Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "August 2001." Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

Ph.D.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

Protein synthesis is a highly conserved process across all domains of life, both structurally and functionally. This cyclic process is catalyzed by numerous soluble protein factors that interact with the ribosome to facilitate efficient protein synthesis. Many canonical translation factors bind and hydrolyze GTP to induce conformational changes that facilitate translation. For example, GTP hydrolysis by EF-Tu is required for the release of aminoacyl-tRNA into the ribosomal A site; GTP hydrolysis by EF-G facilitates the movement of tRNA and mRNA from the A site to the P site of the ribosome. However, protein synthesis seems to also have a requirement for ATP; the essential yeast protein eEF-3 facilitates release of deacyl-tRNA from the ribosomal E site. In Escherichia coli, the protein product of the open reading frame yhih has been suggested to have a similar function. However, the role of this unique prokaryotic protein is not understood. Preliminary characterization of this protein suggests a nucleotide-dependent conformational change occurs in a truncated form of the protein, ΔP541 Yhih. Interestingly, this phenomenon is not observed in ΔL432 Yhih. Both ΔP541 Yhih, and to a lesser extent ΔL432 Yhih, exhibit a ribosome-dependent ATPase activity, suggesting the primary region for binding with the ribosome lies between Leu432 and Pro541. / x, 101 leaves : ill. ; 29 cm.

Plasmodium falciparum is a protozoan parasite responsible for the most severe form of human malaria, with infection often resulting in death. Efforts to control malaria have been hindered by an increased spread of parasite resistance to previously effective antimalarial drugs, leading to an intensified search for novel antimalarial drug targets. A group of proteins suggested as potentially effective targets are the integral membrane transport proteins, since they play key roles in Plasmodium parasite growth and replication. One such membrane protein recently characterised was the P. falciparum copper efflux transporter. Treatment of cultured P. falciparum parasites with the intracellular copper chelator neocuproine inhibited parasite growth, suggesting that additional mechanisms for malaria parasite copper homoeostasis are likely to be present. Copper is an essential trace element involved in enzymatic processes requiring redox-chemistry. In higher eukaryotes copper is transported across the plasma membrane via the copper transport protein, Ctr1, and distributed intracellularly by copper metallochaperones. The mechanisms for copper acquisition and distribution in the Plasmodium parasite are, however, yet to be characterised. An in silico Basic Local Alignment Search Tool for protein (BLASTp) screen of the Plasmodium database (www.plasmodb.org) identified sequences corresponding to a putative copper transporter, and associated copper metallochaperones, in eight species of the Plasmodium parasite. Each of the Plasmodium copper transport protein sequences was found to contain features common to the well characterised copper transporters. These features included predicted copper-binding motifs in the protein's amino terminus, three membrane spanning domains and the characteristic MxxxM and GxxxG motifs located in the second and third transmembrane domains, respectively. Affinity purified anti-peptide antibodies, generated against an immunogenic peptide (CSDKQSGDDECKPILD) in the amino terminus of a putative malaria parasite copper transporter (PY00413), detected the target protein in murine malaria parasites in association with a parasite membrane. The open reading frames corresponding to the amino terminal domains of one P. berghei [PBANKA_130290 (447 bp)] and two P. falciparum [PF14_0211 (132 bp) and PF14_0369 (282 bp)] putative copper transport proteins were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with maltose binding protein (MBP). The resulting sizes for the recombinant proteins were 61kDa for MBP-PbCtrNt, 48kDa for MBP-PfCtr211Ntᵀᴰ and 55kDa for MBP-PfCtr369Ntᵀᴰ, with each protein being recognised by a corresponding anti-peptide antibody. All three recombinant proteins bound copper in vitro and in vivo, with each having a binding preference for the reduced cuprous ion. This preference has been similarly established for the characterised copper transporters. Although the results supported the expression and copper binding ability of a Plasmodium parasite copper transport protein, the directional transport of copper, by this protein, requires experimental confirmation as does its specific location. The identification of a P. falciparum copper transporter, and other copper dependent proteins, implies a parasite metabolic requirement for copper. Mammalian and yeast cells require a Cox17 metallochaperone for copper delivery to cytochrome-c oxidase. Identification of P. falciparum orthologs for Cox17 (PF10_0252) and a number of cytochrome-c oxidase subunits (PF13_0327; PF14_0288; mal_mito_1; mal_mito_2; PFI1365w; PFI1375w), suggests the existence of similar parasite mechanisms for copper delivery. Analysis of the Plasmodium Cox17-like sequences identified essential amino acids conserved in the well characterised yeast and mammalian Cox17. This included the identification of six cysteine residues essential for Cox17 function. A homology model of P. falciparum Cox17, with human Cox17 as the template [PDB ID: 2RN9 (apoCox17); 2RN8 (Cu⁺-Cox17)], suggested that Plasmodium Cox17 orthologs would adopt a similar structural conformation. The open reading frames for full-length P. yoelii [PY03823 (192 bp)] and P. falciparum [PF10_0252 (195 bp)] Cox17 were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with either a His₆-tag or glutathione S-transferase (GST)-tag, respectively. The resulting sizes for the recombinant proteins were 11.6kDa for His₆-PyCox17 and 33.5kDa for GST-PfCox17, with each protein being recognised by a corresponding anti-peptide antibody. Both recombinant Cox17 proteins bound the cuprous ion in vitro and in vivo, similar to mammalian and yeast Cox17. This supported the likely existence of a mitochondrial copper metallochaperone pathway within the malaria parasite; however, this requires further experimental confirmation. Identification of a parasite copper transport protein, and associated metallochaperones, could provide novel targets for drug-based inhibition of parasite growth. Alternatively, the copper transporter may provide a novel mechanism for drug delivery into the Plasmodium parasite. The potential of these malaria parasite proteins being effective drug targets does, however, remain to be confirmed. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties, and proximal limb weakness. The autosomal dominant form of this disease is caused by the expansion of a polyalanine stretch from 10 to 12--17 alanines in the N-terminus of PABPN1. Mutated PABPN1 (mPABPN1) is able to induce the formation of filamentous intranuclear inclusions that are the pathological hallmark of OPMD. PABPN1 is predominantly localized to the nucleus, binds RNA poly(A) tail, forms oliogmers, and is involved in polyadenylation. In this study we first demonstrated that oligomerization of PABPN1 is mediated by two potential oligomerization domains (OD), while inactivating oligomerization of mPABPN1 by deletions of 6--8 residues in either of the ODs prevents intranuclear protein aggregation. Expression of mPABPN1 in COS-7 cells is associated with cell death, whereas preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. We then identified two PABPN1 interacting proteins, hnRNP A1 and A/B, using a yeast two-hybrid library screen. The interaction between PABPN1 and hnRNP A1 or A/B was confirmed by GST pull-down and co-immunoprecipitation assays. When coexpressed with mPABPN1 in COS-7 cells, predominantly nuclear localized hnRNP A1 and A/B co-localize with mPABPN1 to the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. We finally found a nuclear localization signal (NLS) in PABPN1 that is not homologous to any known NLSs. The 18 amino acids 289RGRVYRGRARATSWYSPY 306 in PABPN1 are necessary and sufficient for nuclear translocation. Attaching this sequence to cytoplasmic protein PKM2 completely re-localizes it to the nucleus. Alanine-scanning mutagenesis analysis showed that the last 9 residues 298RATSWYSPY306 are crucial to the function as an NLS. Our studies showed that mPABPN1 induced intran

The process of metastasis is initiated through the acquisition of inherent and autonomous motile and invasive properties by tumor cells. These phenomena are initiated through a balance between forward cancer cell membrane protrusion and tail retraction, and occur via cell cytoskeleton remodeling, actin reorganization, and coordinated focal adhesion assembly and disassembly events. Among the vast network of cytoskeletal proteins, the actin-bundling protein fascin plays a major function in cell cytoskeleton remodeling. It is a 55-kDa protein involved in the formation of filopodia and cell migration, and found to be upregulated in many cancers. We report herein key functions for fascin in the regulation of prostate and breast cancer progression. Fascin expression is upregulated in localized and hormone refractory prostate cancer, responsible for a more aggressive clinical course. In addition, functional dissection of fascin reveals a novel function in the regulation of focal adhesion turnover dynamics, by modulating the phosphorylation state of central focal adhesion proteins through a potential collaboration with the protein tyrosine phosphatase, PEST. Together, our data support the importance of fascin in cancer cell invasion and as a significant prognostic marker and a potential therapeutic target for aggressive cancers.

The Drosophila melanogaster eIF4E binding protein (d4E-BP) inhibits translation initiation and is implicated in cell growth as a downstream effector of the Drosophila insulin signaling pathway. Since d4E-BP null flies show similar growth and development to control flies, the possibility of a conditional phenotype was explored through stress treatments. Adult d4E-BP null flies show sensitivity to oxidative stress, and d4E-BP null larvae die faster than controls under starvation and protein starvation. Expressing a mutant d4E-BP that doesn't bind to eIF4E in the d4E-BP null background does not rescue this stress sensitivity, which suggests that wild-type stress resistance requires binding of d4E-BP to eIF4E. / The Drosophila forkhead transcription factor dFOXO is a transcriptional activator of d4E-BP. There is a strong reduction of d4E-BP peptide in a dFOXO null background. dFOXO null flies are also sensitive to oxidative stress, and rescue of this sensitivity through ectopic expression of UAS-d4E-BP(wt) in a dFOXO null background suggests d4E-BP is a downstream mediator of dFOXO oxidative stress resistance.

Early Drosophila development is a coordinated series of temporal and spatial events leading to specific localized gene expression. The maternally expressed gene Bicaudal-C (Bic-C) encodes a KH-domain RNA binding protein required in the developing oocyte for anterior-posterior patterning and follicle cell migration. The dominant heterozygous phenotype results in the development of embryos with bicaudal and head defects. A two-hybrid screen using BIC-C as "bait" identified the novel protein ANTIMEROS (ATMS) and the SH3-domain containing protein MILE END (MILE). / ATMS is highly conserved between humans and mice, its expression is almost entirely female-specific, and is limited to certain developmental stages. Mutant alleles for atms are able to dominantly enhance the phenotype of Bic-C heterozygotes confirming the Bic-C-atms interaction. Here I show that NOS mislocalization causes the trans-heterozygous phenotype, as introduction of a nos mutation strongly suppresses the bicaudal phenotype. nos transcripts show a hyper-polyandenylation in atms mutant ovaries, an indicator of translational activation, suggesting that ATMS and BIC-C function as translational repressors of nos through changes in its poly(A) tail length. / MILE, contains two highly conserved SH3 domains at the C-terminus. Experiments involving the analysis of mutant alleles and overexpression mile transgenic lines show that MILE is a negative regulator of both Torso and Egfr RTK signaling. Its not clear what functional role BIC-C may have with RTK signaling, but recent evidence suggests that posterior group gene expression influence terminal pole RTK signaling.