- Calcium is the single most important regulator of keratinocyte differentiation. A major goal in keratinocyte biology is to understand how the calcium signal is transduced to drive keratinocyte differentiation. S100 proteins are an important class of protein that are required for calcium signal transduction. They are activated by a calcium binding-dependent conformation change, and then bind to and regulate the function of target proteins. The target proteins include regulators of cell proliferation and differentiation. Although keratinocytes express eight S100 proteins, little information is available regarding S100 protein function in keratinocytes. S100 proteins are thought to function as noncovalently associated homodimers. However, their recent study shows that the calcium-dependent enzyme, transglutaminase (TG), can modify S100 proteins by adding two interprotein covalent links, one linking each end of the antiparallel homodimer. They hypothesize that this regulates S100 function by altering the calcium-dependent conformation change. This hypothesis links transglutaminase, S 100 proteins, and calcium as components of a common regulatory unit. In this model, TG modification of S100 protein structure could provide a trigger to promote differentiation. The overall goals of this study are to characterize the S100 proteins expressed in keratinocytes with respect to (i) ability to act as a TG substrates and the effects of TG modification on function, (ii) subcellular and tissue distribution and the effects of physiological agents in this distribution, (iii) interaction with target proteins, and (iv) alteration in psoriasis. In preparation for these studies they have cloned cDNAs, expressed protein, and produced antibodies against S100A11, S100A10, and S100A7. They show that each of these proteins are TG substrates and in one case, S100A11; they identified the TG reactive sites by amino acid microsequencing. Their study is designed to provide new information on S100 protein function in keratinocytes.
In Specific Aim 1, they clone, express, and generate antibodies to the remaining keratinocyte S100 proteins, S100A2, S100A4, S100A6, S100A8, and S100A9. These reagents are used (i) to determine whether these S100 proteins form TG-dependent covalent multimers in vivo and in cultured keratinocytes, (ii) to determine the structure of crosslinked products, (iii) to identify the amino acids involved in crosslink formation, and (iv) to study S100 protein function in psoriasis. The studies described in Specific Aim 2, are designed to determine whether TG-dependent modification of S100A11, their prototype S100 protein, alters its ability to interact with its target substrate, annexin I. This, they expect, will provide a direct test of their hypothesis that TG modification is a regulatory mechanism designed to alter S100 protein function. Localization can profoundly influence function. Their preliminary studies show that S100A11 is mobilized to the plasma membrane in response to calcium.
In Specific Aim 3 they (i) use anti-S100 protein antibodies to localize the proteins in epidermis (normal and psoriatic), and in cultured cells, (ii) and examine the effects of physiological agents (calcium, etc.) on their subcellular distribution. S100 proteins transduce calcium signals by binding to target proteins; however, the targets are not known in keratinocytes. The goal of Specific Aim 4 is to identify these target proteins using ligand blots, co-immunoprecipitation, affinity chromatography, and protein microsequencing.
