Peptide and protein modification for biological and therapeutic applications

For the first two years of my Ph.D. programme, I conducted research under the supervision of Dr. Kurt Hoogewijs. This research is outlined in Chapter 1. Following Dr. Kurt Hoogewijs departure from the university, I remained at the University of Galway, where I joined the research group of Dr. Eddie Myers. This research is outlined in Chapter 2. Chapter 1 Mitochondria are known as the powerhouse of the cell as they produce 90% of the energy the body needs to sustain life and support organ function. Mitochondrial diseases are a group of disorders caused by mutations in the nuclear or mitochondrial genes, affecting ~ 1 in 4,000 people. At present, there is no effective treatment for these progressive and life-threatening diseases. One of the most common mitochondrial diseases is MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes), which is caused by a point mutation in the gene encoding mitochondrial tRNALeu(UUR)3. Previous studies have shown that the 16-mer LARS2β32_33 peptide derived from mitochondrial leucyl-tRNA synthetase exerts rescuing activity on the mutated mt-tRNA-Leu. In this study, we investigate the structure-affinity relationship of these peptides and identify the truncated form of the LARS2β32_33 peptide that binds to tRNALeu(UUR) by using microscale thermophoresis. Truncation of several N-terminal amino acid residues of LARS2β32_33 provides peptides with retained binding affinity to the tRNA. Alanine scans have highlighted the particular importance of hydrophobic residuesfor binding affinity to the tRNALeu(UUR) . Interestingly, the LARS2 β32_33 peptide also has binding affinity for non-cognate mt-tRNA Lys, Val, Ala and Ala(mouse). This data provides critical insight for the development of peptidomimetic therapeutics based on LARS2β32_33 peptides as potential widespread therapeutics for mitochondrial diseases. Chapter 2 Post-translational modifications (PTMs) have a profound effect on protein structure and function, influencing many cellular processes and disease progression. Lysine residues are known to undergo a particularly diverse range of PTMs, but the function of some of these modifications remains unclear. Accessing pure, well-defined, site-specifically modified proteins is vital for understanding the mechanisms in which they are involved. Owing to the superior nucleophilicity of the cysteine thiolate and the rarity of cysteine in the human proteome (ca. 2%), thisresidue is often exploited forsite-specific installation of PTM mimics2 . γ-Thialysine, the simplest cysteine-derived lysine analogue, exhibits comparable structural and chemical properties, justifying its use as a representative lysine mimic. Herein, we have developed a fast and easily accessible method that enables the transformation of cysteine residues into γ-thialysine derivatives by using cyclic sulfamidates. These reagents demonstrate excellent selectivity and high conversion rates and can be employed under mild reaction conditions. The transformation involves ring-opening of the cyclic sulfamidate to form intermediate sulfamic acid derivatives, which, depending on the nature of the substituent on the nitrogen atom, undergo buffermediated desulfonation to the desired γ-thialysine analogues. We demonstrate the installation of various γ-thialysine PTMs into small molecule, peptide and protein model systems. This work highlights cyclic sulfamidates as privileged reagentsforselective alkylation of cysteine residues and shows promise for subsequent applications in therapeutic or biological research.

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

cbn

Collections

University of Galway Theses (PhD Theses)