Lab Research Focus
My research goals include the development of novel therapeutic strategies that target the translational machinery to allow disease-causing mutations to be suppressed. Currently, compounds such as aminoglycoside antibiotics are being investigated to determine their ability to suppress nonsense mutations and restore functional protein.
Aminoglycosides bind to a region of the small subunit ribosomal RNA known as the decoding site [VanLoock et al., J. Mol. Biol. 285:2069 (1999)]. The decoding site proofreads the interaction between codons located in the ribosomal A site and incoming aminoacyl-tRNAs to verify cognate interactions. Aminoglycosides bound to the decoding site induce a conformational change in the ribosomal RNA that reduces translational proofreading [Fourmy et al., Science 274:1367 (1996); Fourmy et al., J. Mol. Biol. 277:333 (1998); Lynch & Puglisi, J. Mol. Biol. 306:1023 (2001)]. The presence of a single nucleotide difference between the prokaryotic and eukaryotic decoding site reduces aminoglycoside affinity for the eukaryotic ribosome by approximately 100-fold [Recht et al., EMBO J. 18:3133 (1999)]; therefore, aminoglycosides can only slightly reduce translational proofreading in eukaryotes. However, in eukaryotes it has been shown that aminoglycosides can enable an aminoacyl-tRNA that is near-cognate to a premature stop codon to become incorporated into the nascent polypeptide which allows continued translation of the protein in the correct ribosomal reading frame [Fearon et al., J. Biol. Chem. 269:17802 (1994)].
I am currently investigating the feasibility of using aminoglycosides as well as other novel compounds to suppress nonsense mutations that cause Hurler syndrome (MPS-I). Hurler syndrome is a lysosomal storage disease caused by mutations in the a-L-iduronidase gene (IDUA). Lack of a-L-iduronidase results in the lysosomal accumulation of glycosaminoglycans, and eventually leads to the onset of the Hurler syndrome phenotype that includes deterioration of heart, bone, and neurological tissues. Hurler patients usually succumb to the disease in childhood.
I chose to investigate the ability of aminoglycosides to suppress nonsense mutations that lead to Hurler syndrome for the following reasons: 1) the majority of Hurler syndrome patients carry either the Q70X or W402X nonsense mutations; 2) a distinct genotype/phenotype correlation has been established for the MPS-I disorders where milder forms of Hurler syndrome such as Scheie and Hurler/Scheie suggest that very low levels of a-L-iduronidase activity (£ 1% of normal activity) can significantly alleviate the disease phenotype; 3) since Hurler syndrome is a biochemical disorder, the ability of aminoglycosides to suppress IDUA nonsense mutations can be measured quantitatively by the level of a-L-iduronidase protein and enzymatic activity produced; 4) animal models of Hurler syndrome have a similar disease pathophysiology as the human disease.
I am currently developing both a transgenic and a targeted Hurler mouse model in order to test the ability of aminoglycosides and other newly identified compounds to suppress nonsense mutations in vivo and to alleviate the Hurler disease phenotype. This approach is being expanded to include other disease models and other types of mutations such as missense mutations. In addition, potential novel cellular targets for suppression therapy are also currently being identified and characterized in a yeast system.
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