Characterization of the Role of acs-19 in Caenorhabditis elegans Bacterial Infection

The human body attracts a large number of bacterial pathogens, many of which have detrimental effects on the host and add a tremendous economic burden. Caenorhabditis elegans and its interaction with the bacterium Microbacterium nematophilum is a great model system to study various aspects of host-pathogen interactions including bacterial attachment, nematode surface antigen variation, host response to infection, and cellular morphogenesis. M. nematophilum adheres to the post-anal region of the nematode, causing it to display a deformed anal region (DAR) phenotype and experience severe constipation. Previous research has shown that among the genes up regulated in C. elegans in response to M. nematophilum infection is acs-19, a gene encoding acyl-CoA synthetase, which plays a role in lipid metabolism. I have been investigating the specific function of ACS-19 and its role in this interaction. Specifically, I am studying whether its up regulation is dependent on the transcription factor EGL-38. So far, I have observed that the expression of acs-19 goes down in egl-38 mutants with respect to wild type. I am also using RNAi to knock down acs-19 gene expression to investigate whether its up regulation in response to infection is involved in the host’s defense against the pathogen, or if it is something necessary for pathogenicity. I have found that the upregulation of acs-19 reported in the microarray experiments are part of the bacteria’a infection mechanism and that the DAR response is not dependent on egl-38. In the future, I will test the possibility of changes in ACS-19 protein localization in response to infection.ASC Undergrad Research ScholarshipPelotonia Undergraduate Research FellowshipNo embargoAcademic Major: Microbiolog

The Role of BRAP-2 in Caenorhabditis Elegans DNA Damage Induced Germline Apoptosis Development and Germline Health

The DNA damage response protects the genome by executing processes that prevent the disruption of normal cell physiology and the inheritance of mutations. BRCA1 is an essential tumor suppressor gene that facilitates DNA repair, transcription and plays a more complex role in apoptosis. A novel BRCA1 binding protein known as BRAP2/IMP has been characterized as a RAS effector with ubiquitin ligase activity and as a cytoplasmic retention protein. BRAP2 is conserved in C. elegans, and is known as BRAP-2. Previously, we have shown that BRAP-2 is a negative regulator of SKN-1/Nrf2 dependent detoxification gene expression. In addition, brap-2 deletion mutants experience BRC-1 (BRCA1 ortholog) dependent larval arrest when exposed to oxidative stress. BRC-1 function in DNA repair is conserved in the germline, where a loss of brc-1 increases apoptosis. Due to the conservation of BRC-1 function in C. elegans and the genetic link between BRC-1 and BRAP-2 upon oxidative stress, in this study we examined the role of BRAP-2 in DNA damage induced germline apoptosis, C. elegans development and germline health. We found that brap-2 mutants display a reduction in DNA damage induced germline apoptosis and that apoptosis induced by loss of BRC-1 requires BRAP-2. We also found that a loss of PMK-1, SKN-1 and AKT-1 in brap-2 mutants increases apoptosis, indicating that a loss of BRAP-2 limits DNA damage induced germline apoptosis and promotes cell survival through regulation of the PMK-1 activated SKN-1 oxidative stress response pathway and Insulin/Insulin-like growth factor signaling. In addition, brap-2 mutants display defects in development, survival, brood size and germline morphology. Taken together, this suggests that BRAP-2 is required to promote DNA damage induced germline apoptosis by regulating pro-cell survival pathways and that BRAP-2 is required for proper C. elegans growth, development and germline health

The Role of MK-STYX in Stress Granule Assembly/Disassembly

Regulation of cell signaling is critical for determining fundamental processes such as whether a cell will grow, change shape, differentiate or die. Phosphorylation is a post-translational modification of proteins that is key for transducing signaling messages. The major players in phosphorylation cascades are proteins called kinases and phosphatases. Pseudokinases and pseudophosphatases are catalytically inactive proteins that add complexity to the regulation of cell signaling. In this thesis, the pseudophosphatase MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] is implicated in the stress response pathway. MK-STYX interacts with G3BP-1 (Ras-GTPase activating protein SH3 domain binding protein-1), and inhibits stress granule assembly. Stress granules, cytoplasmic storage sites for mRNA, form as a protective mechanism against stressors such as UV irradiation, hypoxia, and heat shock. In addition, the overexpression of G3BP-1 induces stress granule assembly. The initial hypothesis for how MK-STYX attenuates stress granule assembly was through a mechanism dependent on the phosphorylation status of G3BP-1 at serine 149. However, data with a G3BP-1 phosphomimetic mutant suggests differently. The eukaryotic initiation factor 2 alpha (eIF2α) is investigated for playing a role in the reduction of stress granules by MK-STYX. eIF2α initiates translation by forming a ternary complex with methionine bound to tRNA. Phosphorylation of eIF2α arrests translation and results in stress granule formation, whereas dephosphorylation promotes polysomes. To determine if MK-STYX inhibits stress granule assembly via upstream interactions, we investigated its effects on eIF2α phosphorylation. HeLa cells were transfected with pMT2-FLAG-MK-STYX-FLAG or pMT2 expression vectors, and heat shocked for an hour to induce stress. Cells were lysed and immunoblots for phosphorylation of eIF2α were performed. We show that the presence of MK- STYX reduced the phosphorylation of eIF2α. Also, MK-STYX was immunoprecipitated with eIF2α, suggesting that MK-STYX and eIF2α interact. These data are significant because they suggest MK-STYX may play a role in stress granule assembly. MK-STYX involvement in disassembly was also investigated by immunoblotting for Hsp70. Hsp70 is thought to be necessary for stress granule disassembly and reduces protein aggregation. The overexpression of MK-STYX correlates with increased expression of Hsp70, suggesting MK-STYX may also play a role in stress granule disassembly. This thesis implicates MK-STYX, a pseudophosphatase, as a regulator of cellular processes

Role of local electrostatic fields in protein-protein and protein-solvent interactions determined by vibrational Stark effect spectroscopy

textThis examines the interplay of structure and local electrostatic fields in protein-protein and protein-solvent interactions. The partial charges of the protein amino acids and the polarization of the surrounding solvent create a complex system of electrostatic fields at protein-protein and protein-solvent interfaces. An approach incorporating vibrational Stark effect (VSE) spectroscopy, dissociation constant measurements, and molecular dynamics (MD) simulations was used to investigate the electrostatic interactions in these interfaces. Proteins p21Ras (Ras) and Rap1A (Rap) have nearly identical amino acid sequences and structures along the effector-binding region but bind with different affinities to Ral guanine nucleotide dissociation stimulator (RalGDS). A charge reversion mutation at position 31 alters the binding affinity of Ras and Rap with RalGDS from 0.1 [mu]M and 1 [mu]M, to 1 [mu]M and 0.5 [mu]M, respectively. A spectral probe was placed at various locations along the binding interface on the surface of RalGDS as it was docked with Ras and Rap single (position 30 or 31) and double mutants (both positions). By comparing the probes' absorption energies with the respective wild-type (WT) analogs, VSE spectroscopy was able to measure molecular-level electrostatic events across the protein-protein interface. MD simulations provided a basis for deconvoluting the structural and electrostatic changes observed by the probes. The mutation at position 31 was found to be responsible for both structural and electrostatic changes compared to the WT analogs. Furthermore, previous identification of positions N27 and N29 on RalGDS as "hot spots" that help discriminate between structurally similar GTPases was supported. The RalGDS probe-containing variants and three model compounds were placed in aqueous solvents with varying dielectric constants to measure changes in absorption energy. We investigated the ability of the Onsager solvent model to describe the solvent induced changes in absorption energy, while MD simulations were employed to determine the location and solvation of the probes at the protein-solvent interface. The solvent accessible-surface area, a measure of hydration, was determined to correlate well with the change in magnitude of the probe's absorption energy and the displaced solvent by the probe.Chemistr

Investigating the Effects of a p53 Mutation and Various Tyrosine Kinase Inhibitors on Glioma Progression and Therapy Resistance in Drosophila

Gliomas, which are brain tumors that arise from glial cells, are some of the most aggressive and lethal types of tumors. These brain tumors are difficult to treat because not enough information regarding the mutations present in these tumors exists. This project studies effects of a p53 mutation on Drosophila glioma progression and then will test to see if this results in resistance to current chemotherapy. The main goal of this endeavor is to investigate the numerous defects occurring at the cellular and biochemical level in gliomas, which will give insight into why these types of tumors are so difficult to treat. Additionally, this document also discusses some promising chemotherapeutic agents found through a drug screen project. The effects of five different Tyrosine Kinase inhibitors on glioma development are presented here

EXPANDING THE POTENTIAL PRENYLOME: PRENYLATION OF SHORTENED TARGET SUBSTRATES BY FTASE AND DEVELOPMENT OF FRET-BASED SYSTEM FOR DETECTING POTENTIALLY “SHUNTED” PROTEINS

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal CaaX sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid Cxx C-terminal sequences using both genetic and biochemical assays. Surprisingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. The work herein expands the substrate pool of sequences that can be potentially prenylated, further refines our understanding of substrate recognition by FTase and GGTase-I and suggests the possibility of a new class of prenylated proteins within proteomes. To identify potential new Cxx substrates in human proteomes, we explored a FRET-based system using phosphodiesterase delta subunit (PDE) as the acceptor protein for potentially prenylated Cxx sequences. While not conclusive, this work lays the foundation for an assay not dependent on membrane localization as a signal for prenylation inside cells and suggests future studies to improve upon the utility of this assay. Lastly, this work demonstrates FTase’s flexibility in accepting a prenyl donor analogue with an azobenzene moiety that can be modulated with light. This establishes a potential new avenue for mediating membrane localization behavior of prenylated proteins

Reiterative FGF signaling determines the identity and morphology of the lacrimal gland

Indiana University-Purdue University Indianapolis (IUPUI)The lacrimal gland plays an essential role in protection of the ocular surface by secreting the aqueous component of the tear film. Deficiency in the lacrimal gland is the main cause of dry eye disease, but existing treatments only alleviate the symptoms without curing the underlying disease. To develop curative measures, a thorough understanding of lacrimal gland development is needed. Lacrimal gland is formed as a result of interaction between the neural crest-derived mesenchyme and the conjunctival epithelium. The mesenchyme secretes the chemo-attractive signal of Fgf10, which binds to epithelial Fgfr2b and co-receptor heparan sulphate proteoglycans, to promote budding and branching morphogenesis of the lacrimal gland. However, the mechanism by which Fgf10 expression is regulated within the neural crest and the direct downstream targets of Fgf signaling in the epithelium are currently unknown. In this study, we show that FGF signaling mediated by protein phosphatase Shp2 is required for the proper patterning and differentiation of the neural crest-derived mesenchyme to produce Fgf10. Genetic evidence further demonstrates that Shp2 is recruited by Frs2α to activate Ras-MAPK signaling downstream to Fgfr1 and Fgfr2 but not to Pdgfrα in the neural crest. By differential gene expression analysis, we identified homeodomain transcription factor Alx4 as the key effector of Shp2 signaling to control expression of Fgf10 in the periocular mesenchyme. Loss of function ALX4/Alx4 mutation disrupted lacrimal gland development in both human and mouse. Our results reveal a FGF-Shp2-Alx4-Fgf10 axis in regulating neural crests during lacrimal gland development. In addition, we also show that Fgf signaling cascade mediated by Pea3 family of transcription factors are critical for lacrimal gland duct elongation and branching. High-throughput gene expression analysis revealed that Pea3 genes were important for establishing the tissue identity of the lacrimal gland. Loss of Pea3 resulted in upregulation of Notch signaling with the concomitant loss in the expression of the members of Six family of transcription factors and a switch of cell fate to the epidermal skin-like cells. These findings show that Fgf signaling is used reiteratively to establish the identity of both the epithelium and mesenchyme of the lacrimal gland.2 year

Determination of the functional role of Rab-GGT in Physcomitrium patens.

Protein prenylation, a common lipid post-translational modification, is required for growth and development in eukaryotes. Rab geranylgeranylation involves the addition of one or two 20-carbon geranylgeranyl moieties to Rab-GTPase target proteins, which regulate intracellular vesicle trafficking. The reaction is carried out by heterodimeric Rab geranylgeranyltransferase (Rab-GGT), which is composed of two associated α- and β-subunits, with the assistance of an additional protein called Rab escort protein (REP). Loss of function of the Rab-GGT α subunit RGTA1 has not been reported in any plant. While knockout of either of the two β subunits RGTB1 or RGTB2 results in no phenotype in the moss Physcomitrium patens (P. patens, formerly Physcomitrella patens), in the flowering plant Arabidopsis thaliana, knockout of RGTB1 results in loss of apical dominance and photomorphogenic and gravitropic defects, and knockout of RGTB2 results in a subtle growth defect in certain cells. These results showed that both Rab-GGT β subunits in P. patens are redundant, but RGTB2 in Arabidopsis could not fully compensate for the loss of function of RGTB1 and vice versa. Previous studies in our lab showed that complete knockout of any P. patens Rab-GGT components (RGTA1, RGTB1 & RGTB2, REP) appears to be lethal, since no viable single mutant plants of RGTA1 or REP and double mutant plants of RGTB1 and RGTB2 were recovered. Therefore, the biological function of Rab-GGT remains largely unknown. Here we have generated P. patens transgenic plants containing artificial miRNA constructs targeting each Rab-GGT component by an inducible knockdown system, and systematically analyzed the phenotypes upon induction. The results showed that knockdown of either RGTA1 or REP, or knockdown of RGTB1 in a rgtb2 knockout background (or vice versa) resulted in defects in tip growth (polar cell elongation), reduced or incomplete caulonema differentiation, and an altered response towards exogenous phytohormones. This dissertation may help to elucidate the functional role of Rab-GGT in P. patens, as well as provide fundamental insights into key P. patens developmental processes and environmental responses

Characterizing the interaction between RanBPM and c-Raf

RanBPM/RanBP9 is a multi-domain nucleocytoplasmic protein which has been linked to numerous cellular processes including cell adhesion, migration, transcription and apoptosis. Although RanBPM is a member of the mammalian CTLH complex, the counterpart of a conserved yeast E3 ubiquitin ligase complex, its exact function remains unknown. Previous work in our laboratory has shown that RanBPM inhibits the ERK pathway by interacting with the kinase c-Raf and downregulating c-Raf levels. Here, we show that the N-terminus, LisH/CTLH and CRA domains of RanBPM are required for downregulation of c-Raf and that RanBPM interacts directly with c-Raf through its CRA domain. We also provide evidence that MAEA, another CTLH complex member, associates with c-Raf. Therefore, we propose a mechanism by which RanBPM downregulates c-Raf in a CTLH complex-dependent manner. This work contributes to our knowledge of the function of RanBPM and clarifies the relationship between RanBPM and c-Raf, two important proteins in oncogenesis