Neuroepithelial circuit formed by innervation of sensory enteroendocrine cells.

Satiety and other core physiological functions are modulated by sensory signals arising from the surface of the gut. Luminal nutrients and bacteria stimulate epithelial biosensors called enteroendocrine cells. Despite being electrically excitable, enteroendocrine cells are generally thought to communicate indirectly with nerves through hormone secretion and not through direct cell-nerve contact. However, we recently uncovered in intestinal enteroendocrine cells a cytoplasmic process that we named neuropod. Here, we determined that neuropods provide a direct connection between enteroendocrine cells and neurons innervating the small intestine and colon. Using cell-specific transgenic mice to study neural circuits, we found that enteroendocrine cells have the necessary elements for neurotransmission, including expression of genes that encode pre-, post-, and transsynaptic proteins. This neuroepithelial circuit was reconstituted in vitro by coculturing single enteroendocrine cells with sensory neurons. We used a monosynaptic rabies virus to define the circuit's functional connectivity in vivo and determined that delivery of this neurotropic virus into the colon lumen resulted in the infection of mucosal nerves through enteroendocrine cells. This neuroepithelial circuit can serve as both a sensory conduit for food and gut microbes to interact with the nervous system and a portal for viruses to enter the enteric and central nervous systems.

Structural and Biochemical Dissection of the Trehalose Biosynthetic Complex in Pathogenic Fungi

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

Predicting Transcript Production Rates in Yeast With Sparse Linear Models

To provide biological insights into transcriptional regulation, a couple of groups have recently presented models relating the promoter DNA-bound transcription factors (TFs) to downstream gene’s mean transcript level or transcript production rates over time. However, transcript production is dynamic in response to changes of TF concentrations over time. Also, TFs are not the only factors binding to promoters; other DNA binding factors (DBFs) bind as well, especially nucleosomes, resulting in competition between DBFs for binding at same genomic location. Additionally, not only TFs, but also some other elements regulate transcription. Within core promoter, various regulatory elements influence RNAPII recruitment, PIC formation, RNAPII searching for TSS, and RNAPII initiating transcription. Moreover, it is proposed that downstream from TSS, nucleosomes resist RNAPII elongation.

Here, we provide a machine learning framework to predict transcript production rates from DNA sequences. We applied this framework in the S. cerevisiae yeast for two scenarios: a) to predict the dynamic transcript production rate during the cell cycle for native promoters; b) to predict the mean transcript production rate over time for synthetic promoters. As far as we know, our framework is the first successful attempt to have a model that can predict dynamic transcript production rates from DNA sequences only: with cell cycle data set, we got Pearson correlation coefficient Cp = 0.751 and coefficient of determination r2 = 0.564 on test set for predicting dynamic transcript production rate over time. Also, for DREAM6 Gene Promoter Expression Prediction challenge, our fitted model outperformed all participant teams, best of all teams, and a model combining best team’s k-mer based sequence features and another paper’s biologically mechanistic features, in terms of all scoring metrics.

Moreover, our framework shows its capability of identifying generalizable fea- tures by interpreting the highly predictive models, and thereby provide support for associated hypothesized mechanisms about transcriptional regulation. With the learned sparse linear models, we got results supporting the following biological insights: a) TFs govern the probability of RNAPII recruitment and initiation possibly through interactions with PIC components and transcription cofactors; b) the core promoter amplifies the transcript production probably by influencing PIC formation, RNAPII recruitment, DNA melting, RNAPII searching for and selecting TSS, releasing RNAPII from general transcription factors, and thereby initiation; c) there is strong transcriptional synergy between TFs and core promoter elements; d) the regulatory elements within core promoter region are more than TATA box and nucleosome free region, suggesting the existence of still unidentified TAF-dependent and cofactor-dependent core promoter elements in yeast S. cerevisiae; e) nucleosome occupancy is helpful for representing +1 and -1 nucleosomes’ regulatory roles on transcription.