Unraveling the function of bacterial-type phosphoenolpyruvate carboxylase in vascular plants

Two distinct phosphoenolpyruvate carboxylase (PEPC) isozymes occur in vascular plants and green algae: plant-type PEPC (PTPC) and bacterial-type PEPC (BTPC). PTPC polypeptides typically form a tightly regulated cytosolic Class-1 PEPC homotetramer. BTPCs, however, appear to be less widely expressed and to exist only as catalytic and regulatory subunits that physically interact with co-expressed PTPC subunits to form hetero-octameric Class-2 PEPC complexes that are highly desensitized to Class-1 PEPC allosteric effectors. Yeast two-hybrid studies indicated that castor plant BTPC (RcPPC4) interacts with all three Arabidopsis thaliana PTPC isozymes, and that it forms stronger interactions with AtPPC2 and AtPPC3, suggesting that specific PTPCs are preferred for Class-2 PEPC formation. In contrast, Arabidopsis BTPC (AtPPC4) appeared to interact very weakly with AtPPC2 and AtPPC3, suggesting that BTPCs from different species may have different physical properties, hypothesized to be due to sequence dissimilarities within their ~10 kDa intrinsically disordered region. Recent RNA-seq and microarray data were analyzed to obtain a better understanding of BTPC expression patterns in different tissues of various monocot and dicot species. High levels of BTPC transcripts, polypeptides and Class-2 PEPC complexes were originally discovered in developing castor seeds, but the analysis revealed a broad range of diverse tissues where abundant BTPC transcripts are also expressed, such as the developing fruits of cucumber, grape, and tomato. Marked BTPC expression correlated well with the presence of ~116 kDa immunoreactive BTPC polypeptides, as well as Class-2 PEPC complexes in the immature fruit of cucumbers and tomatoes. It is therefore hypothesized that in vascular plants BTPC and thus Class-2 PEPC complexes maintain anaplerotic PEP flux in tissues with elevated malate levels that would potently inhibit ‘housekeeping’ Class-1 PEPCs. Elevated levels of malate can be used by biosynthetically active sink tissues such as immature tomatoes and cucumbers for rapid cell expansion, drought or salt stressed roots for osmoregulation, and developing seeds and pollen as a precursor for storage lipid and protein biosynthesis.

The pathophysiology of eosinophilic esophagitis: Altered mechanisms of antigen detection in the esophagus

Recently, a chronic idiopathic disease of the esophagus has emerged, which is now known as eosinophilic esophagitis (EoE). Incomplete knowledge regarding the pathogenesis of EoE has limited treatment options. EoE is known to be a Th2-type immune-mediated disorder. Based on previous studies in both patients and experimental models, it is possible that an abnormal reaction to antigen mediates the pathophysiology of EoE. In this thesis, symptoms and signs unique to EoE were identified by an age-matched, case-controlled study of 326 patients with EoE and gastroesophageal reflux disease. The molecular mechanisms involved in antigen detection in the esophagus, in relation to EoE were then investigated. Esophageal epithelial cells were found, for the first time, to be capable of acting as non-professional antigen presenting cells, with the ability to engulf, process and present antigen on MHC class II to T helper lymphocytes. Antigen presentation by esophageal epithelial cells was induced by interferon-γ, which is increased in biopsies from patients with EoE. Next, it was discovered that esophageal epithelial cell lines expressed functional toll-like receptor (TLR) 2 and TLR3, but in esophageal mucosal biopsies only infiltrating immune cells (including eosinophils) expressed TLR2 and TLR3. Finally, the potential involvement of IgE in the pathogenesis of esophageal inflammation was investigated. IgE in the esophagus was found to be present on mast cells, which are increased in density in the esophageal mucosae of patients with EoE and especially those with a history of atopy. Mechanisms of antigen detection may mediate the pathophysiology of EoE in the esophagus through antigen presentation by epithelial cells, detection by TLRs on immune cells and detection through IgE on mucosal mast cells. Together, these findings demonstrate that mechanisms of antigen detection may actually contribute to the pathophysiology of EoE. Through increased understanding of the mechanisms of EoE, the results of this thesis may contribute to future therapy.