GHK-Cu (Copper Peptide) Research & Studies

Copper peptide, a low molecular weight peptide composed of glycyl-L-histidyl-l-lysine-copper, possesses anti-aging, anti-inflammatory, and wound healing properties. In this study, we investigated the effects of a combination agent CP-AcT, composed of palmitoyl copper peptide (pal-GHK-Cu) and acetyl tyrosine (N-Acetyl-l-tyrosine), on melanin production in the human malignant melanoma cell line A375 and the mouse melanoma cell line B16. Firstly, the cytotoxicity of CP-AcT at various concentrations (0-8 μg/mL) on HaCat, HFF, A375, and B16 cells was evaluated. Subsequently, the effects of the CP-AcT on tyrosinase activity both extracellular and intracellularly, as well as on melanin production in two melanoma cell lines, were evaluated under conditions that did not compromise cell viability. Additionally, quantitative gene plex (QGP) combined with branched DNA (bDNA) technology was used to analyze the effects of CP-AcT on the expression of melanin-related genes in A375 cells, with a focus on five specific genes. Finally, the effects of the CP-AcT on the expression of three proteins involved in the biosynthesis pathway of melanin: tyrosinase (TYR), dopachrome tautomerase (DCT), and endothelin 3 (EDN3) were analyzed. The results indicate that the complex CP-AcT effectively promotes melanin production in both types of melanoma cells.

Peptides are recognized as highly effective and safe bioactive ingredients. However, t their practical application is limited and hampered by harsh conditions for practical drug delivery. Hence, a novel peptide nanocarrier of copper peptide (GHK-Cu) encapsulation developed by liposome technology combined with the classical Chinese concept of rigidity and flexibility. Different polyols were selected as modification ligands for phospholipid bilayers to construct a nano drug-carrying system with high loading rate, good stability and biocompatibility. In vitro, this complex not only significantly retarded the release ability of copper peptides, but also enabled copper peptides to be effectively resistant to enzymatic degradation. Furthermore, cellular experiments showed that this system mainly regulates Nrf2, SIRT1, and PEG2/COX-2-related signaling pathways, thus effectively counteracting cellular inflammation, senescence, and apoptosis from oxidative damage. Interestingly, a green, non-toxic, efficient and convenient antioxidant system was developed for the prevention and deceleration of skin aging.

Wound healing in diabetes is retarded by the dysfunctional local microenvironment. Although there are many studies using hydrogels as substitutes for natural extracellular matrices (ECMs), hydrogels that can mimic both the structure and functions of natural ECM remain a challenge. Self-assembling peptide RADA16 nanofiber has distinct advantages to provide a biomimetic extracellular matrix nanofiber structure. However, it still lacks biological cues to promote angiogenesis that is of vital significance for diabetic wound healing. With a customized copper peptide glycyl-histidyl-lysine (GHK) functionalized RADA16, an integrated approach using functionalized RADA16 nanofiber to chelate copper ion, is innovatively proposed in this present study. The acquired composite hydrogel holds the biomimetic nanofiber architecture, and exhibits promoting angiogenesis by both enhancing adhesion and proliferation of endothelial cells (EC) in vitro and neovascularization in vivo. It shows that the functionalized nanofiber scaffolds significantly accelerated wound closure, collagen deposition, and tissue remodeling both in healthy and diabetic mice. Furthermore, immunohistochemical analysis give evidence that an upregulated expression of eNOS and CD31 in the copper peptide-functionalized RADA16 treated group. It can be envisioned that this scaffold can serve as a promising dressing for diabetic wound healing.

We report ligand field molecular mechanics, density functional theory and semi-empirical studies on the binding of Cu(II) to GlyHisLys (GHK) peptide. Following exhaustive conformational searching using molecular mechanics, we show that relative energy and geometry of conformations are in good agreement between GFN2-xTB semi-empirical and B3LYP-D DFT levels. Conventional molecular dynamics simulation of Cu-GHK shows the stability of the copper-peptide binding over 100 ps trajectory. Four equatorial bonds in 3N1O coordination remain stable throughout simulation, while a fifth in apical position from C-terminal carboxylate is more fluxional. We also show that the automated conformer and rotamer search algorithm CREST is able to correctly predict the metal binding position from a starting point consisting of separated peptide, copper and water.

Copper peptide (GHK-Cu) plays an important role in skin regeneration and wound healing. However, its skin absorption remains challenging due to its hydrophilicity. Here we use polymeric microneedle array to pre-treat skin to enhance GHK-Cu skin penetration.

Aggregation of the beta-amyloid peptide (Abeta) to amyloid plaques is a key event in Alzheimer's disease. According to the amyloid-cascade hypothesis, Abeta aggregates are toxic to neurons through the production of reactive oxygen species (ROS). Copper ions play an important role, because they are able to bind to Abeta and influence its aggregation properties. Moreover, Cu-Abeta is supposed to be directly involved in ROS production. To get a better understanding of these reactions, we measured the production of HO(.) and the redox potential of Cu-Abeta. The results were compared to other biological copper-peptide complexes in order to get an insight into the biological relevance. Cu-Abeta produced more HO(.) than the complex of copper with Asp-Ala-His-Lys (Cu-DAHK), but less than with Gly-His-Lys (Cu-GHK). Cyclic voltammetry revealed that the order for reduction potential is Cu-GHK>Cu-Abeta>Cu-DAHK, but for the oxidation potential the order is reversed. Thus, easier copper redox cycling correlated to higher HO(.) production. The copper complex of the form Abeta1-42 showed a HO(.) production five-times higher than that of the form Abeta1-40. Time-dependence and aggregation studies suggest that an aggregation intermediate is responsible for this increased HO(.) production.

[Cu(2+).Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 microM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 microM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 microM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu(2+)-amino-terminal copper and nickel binding motif (lambda (max) approximately 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu(2+) charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (DeltaE = 92 mV) for the Cu(3+/2+) redox couple obtained for [Cu(2+).Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu(2+).Lys-Gly-His-Lys](+) (0.84 V, DeltaE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu(2+).Lys-Gly-His-Lys](+) homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O(2)) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k (obs) = 7.7 x 10(-2) min(-1), yielding a second-order rate constant of (7.7 +/- 0.9) x 10(4) M(-1) min(-1). Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.