3.3 PTM-enzyme assays beyond proteases.
In principle, any PTM-enzyme whose activity can be detected on the yeast cell surface and whose activity is minimally obstructed by an endogenous enzyme can be assayed in YESS. Thus, YESS-based enzyme substrate profiling has extended to tyrosine kinases and histone acetyltransferases. Taft and coworkers first showed that tyrosine phosphorylation could be measured in YESS and performed two interesting experiments (Taft et al., 2019). First, they profiled the substrate specificity of human SRC, LYN, and ABL kinases and implemented a machine-learning algorithm utilizing amino acid covariances to predict ABL1 kinase peptide substrates. Second, and most importantly, they used YESS to screen a randomized library of ABL1 kinase domain and isolated ABL1 mutants resistant to the clinically used inhibitors dasatinib and ponatinib. This drug resistance screening strategy recapitulated all validated BCR-ABL1 mutations leading to clinical resistance to dasatinib, in addition to identifying other mutations previously observed in patients. Importantly, Taft showed that ponatinib remained effective against most single mutants of ABL1 kinase, with drug resistance starting only with rare compound mutations. Stern and colleagues recently harnessed ER sequestration to measure tyrosine phosphorylation cascades using full-length protein substrates (Ezagui et al., 2022). Using a modified version of the YESS plasmid containing a ribosomal skipping sequence (T2A), the protein-tyrosine kinase (LCK) is expressed and activates ZAP-70, an intermediate enzyme, through phosphorylation of its catalytic loop. Activated ZAP-70 is shuttled through the ER sequestration signal, where it interacts and phosphorylates LAT, the peptide sequence that is shuttled to the cell’s surface for YSD analysis. This cascade of phosphorylation-activated enzymatic activity provides an experimental framework for understanding the kinase interactome in an orthogonal host.
Mapping epigenetic changes in a cell typically involves expensive and complicated recombinant proteins and cell-based assays followed by chromatography and mass spectrometry. Furthermore, crosstalk between epigenetic modifications, even catalyzed by the same enzyme, remains unclear. Keung and Rao adapted the YESS system to show that one can study human histone acetyltransferases (HAT) in yeast and investigate inter-residue communication in histone lysine modification (Waldman et al., 2021). They chose the HAT p300 as the writer and histones H3 and H4 as the substrates. Using yeast in this capacity proved a reliable platform to test the binding affinity and specificity of several commercial anti-lysine acetylation antibodies. Furthermore, this assay proved robust in mapping residue crosstalk between histone lysine residues, particularly the H3 and H4 sites. Among other interesting findings, it was observed that the strong acetylation preference of p300 at H4K20, H4K8, and H4K16 was significantly diminished when H4K20 was mutated to arginine. Interestingly, this was not observed when an arginine mutation was introduced at H4K8 or H4K16. Tuning interaction time between the epigenome writers and histone regions provides a robust, adaptable avenue to gain insights into epigenetic mechanisms. In theory, YESS can extend to processes such as protein methylation and other PTMs.