Calcium is a key regulator in the normal differentiation of epidermal keratinocytes. The calcium signal is transduced via calcium-binding proteins. The S100 family of calcium-binding proteins are strongly implicated in tissue-specific mediation of the calcium signal. Given that calcium is an important regulator of differentiation and that keratinocytes synthesize several S100 proteins, aberrant differentiation of keratinocytes may, in part, be due to altered expression of this important class of calcium-binding proteins. In support of this idea, psoriatic keratinocytes display an altered expression of this important expression with S100A8, S100A9 and S100A7 being upregulated. In this proposal, the role of one of these proteins, S100A7, or psoriasin, in psoriasis will be examined. S100A7 has a limited tissue distribution, being present in fetal skin, ear, and tongue. This S100 protein is not synthesized in normal human interfollicular skin, however it is highly expressed in psoriatic lesional plaques. The mechanism by which S100A7 influences abnormal keratinocyte differentiation is not known. In general, S100 proteins mediate calcium signal through a calcium-dependent conformational change. The calcium-activated S100 protein binds to, and modifies the activity of, specific target proteins. While many targets of S100 proteins have been identified, the target protein(s) of S100A7 is not known. In addition, some of the S100 proteins are post-translationally modified, which potentially affects their physiological function. For example, S100A11 and S100A10 are covalently cross-linked by epidermal transglutaminases (TGs) in vivo. Based on the shared sequence and structural homology within the S100 protein family, it is likely that S100A7 is similarly modified by TG. It is suspected that this modification could prove to be a critical step in regulation of differentiation since it links together calcium (a key regulator of the process), S100 protein (a component of the transduction pathway) and TG (the enzyme responsible for the formation of the cornified envelope- a terminal differentiation product). The overall goals of this study are to (i) characterize, in vivo and in vitro, the ability of TGs to covalently modify S100A7 and (ii) to identify the target protein(s) of S100A7. In preparation for these studies, the coding region of S100A7 has been cloned into a prokaryotic expression vector. The antibody elicited against rhS100A7, however, cross-reacts with other S100 proteins.
In specific aim 1 a antibody directed against the V8 protease released carboxyl-terminal peptide of S100A7 will be produced. This peptide is from an area of divergence within the S100 family.
In specific aim 2 the ability of TGs to cross-link S100A7 in vitro and in vivo will be investigated. rhS100A7 will be incubated in vitro with TG and the products analyzed by polyacrylamide gel. Specific sites of cross-link formation will be identified after trypsin digestion of S100A7, peptide purification and sequence analysis. Keratinocyte extracts, from cell culture and from normal and psoriatic epidermis, will be analyzed by western blot to determine if S100A7 is modified in vivo by TG. The goal of Specific Aim 4 is to identify S100A7 target protein(s). This fundamentally important question will be answered using ligand blots, co-immunoprecipitation assays, affinity chromatography and protein microsequencing.
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