Cutaneous ageing is an important extrinsic process that modifies the pigmentary system. Because cellular senescence is a fundamental ageing mechanism, we examined the role of senescent cells in ageing pigmentation.Methods: Biopsies obtained from senile lentigo and perilesional normal skin were assayed for a marker of cellular senescence, p16INK4A. To determine the secretory phenotypes of senescent fibroblasts, we performed microarray, RNA sequencing and methylation array analyses in senile lentigo and senescent fibroblasts. To further investigate the impact of senescent cells on ageing-related pigmentation, an intervention that targeted senescent cells using radiofrequency was performed.Results: In vivo, senescent fibroblasts accumulated at the sites of age-related pigmentation. Phenotype switching of the cells resulted in the repression of stromal-derived factor 1 (SDF1) by promoter methylation. SDF1 induced melanocyte differentiation via stromal-epithelial interactions, ultimately driving skin pigmentation. Furthermore, the elimination of senescent fibroblasts from pigmented skin using radiofrequency was accompanied by skin lightening, rendering it a potential target for treatment.Conclusion: Aged pigmented skin contains an increasing proportion of senescent fibroblasts. Cells with phenotype switching exhibited a loss of SDF1, which stimulates the melanogenic process and thereby contributes to aging pigmentation. These data may promote the development of new therapeutic paradigms, such as a stroma-targeting therapy for pigmentary disorders.

Background: Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds. Results: SrtA was used to backbone-cyclize disulfide-rich peptides, including kalata B1, ␣-conotoxin Vc1.1, and SFTI-1. Conclusion: SrtA-mediated cyclization is applicable to small disulfide-rich peptides. Significance: SrtA-mediated cyclization is an alternative to native chemical ligation for the cyclization of small peptides of therapeutic interest.

Ultraviolet (UV)-associated hyperpigmented skins are characterized with increased vasculature underlying pigmentation, suggestive of the possible biological role of endothelial cells in the regulation of skin pigmentation during UV irradiation. In this study, we showed that UV-irradiated endothelial cells significantly increased the pigmentation of melanocytes through epithelial-mesenchymal crosstalk. The stimulatory effect of endothelial cells was further demonstrated using ex vivo human skin. RNA sequence analysis and enzyme-linked immunosorbent assay showed that endothelial cells secrete more stem cell factor (SCF) upon UV irradiation than non-irradiated cells. The increased pigmentation elicited by endothelial cells was abrogated following inhibition of SCF/c-KIT signaling. Together these results suggest that endothelial cells are activated upon UV exposure to release melanogenic factors such as SCF, which contributes to the development of skin hyperpigmentation during chronic sun exposure.

It has been proposed that melasma is a photoageing skin disorder. The photoaged fibroblasts have been suggested as an important source of melanogenic factors which are involved in the regulation of pigmentation. To investigate whether melasma includes senescent cells, lesional and perilesional normal skin from 38 melasma patients was assessed using a cell senescence marker, p16INK4A. The results showed that lesional dermal skin had more p16INK4A‐positive senescent cells than perilesional skin. The impact of senescent fibroblasts was further investigated in a pilot study using radiofrequency (RF) intervention for melasma. It showed that the RF therapy decreased the number of senescent cells with increased expression of procollagen‐1, which were associated with reduced epidermal pigmentation. This leads us to the speculation that senescent fibroblasts may contribute to drive melasma and might be considered as a potential therapeutic target.

Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules.