Cracking the Post-Translational Modification Crosstalk Code in S. cerevisiae

Introduction

Post-translational modifications (PTMs) regulate all aspects of protein functionality. Dysregulation of PTMs or the enzymes that regulate them is frequently associated with disease. Far from working in isolation, PTMs crosstalk with each other, creating decision-making circuits that regulate the cellular adaptation to fluctuating environments.

Importance of PTM Crosstalk

PTM crosstalk is a regulatory layer of the proteome across the tree of life. However, to what extent and how PTM crosstalk regulates biological processes is a long-standing question in biology that is experimentally challenging to tackle. The challenges lie in the natural complexity and infinite combinatorial possibilities of PTMs, and the lack of unbiased systematic methods to study their function.

Methodology

We will use a combination of state-of-the-art CRISPR-based editing methods to construct 30,000 PTM-mutants in all the proteins involved in the signalling-to-chromatin pathways. Additionally, we will employ a scalable systems biology approach based on reverse genetics and chemical genomics to profile these mutants under 150 stress conditions.

Functional Associations

Mutants with similar phenotypic profiles are functionally associated, allowing us to infer functional relationships for PTMs, regulators, and conditions. These functional associations will provide conditional regulatory PTM crosstalk events at an unprecedented scale.

Mechanistic Dissection

We will further mechanistically dissect the function of selected PTM crosstalk events by interrogating:

1. Protein activity
2. Cellular localization
3. Interactions

Moreover, we will dissect their directionality and modularity.

Expected Outcomes

We expect these findings to be transferable to other eukaryotes due to the high degree of conservation in these core biological processes. Overall, this project will revolutionize the PTM field, providing ground-breaking insights into the functional understanding of PTM crosstalk and their regulation in S. cerevisiae, and unlocking the door for cracking the PTM code within and across biological processes.