The role of palmitoylation in regulating the development of the embryo and the epidermis

Abstract

Palmitoylation is a reversible post-translational modification, involving the addition of the fatty acid palmitate to cysteine residues of target proteins. In recent years, this modification has emerged as orchestrating the subcellular localisation of protein substrates and regulating signalling pathways. Palmitoylation is mediated by the palmitoyl acyl transferases (PATs), which represent the major palmitoylation enzymes in yeast and mammals. This protein family has been linked to multiple developmental diseases, indicating that these enzymes have a significant role in the development of the organism. In this thesis, the expression of PATs in the developing mouse embryo was investigated. It was found that several of these genes display specific expression at the later stages of organogenesis, indicating a developmental role for these proteins. Expression was detected in the following organs: the limb, the eye, the lung, somites and hair follicles. The role of palmitoylation in mammalian development was analysed further by generating a knockout mouse targeting the PAT Zdhhc2. This mouse did not display any physiological or neurological defects, indicating that the role of Zdhhc2 in development is redundant. To further examine the role of PATs in organ homeostasis, the mouse mutant depilated (dep) was studied. dep is caused by a spontaneous mutation in the PAT Zdhhc21 and its phenotype is comprised of progressive hair loss, greasy coat and pigmentation defects. Zdhhc21 maps within a locus associated with craniofacial malformation in humans, however, analysis of P1 dep pups revealed no craniofacial defects. Given that the dep mouse displays a skin phenotype, this mutant was used to investigate the role of Zdhhc21 in the development and maintenance of the epidermis and hair follicle. The number and location of follicular stem cells was analysed and whilst dep hair follicles contained similar number of these cells compared to wildtype, the intra-follicular distribution tended to be altered. Furthermore, subjecting the dep mouse to cutaneous wounding captured an initial delay in wound-healing, with a trend of decreased proliferation. This suggests a potential role for Zdhhc21 in regulating the activity of epidermal stem cell. In order to further understand the molecular basis of the dep phenotype, potential protein targets of Zdhhc21 were isolated and identified using a proteomic approach involving click chemistry. A candidate list was generated and four candidates (CD81, Claudin1, GNAQ and Rab1b) were chosen to be further analysed as targets of Zdhhc21. Three of these candidates were found to be mislocalised in dep keratinocytes, indicating that Zdhhc21 regulates the intracellular trafficking of these proteins in primary keratinocytes. These candidates were also found to be expressed in the same epidermal structure as Zdhhc21, suggesting that these proteins may be involved in the development of the dep phenotype. Furthermore, these candidates have also been shown to be implicated in the ERK signalling cascade, which was found to be upregulated in dep hair follicles.