Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Biochemistry

Supervisor

Heinemann, Ilka

Abstract

Aminoacyl-tRNA synthetases ligate amino acids to their cognate tRNAs during protein synthesis­. Mutations in the catalytic domain of histidyl-tRNA synthetase (HARS) lead to an incurable neurodegenerative disease Charcot Marie Tooth Disease Type 2W (CMT2W), with the molecular basis of many disease-causing mutations unknown. I generated a yeast model for CMT2W-causing HARS mutations V133F, V155G, Y330C, and S356N. All human HARS variants complemented genomic deletion of yeast ortholog histidyl tRNA synthetase 1 (HTS1) but with reduced growth. HARS V155G and S356N lead to global insoluble protein accumulation, with their growth defect and perturbed proteome rescued by histidine supplementation. HARS V133F and Y330C decreased HARS abundance and histidine supplementation further reduced viability, indicating histidine toxicity. As histidine is in clinical trials for treating patients with HARS mutations, our data will inform treatment options for CMT patients, where histidine supplementation may either have a toxic or compensating effect depending on the nature of the causative HARS variant.

Summary for Lay Audience

Histidyl tRNA synthetase (HARS) is an enzyme that ligates the amino acid histidine to tRNAs, which is a molecule that brings amino acids into the cell’s protein-making machinery to make proteins. Mutations in the human HARS gene cause two hereditary diseases named Usher Syndrome type 3B (USH3B) or Charcot Marie Tooth 2W (CMT2W) disease. In USH3B, patients develop blindness, deafness, and hallucination during fevers. CMT2W is a disease that affects the peripheral nerves, leading to motor and sensory defects and weaker muscles. It is unclear what changes these mutations cause on the cellular and molecular level to lead to disease. This study aimed to study mutations V133F, V155G, Y330C, and S356N in HARS that lead to CMT2W. I generated a yeast model where I replaced the yeast version of HARS gene with the human HARS gene. Yeast is a powerful model system for human disease as it shares many similar cellular processes with humans, yet it is also a simple eukaryotic organism to manipulate. I found that yeast with mutant HARS grew slower compared to normal HARS. Mutants V155G and S356N caused problems in protein production throughout the cell, leading to insoluble protein accumulation, which could mean that proteins are not being made properly, and therefore not being folded properly. Supplementing these two mutants with extra histidine allowed them to growth better, and restored proper protein synthesis. On the other hand, mutants V133F and Y330C lead to less expression of the HARS protein itself. Giving these two mutants extra histidine made their growth defect even worse. Currently there is a clinical trial for oral histidine supplements to treat USH3B. This study is important in showing that we need to be cautious when giving extra histidine to treat patients with CMT2W as it could make the patients worse. This study also proposes the molecular mechanisms of how mutations in HARS lead to growth defects in yeast.

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