abstract: Many Fic domain proteins, through catalyzing post translational modifications (PTM) of protein substrates, functionally contribute to bacterial pathogenesis and the regulation of bacterial growth. Furthermore, one form of Fic-mediated regulation is the Fic toxin-antitoxin system, whereby an antitoxin interacts with and inhibits the Fic toxin. This study sought to determine the functional importance of Mycobacterium tuberculosis Fic and its putative antitoxin protein, Rv3642c. Using M. tuberculosis H37Rv genetic deletion mutants, fic and Rv3642c were demonstrated to promote intracellular survival in human THP-1 macrophage-like cells. Unlike other Fic toxins, of Fic toxin-antitoxin systems, Fic did not inhibit bacterial growth in vitro in the absence of Rv3642c. Notably, Fic demonstrated in vitro AMPylation of a THP-1 cell extract protein as shown by immunodetection. Fic also exhibited auto-AMPylation activity. Interestingly, a mutation of the conserved histidine in the Fic domain motif, a residue previously shown to be critical for AMPylation, had no effect on Fic-mediated ATP hydrolysis or AMPylation activity. Rv3642c was demonstrated to form a complex with Fic when co-expressed in Escherichia coli, indicating a toxin-antitoxin interaction. Screening M. tuberculosis protein fractions and culture filtrate with α-Fic and α-Rv3642c rabbit antisera did not detect monomers of Fic or Rv3642c, thus the cellular localization of Fic and the Rv3642c-Fic complex remains unclear. The results of this study provide insight into the function of M. tuberculosis Fic, and suggest that Fic and Rv3642c are important for M. tuberculosis survival in the intracellular macrophage environment. Furthermore, these findings challenge the current dogma that Fic domain catalysis is dependent on the conserved histidine of the Fic motif. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2017

<p>  </p> <p>The heat shock protein HSPA5 or BiP serves as a sentinel for ER (endoplasmic reticulum) stress and regulates proteostasis by chaperoning unfolded and misfolded proteins. We and others recently discovered an additional level of proteostatic control, whereby the Fic protein, HYPE/FicD, regulates BiP’s chaperoning capacity by the reversible, post-translational addition of AMP to BiP. Wild-type (WT) HYPE is predominantly a de-AMPylating enzyme that is intrinsically inhibited for AMPylation and shows only basal adenylyltransferase activity relative to a constitutively active mutant (E234G HYPE). </p> <p>Proteostatic dysfunction is often seen in neurodegenerative diseases associated with protein aggregation. Accordingly, we observed that HYPE can directly AMPylate the misfolded presynaptic protein αSynuclein (αSyn) implicated in neurodegenerative phenotypes associated with Parkinson’s disease (PD). Interestingly, HYPE-mediated AMPylation ameliorates many of the in vitro neurotoxic phenotypes of αSyn such as αSyn fibrillation and increased membrane permeability. These potentially cytoprotective phenomena conferred by HYPE’s adenylyltransferase activity make it an attractive point of intervention for PD therapy and for interrogating proteostasis. To this end, we conducted a pilot screen of FDA-approved, natural, and semi-synthetic small-molecule compound libraries towards the identification of activators of WT HYPE and inhibitors of E234G HYPE AMPylation. Employing fluorescence polarization of a labelled ATP analogue on a 384-well microplate platform, we’ve developed a robust, high-throughput screening (HTS) assay suitable for monitoring changes in AMPylation. First-pass selection of our pilot screen yielded multiple compounds capable of manipulating AMPylation in vitro. After expanding our HTS to over 30,000 novel compounds (including those with blood brain barrier penetrance to target PD), we obtained several promising lead candidates. Ongoing cellular validations of these lead compounds to determine their efficacy against intracellular AMPylation of HYPE substrates will be discussed. Challenging neuronal cell culture models of PD with these hits will provide molecular insights into αSyn-induced neurotoxicity, paving the path for novel therapeutic strategies in combating PD during early stages of disease onset.</p>

<p dir="ltr">This dissertation delves into two research projects that aim to characterize post-translational modifications in two distinct proteins, each originating from a different species – one from the eukaryotic sea slug Aplysia californica and the other from the bacterial pathogen Bordetella bronchiseptica.</p><p dir="ltr">Aplysia have an unusually large neuron and therefore serve as an excellent model for studying cell signaling regulating neuronal chemotaxis. Cortactin is an actin binding protein that is regulated by post-translational modifications, including acetylation and phosphorylation. Studies have shown that Src2 tyrosine kinase phosphorylates cortactin to regulate lamellipodia protrusion and filopodia formation in Aplysia bag cell neurons. However, these in vivo phenotypes have not been tested mechanistically in vitro. To this end, the goal of my thesis work was to validate in vivo observations. The following work describes the methodology we developed to purify homogenous non-phosphorylated proteins. Our collaborative results show that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro.</p><p dir="ltr"> Filamentation induced by cAMP (Fic) proteins constitute a recently characterized family of enzymes that are being recognized to regulate diverse cellular processes in bacteria and metazoans. While Fic proteins predominantly utilize adenosine triphosphate (ATP) to post-translationally modify target proteins via a covalent addition of AMP, two Fic proteins have been reported that utilize uridine triphosphate (UTP) and cytidine diphosphate-choline (CDP-choline) to alter the activity of their target. In this dissertation, we report the discovery of the first guanosine triphosphate (GTP) specific Fic protein – BB0907 (BbFic) from Bordetella bronchiseptica. BbFic displays weak to no binding to ATP; instead has a 10-fold increased preferential usage for GTP. We identify key residues involved in GTP recognition. Additionally, sequence similarity network (SSN) analyses reveal that BbFic represents a distinct clade of Fic proteins, highlighting BbFic as a representative new class of guanylyltransferase. Our discovery adds to the functional diversity of the growing Fic protein family and frames the groundwork for understanding Fic-mediated GMPylation as a novel signaling paradigm. </p><p dir="ltr">Taken together, my thesis work provides novel insights into biological consequences of Fic-mediated GMPylation in bacteria and Src-mediated phosphorylation in filopodia formation.</p><p><br></p>