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.