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DECIPHERING THE ROLE OF ⍺-N-TERMINAL METHYLATION IN MODULATING YEAST PROTEIN FUNCTION INCLUDING THE MULTITASKING STRESS RESPONSE PROTEIN, HSP31

thesis
posted on 29.04.2022, 04:28 authored by Panyue ChenPanyue Chen

  

Protein methylation is one of the most common protein posttranslational modifications (PTMs), within which protein α-N-terminal methylation is largely underexplored. Protein α-N-terminal methylation has been implicated in disease development, including cancer and neurodegenerative diseases, but the physiological and pathological roles of this PTM is not well understood. Protein α-N-terminal methylation modifies the free α-amino group on the protein N-termini and adds between one and three methyl groups by α-N-terminal methyltransferases. It has been shown that protein α-N-terminal methylation is conserved across prokaryotes and eukaryotes. The identification and characterization of the two α-N-terminal methyltransferases in humans, NTMT1 and NTMT2, and their homolog in yeast, Tae1, shows a high conserved substrate recognition and possible shared biological roles. α-N-terminal methyltransferases in humans and yeast recognize substrates with a canonical N-terminal motif, X1-P2-[K/R]3 (X=A, S, P or G after the initial M is cleaved). However, most of the proteins containing the canonical motif have not been studied and identified as substrates. In this study, we use a yeast as a model to explore the substrate members in the protein α-N-terminal methylome and understand the potential regulatory mechanisms. 

We characterized the yeast phenotypes associated with a TAE1 deletion strain, including increased resistance to heat stress, oxidative stress and paromomycin, and increased sensitivity to benomyl. We also extended the substrate repertoire by validating the presence of α-N-terminal methylation on six substrates by mass spectrometry. Furthermore, we investigate how α-N-terminal methylation could regulate Hsp31, a multifunctional heat shock protein that is associated with yeast heat response and oxidative response. Results suggest that methylation might regulate the localization of Hsp31, rather than directly regulating Hsp31 chaperone activity or methylglyoxalase activity. Alternatively, we developed another methodology to explore the α-N-terminal methylome without motif restriction by repurposing public mass spectrometry datasets for α-N-terminal methylation events in both yeast and humans. We found about 1-2 % of the total proteome are α-N-terminally methylated. Interestingly, the majority of the α-N-terminal methylation events were not on the canonical motif sequence. This indicates a more prevalent existence of α-N-terminal methylation.

Funding

MCMP Graduate Student Endowment Award

Richard and Anne Borch MCMP Research incentive Award

CoP Summer Research Grant

College of Pharmacy Graduate Travel Award

PGSG Graduate Travel Award

History

Degree Type

Doctor of Philosophy

Department

Medicinal Chemistry and Molecular Pharmacology

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Tony Hazbun

Additional Committee Member 2

Chang-Deng Hu

Additional Committee Member 3

Rong Huang

Additional Committee Member 4

Vincent Jo Davisson

Additional Committee Member 5

Scott D. Briggs