinephrine, PTD-pHSP20 but not PTD-scHSP20 led to relaxation. There was no significant difference in smooth muscle cell proliferation in cells treated with PTD-pHSP20 compared with PTD-scHSP20. Treatment with PTD-pHSP20 significantly inhibited cellular migration compared with PTD-scHSP20. Control, untreated, and PTD-scHSP20-treated saphenous veins had significant increases in intimal thickness after culture. This intimal thickening was completely inhibited by treatment with PTD-pHSP20. CONCLUSIONS: Protein transduction of biologically active motifs of HSP20 can affect pathologic and physiologic responses of HSV and represents a novel proteomic-based therapeutic approach. CLINICAL RELEVANCE: We have been a part of the genomics era and are now viewing the emergence of "proteomics." The genome is linear and relatively easy to examine; however the proteome is much more complex and dynamic. In essence, the purpose of gene therapy is to manipulate the genome to produce a particular protein. This manuscript describes a new proteomic approach in which the biologically active part of a protein is directly introduced into vascular cells. Peptides were synthesized which contained a total of 24 amino acids, 11 of which represent a protein transduction domain or "carrier" while the other 13 are the biologically active "cargo." These synthetic peptides prevent spasm (contraction) and intimal hyperplasia in segments of human saphenous vein treated ex vivo. Preclinical development is currently underway to develop these molecules as a proteomic-based vein harvest solution to enhance vein-graft patency.