University of Louisiana at Monroe
Mechanism of translation initiation in protozoan parasite Giardia lamblia
Yong-Hwan Lee, LSU
Full Project (May 1, 2019 - April 30, 2022)
Pilot Project (May 1, 2017 - April 30, 2018)
Startup Project (May 1, 2015 - April 30, 2017)
Giardia lamblia is a flagellated protozoan parasite that causes gastrointestinal disease giardiasis in humans and is responsible for major waterborne outbreaks of diarrhea in the United States. Metronidazole (Flagyl) has been the drug of choice for treating giardiasis, however adverse side effects and development of drug resistance strains have necessitated search for new drugs. Translation machinery of Giardia is significantly different from mammals and yeast and hence could serve as a potential drug target. The long-term goal of this project is to understand the mechanism of translation initiation of Giardia.Giardia mRNAs have very short 5'UTRs (0 - 6 nts) and lacks some key translation initiation factors. In mammals and yeast, translation initiation factor eIF4G forms a trimeric complex eIF4F by binding with two proteins, a 5' cap-binding protein eIF4E and RNA helicase eIF4A. Initiation factor eIF4G is a key player in binding pre-initiation complexes to 5' end of mRNA by interacting with eIF3. Homology searches of Giardia genome database have identified homologues for eIF4E and eIF4A but not eIF4G. Therefore, it is not clear as to how pre-initiation complexes are recruited to the mRNA in the absence of eIF4G. Since GleIF4E2 (cap-binding homolog of eIF4E) and GleIF4A (RNA helicase homolog of eIF4A) bind in close proximity to the initiation codon due to short 5'UTRs, we hypothesized that GleIF4E2 and GleIF4A could directly interact with the components of the pre-initiation complex and facilitate its binding to the initiation codon. This hypothesis was supported by our preliminary studies using yeast two hybrid assays that have identified two novel protein-protein interactions; (a) interaction of GleIF4E2 with GleIF2β, a subunit of GleIF2 and (b) interaction of GleIF4A with GleIF3i, a subunit of GleIF3 complex. Both GleIF2 and GleIF3 are key components of pre-initiation complex. These results suggest that the recruitment of pre-initiation complexes to the 5'end of the mRNA may follow a different pathway compared to that of mammals and yeast. The short-term goal is to assess the role of these novel protein-protein interactions in translation initiation of Giardia. In addition, we will examine the composition of GleIF3 complex of Giardia as homologues of several key subunits required for translation initiation in mammals and yeast could not be identified in Giardia genome database. Specific aim #1: To validate protein-protein interactions identified by yeast two-hybrid assays. To validate the interactions observed between the two pairs of proteins in yeast two-hybrid assays, we will use proximity-dependent biotin ligation assays, co-immunoprecipiation (Co-IP) assays and proximity dependent ligation (PLA) assays. Specific aim #2: To investigate the role of protein-protein interactions between GleIF4E2 and GleIF2β in translation initiation. Computational methods (SWISS-MODEL and ZDOCK) will be used to identify amino acid residues of GleIF4E2 involved in interaction with GleIF2β. Site-directed mutagenesis will be performed to alter predicted amino acids to alanine. The mutated GleIF4E2 will be checked for its interaction with GleIF2β using yeast two-hybrid assay. Alternatively, a genetic screen of randomly mutagenized clones of GleIF4E2 will be performed to identify mutants that lack the ability to interact with GleIF2. These mutants will be then further confirmed for their lack of interactions using proximity dependent biotin ligation assay, co-immunoprecipitation assays and PLA assay. The GleIF4E2 mutants that failed to interact with GleIF2β will be overepxressed to test their effect on translation of a reporter gene in Giardia. Specific aim #3: To investigate the role of protein-protein interactions between GleIF4A and GleIF3i in translation initiation. Similar to specific aim #2, to identify amino acids residues of GleIF4A involved in binding to GleIF4A, site-direct mutagenesis and genetic screen of randomly mutagenized clones will be used. The mutants that fail to interact with GleIF3i will be overexpressed to test their effect on translation of a reporter gene in Giardia. In addition, we will determine whether GleIF3i stimulate the ATPase and helicase activity of GleIF4A in vitro. Specific aim #4: To characterize the components of GleIF3 complex from Giardia lamblia. We will affinity purify the GleIF3 complexes from Giardia cells using PTP tagged GleIF3 subunits and identify the components by mass spectrometry. In addition we will perform yeast two hybrid assays to determine the binary interactions between six subunits identified by homology searches of Giardia genome database.