graduate student Purdue University West Lafayette, Indiana, United States
Panyue Chen (Purdue University)| Guangping Dong (Purdue University)| Rong Huang (Purdue University)| Tony Hazbun (Purdue University)
As the one of the most common protein post-translational modifications (PTMs), protein methylation, especially a-N-methylation is largely underexplored. a-N-methylation is conserved in both eukaryotes and prokaryotes. Identification of the two a-N-methyltransferases in humans, NTMT1 and NTMT2, and their homologue of high sequence similarity in yeast, Tae1, indicated a shared novel protein regulation mechanism in both species. Recently, NTMT1/2 are shown important functions in many biological pathways including cell division and dysregulation is implicated in cancer development. Structural studies and in vitro analysis support a canonical motif, X1-P2-K3 (X=A, S, P or G after initiating M is cleaved) on substrate proteins as the determinant for recognition by NTMT1/2 and Tae1. However, a-N-methylation on most of the potential substrates with the motif are not substantiated. The object of this study is using yeast as an ideal model to investigate the a-N-terminal methylome, taking advantage of its well observed phenotypes, fully sequenced genome, tractable genetics, and comparatively simplified N-terminal methyltransferase network.
We combine bioinformatic searching, mass spectrometry PTM mapping, yeast phenotypic assay and biochemical assays in this study to approach our aims: (1) Screen for potential substrates and identify methylation events using a proteomic dataset repurposing approach, (2) Confirm a-N-methylation experimentally, (3) Investigate the Tae1 relevant phenotypes and biological pathways. (4) Exploration of mechanism in regulating substrate function. Initial bioinformatic research by motif mapping indicates that 45 proteins might be regulated by Tae1 through a-N-methylation. We have verified a-N-methylation on 6 proteins including Heat shock protein 31, Hsp31. Hsp31 and its three paralogues are members of the DJ-1/ThiJ/PfpI family with the human protein implicated in Parkinson’s Disease. We demonstrate that TAE1 deficient yeast show susceptibility to inhibitors of protein synthesis or microtubule assembly. However, the same strain demonstrates resistance to heat stress and oxidative stress. Hsp31 is a multifunctional protein with chaperone, deglycase and methylglyoxalase activity. Thus, we hypothesize that Tae1 regulates yeast response to heat stress and oxidative stress potentially through its substrate, Hsp31. Further studies confirmed that Tae1 is responsible for a-N-methylation of Hsp31. Overexpression of Hsp31 N-terminal mutants with decreased methylation potential show elevated protective effect under both heat shock and methylglyoxalase (MGO) stress. However, MGO stress treatment on yeast show limited effect on methylglyoxalase activity of purified Hsp31. The exact mechanism of how a-N-methylation affects protein function is currently under assessment for Hsp31 and other potential substrates.
The results of this study will reveal the more global view of N-terminal methyltransferase substrates, facilitate the translation to human substrates and further assist in investigating N-terminal methylation under various disease contexts and assist in optimizing drug design of disease targets.