Structure and Mechanism of Class II Aminoacyl-tRNA Synthetases All cells require protein synthesis to maintain their core physiology, as well as permit orderly cell division and regulated differentiation. Mutations and expression changes in enzymes of the translation apparatus can present with complex phenotypes in human diseases, representing a major gap in knowledge. The limited tissue and organ specific nature (e,g, the brain) of these diseases is also a challenge. The long term objective of this work is to discover how specific changes, both genetic and at the level of expression, in the function of conserved components of the translational apparatus bring about human disease. A better understanding of this gene phenotype relationship as it relates to translation factors will have major implications for human health. The immediate goal of this application is to understand how two aminoacyl-tRNA synthetases- histidyl- tRNA synthetase (HARS) and threonyl-tRNA synthetase (TARS)- undergo functional switching between their canonical role in protein synthesis and secondary roles in other physiological processes. Alterations in this switching in HARS and TARS may be linked to Type IIIb Usher syndrome and cancer, respectively. Here, we will test specific hypotheses to account for these secondary functions. For HARS and the Type IIIb Usher syndrome, we hypothesize that the mutant substitution linked to the disease decreases histidyl-tRNAHis levels, leading to near codon suppression or nutritional deficit dependent stress responses. Alternatively, protein- protein interactions or signaling pathways associated with a previously undiscovered secondary function may be perturbed. For TARS, we hypothesize that its association with cancer requires a switch to a non-canonical angiogenesis related function and its subsequent export from cells via an exosome dependent pathway. The application contains two multi-part Aims: (1) Determine the molecular basis of the link between Y454S human histidyl-tRNA synthetase and type III Usher syndrome by (i) measuring the contribution of HisRS under-acylation/miscoding to the Usher IIIB phenotype;(ii) determining the extent to which Type IIIb Usher Syndrome is the result of a loss a HARS-protein protein interaction;and (iii) determining the association of Y454S HisRS with altered signaling by determining the phosphorylation state of HARS. (2) Determine the mechanism by which human threonyl-tRNA synthetase switches between its canonical aminoacylation function and its novel angiogenic function by (i) Determining how the intracellular location of TARS changes in response to drug, hypoxia, nutritional deficit, and cytokines that elicit secretion (TNF-a);(ii) determining the extent to which TARS secretion and pro-angiogenic functios are associated with exosomes; and (iii), determining the role of TARS's cryptic GTPase function in pro-angiogenic functions. The research is significant for the knowledge gained regarding the underlying basis of protein functional diversity and flexibility, and for the high likelihood that further insights into the mechanisms of sensorineural disease and cancer will be gained.

Public Health Relevance

Structure and Mechanism of Class II Aminoacyl-tRNA Synthetases A better understanding of the molecular basis of disease has potential benefits to public health. By distinguishing between mechanisms that involve either altered decoding or altered signal transduction associated with histidyl-tRNA synthetase, physiologically rationale treatments for Usher Syndrome might be designed. Similarly, understanding how threonyl-tRNA synthetase executes its pro- angiogenic functions is likely to aid in the diagnosis and treatment of angiogenesis-dependent cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054899-17
Application #
8629104
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Barski, Oleg
Project Start
1997-05-01
Project End
2018-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
17
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Burlington
State
VT
Country
United States
Zip Code
05405
Lounsbury, Karen M; Francklyn, Christopher S (2016) Aminoacyl-Transfer RNA Synthetases: Connecting Nutrient Status to Angiogenesis Through the Unfolded Protein Response. Arterioscler Thromb Vasc Biol 36:582-3
Fang, Pengfei; Yu, Xue; Jeong, Seung Jae et al. (2015) Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase. Nat Commun 6:6402
Mirando, Adam C; Fang, Pengfei; Williams, Tamara F et al. (2015) Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor. Sci Rep 5:13160
Novoa, Eva Maria; Camacho, Noelia; Tor, Anna et al. (2014) Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo. Proc Natl Acad Sci U S A 111:E5508-17
Mirando, Adam C; Francklyn, Christopher S; Lounsbury, Karen M (2014) Regulation of angiogenesis by aminoacyl-tRNA synthetases. Int J Mol Sci 15:23725-48
Wellman, Theresa L; Eckenstein, Midori; Wong, Cheung et al. (2014) Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer. BMC Cancer 14:620
Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M (2014) Transfer RNA and human disease. Front Genet 5:158
Li, Li; Francklyn, Christopher; Carter Jr, Charles W (2013) Aminoacylating urzymes challenge the RNA world hypothesis. J Biol Chem 288:26856-63
Williams, Tamara F; Mirando, Adam C; Wilkinson, Barrie et al. (2013) Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis. Sci Rep 3:1317
Puffenberger, Erik G; Jinks, Robert N; Sougnez, Carrie et al. (2012) Genetic mapping and exome sequencing identify variants associated with five novel diseases. PLoS One 7:e28936

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