Post-Aire Maturation of Thymic Medullary Epithelial Cells Involves Selective Expression of Keratinocyte-Specific Autoantigens

The autoimmune regulator (Aire)-directed ectopic expression of tissue-specific antigens (TSAs) by mature medullary thymic epithelial cells (mTECs) has been viewed as an essential mechanism in the induction of central tolerance. Recent data suggest that the survival of mTECs extends beyond the Aire+ cell population to form the post-Aire mTEC population and Hassall’s corpuscles (HCs). The nature and function of these post-Aire epithelial cells and structures, however, have remained unidentified. In this study, we characterized in detail the end-stage development of mTECs and HCs in both Aire-sufficient and Aire-deficient mice. In addition, using a transgenic mouse model in which the LacZ reporter gene is under the control of the endogenous Aire promoter, we purified and analyzed the post-Aire mTECs to characterize their function. We showed that the end-stage maturation of mTECs closely resembles that of keratinocytes and that the lack of Aire results in a marked block of mTEC differentiation, which is partially overcome by ligands for RANK and CD40. We also provide evidence that, during mTEC development, Aire is expressed only once and during a limited 1–2 day period. The following loss of Aire expression is accompanied by a quick downregulation of MHC class II and CD80, and of most of the Aire-dependent and Aire-independent TSAs, with the exception of keratinocyte-specific genes. In the final stage of maturation, the mTECs lose their nuclei to become HCs and specifically express desmogleins (DGs) 1 and 3, which, via cross-presentation by APCs, may contribute to tolerance against these pemphigus vulgaris-related TSAs

Germacrene A Synthase in Yarrow (Achillea millefolium) Is an Enzyme with Mixed Substrate Specificity: Gene Cloning, Functional Characterization and Expression Analysis

Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes