BMB Honors Research Students
Alexander Andrew Tong ′11.
The Role of Glycogen Synthase Kinase 3 Beta in Regulating the Function of PAX3-FOXO1 by Phosphorylation.
Alveolar rhabdomyosarcoma (ARMS) is a type of rhabdomyosarcoma , which is the most common soft tissue sarcoma in pediatric patients. Nearly 70% of ARMSs express the fusion protein PAX3-FOXO1, which is linked to poor prognosis and increased tumor aggressiveness. Previous studies have shown that the glycogen synthase kinase 3 beta (GSK3B) inhibitor TWS119 can inhibit cell proliferation in alveolar rhabdomyosarcoma cells, and that GSK3B can phosphorylate PAX3-FOXO1 in vitro. However, the specific nature of these phosphorylation events and physiological relevance of these events for TWS119 activity are not known. In this study, site-directed mutagenesis is used to evaluate a putative phosphorylation site located at the junction of the PAX3 and FOXO1 domains in the fusion protein for the site′s importance in PAX3-FOXO1 functional activity and strongly suggests it may be phosphorylation site for GSK3B. These studies provide insight to the role of PAX3-FOXO1 function in ARMS cells, important since this fusion protein is indicative of a more aggressive cancer phenotype that is resitant to conventional chemotherapy and radiotherapy. Novel strategies in treating these aggressive ARMS types might include modulating the activity of the fusion protein′s target genes as well as the fusion protein′s transcriptional activity, particularly through inhibiting GSK3B.
Laura R. Johnson ′10.
The C-terminal domain of the GDP-mannose transporter, GmtA, is essential for localization to the Golgi apparatus in Aspergillus nidulans.
GDP-mannose transporters (Gmt) carry nucleotide sugars from the cytosol across the Goolgi apparatus membrane in many eukaryotic organisms. Some fungal species like Saccharomyces cerevisiae express a single Gmt, while others including A. nidulans express two (GmtA and GmtB). We have shown that fluorescent versions of GmtA and GmtB (fusion with green fluorescent protein, GFP) localize to the Golgi apparatus. Gmt in S. cerevisiae localizes to the Golgi as well, and proper localization is dependent on homodimerization, which is facilitated by the C-terminus. Through localization studies of GmtA C-terminal truncations (fused to GFP), we determined that the region between 30 amino acids and 45 amino acids from the C-terminus is essential for proper localization to the Golgi. The full C-terminus truncated version of GmtA mislocalized to the Endoplasmic Reticulum. In addition, progress has been made toward determining if GmtA and GmtB form oligomers. Using split-YFP(Yellow Fluorescent Protein) to measure protein interaction, we did not observe heterodimerization of GmtA and GmtB or homodimerization of GmtA.
John L. Musgrove ′10.
Regulated expression of the SccA gene by the inducible AlcA promoter affects both development and cell wall integrity in Aspergillus nidulans.
The filamentous fungus Aspergillus nidulans is the most genetically tractable member of the Aspergillus genus, which includes species of industrial and medical significance. Pathogenicallly, these fungi cause superficial, allergic and invasive aspergillosis. The research describes a novel gene designated SccA, which affects cell wall integrity in A. nidulans. Plasmid-borne extra copies of SccA can suppress the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin-binding agent Calcofluor White (CFW). In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity. The hypothetical translated product of SccA is a 271-a.a. protein (unprocessed), with a probable transmembrane domain and an extracellular domain rich in serine and threonine. A SccA::GFP hybrid localizes to the plasma membrane of vegtative hyphae. When SccA is placed under the tightly regulatable AlcA promoter (AlcAP), vegetative growth is normal under both inductive and repressive conditions (growth on glycerol and glucose containing medium, respectively), but asexual sporulation is inhibited during growth on glycerol. This sporulation defect is osmotically remediable by supplementing the glycerol medium with 0.6M KCL. SccA repression enhances CFW sensitivity, whil SccA induction decreases sensitivity. We hypothesize that this phenotype is representative of a signal transduction protein functioning in the cell wall integrity pathway.
Jacqueline M. Ward ′10.
Analysis of the C-terminal domain of SccA, a putative cell wall stress receptor in Aspergillus nidulans.
Aspergillus nidulans is a filamentous fungus that has been used as a model organism for many eukaryotic processes. Its research implications are far-reaching because fungi have both negative and positive effects on many aspects in medicine, industry and the environment. Studying the cell wall specifically is important as this structure is not present in animal cells and could potentially serve as a target for medical treatment in many threatening fungal infections. This research aims to elucidate the function of the novel gene designated SccA which affects cell wall integrity in A. Nidulans. Plasmid-based, overexpression of SccA can suppress the calC2 mutation in protein kinase C (PkcA), which results in hypersensitivity to the chitin-binding agent Calcofluor White (CFW). It has previously been shown that hypersensitivity to CFW is indicative of cell wall integrity defects in both yeast and filamentous fungi. SccA is predicted to have a single transmembrane domain, an extracellular domain rich in serine and threonine amino acids, and a short cytoplasmic C-terminus of 59 amino acids. No sequence homologues exist in yeast, but structurally there is great similarity between SccA and other stress receptors. The C-terminal domains of yeast stress receptors like Wsc1 and Mid2 have been shown to play important roles in signal transduction upstream from Pkc. In an analysis of C-terminal truncation mutants, we have shown for the first time in A. nidulans that the C-terminal domain of SccA is also essential in proper cell wall integrity function.
BMB Research Students (BMB 451 & 452)
Lindsey Bierle ’12. Budding yeast CLN3 function in cell cycle progression requires the mRNA transport protein NPL3. (Dr. Mary Miller)
Lindsey Gurkovich ’11. Heterologous expression of the yeast Mid2 gene in the filamentous fungus Aspergillus nidulans. (Drs. Darlene Loprete, Terry Hill, and Loretta Jackson-Hayes)
Rachel Hickey ‘11. Promoter replacement of the gene encoding the SccA protein in the filamentous fungus Aspergillus nidulans. (Drs. Darlene Loprete, Terry Hill, and Loretta Jackson-Hayes)
Mary Elizabeth Huddleston ’10 Nucleopore dependent G1 progression in budding yeast. Fall and Spring Research through the Rhodes Student Associate Program (Dr. Mary Miller)
Stephen Juel ’10. Role of EBA-175 dimerization in tight junction formation with red blood cells. (Dr. Laura Luque de Johnson)
John Musgrove ’10. Characterization of Aspergillus nidulans mutants expressing the gene SccA under control of the AlcA promoter. (Dr. Terry Hill)
Michael Pluta ‘11. GFP tagging of genes regulating cytokinesis in the filamentous fungus Aspergillus nidulans. (Drs. Terry Hill, Loretta Jackson-Hayes, and Darlene Loprete)
Miranda White ‘12. Promoter replacement of genes regulating cytokinesis in the filamentous fungus Aspergillus nidulans. (Drs. Loretta Jackson-Hayes, Terry Hill, and Darlene Loprete)
BMB URCAS (Undergraduate Research and Creative Activity Symposium) Presentations
John Musgrove ’10 and Erinn Ogburn ’11. Regulated expression of the SccA gene by the inducible AlcA promoter affects both development and cell wall integrity in Aspergillus nidulans. Presented at URCAS 2010.
Alex Tong ’11. Protein Phosphatase 2Cbl Regulates Human Pregnane X Receptor-Mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells. Presented at URCAS 2010.
Jackie Ward ’10. Analysis of the C-terminal Domain of a Putative Cell Wall Stress Receptor in Aspergillus nidulans. Presented at URCAS 2010.