This book chapter highlights the development, interpretation, and caveats underlying ML models for functional PTM site prediction.

Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O₂^−) by catalyzing its conversion to dioxygen (O₂) and hydrogen peroxide (H₂O₂). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major new aspect of the antioxidant function of Sod1 is to integrate O₂ availability to promote NADPH production. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H₂O₂ is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

The Gγ subunit is the smallest member of the heterotrimeric G protein, and it may have more functional roles beyond its important role in anchoring the Gβγ dimer to the plasma membrane. Nassiri Toosi et al. showed that the N-terminal region of the yeast Gγ subunit Ste18 contains two serine residues within an intrinsically disordered region that were phosphorylated by different kinases in response to distinct stimuli. These combinatorial phosphorylation events likely altered the structure of Gγ and modulated the activation of a downstream effector. Together, these results suggest that distinct phosphorylation events in the Gγ intrinsically disordered region determine functional outcomes.

In this study, potentially functional phosphorylation hotspots in the linker histone protein family were identified using SAPH-ire - our machine-learning tool for functional PTM prioritization. Five hotspots ranging from low to high SAPH-ire probability score were investigated using the yeast linker histone, Hho1, as a model. Each phosphosite was mutated to nullify (S/T-A) or mimic (S/T-E) phosphorylation in vivo and then tested in parallel to quantify effects of each on DNA double strand break (DSB) repair. These results identified a strong dependence of DSB repair on site-specific phosphorylation of the linker histone protein.

Mutations enriched in metastatic leader versus follower cells from H1299 non-small cell lung cancer cells were investigated individually to understand their essentiality for such invasive phenotypes. As part of this analysis, we utilized SAPH-ire to understand PTMs in Arp3 - one of the primary candidates found mutated in these studies.

Here we worked with the Lieberman lab to map transient structural interactions between myocilin and the heat shock protein Grp94 using cross linking mass spectrometry. Interaction between these two proteins serves as an important drug target for the treatment of glaucoma. 

Here we used mass spectrometry to discover citrullination sites on fibronectin, the role of this PTM on cell motility and in the immune response of patients with rheumatoid arthritis.

In this study, we summarize physiologic roles for RGS proteins and links to human diseases/traits and report rare variants found within each human RGS protein exome sequence derived from global population studies. Each RGS sequence is analyzed using recently described bioinformatics and proteomic tools for measures of missense tolerance ratio paired with combined annotation-dependent depletion scores, and protein post-translational modification (PTM) alignment cluster analysis. We highlight selected variants within the well-studied RGS domain that likely disrupt RGS protein functions and provide comprehensive variant and PTM data for each RGS protein for future study.

This review covers a wide range of 14-3-3 regulatory mechanisms including PTM. We used SAPH-ire to thoroughly predict the function potential of experimental PTMs observed on 14-3-3 proteins across eukaryotes - only some of which have been studied previously. Among the more interesting discoveries was that the family harbors 4 distinct clusters of PTM - two of which were unrealized previously.

This work demonstrates for the first time that G gamma (Gg) subunits, besides acting as anchors for their obligate G beta (Gb) subunits, have more complex roles in regulating G protein signaling. Furthermore, they show that this tuning of G protein signaling by the phosphorylated Gg N-terminal tail is achieved by altering the interaction between Gbg and downstream effectors in a PTM-dependent manner.

Dr. Maneesha Aluru and team discovered that specific MAPKs thought to have very restricted roles in the process of yeast mating are in fact important regulators of cellular longevity. 

In this project, we utilized several elements of SAPH-ire to explore the relationships and functional implications of plant and mammalian PTMs found on Regulator of G protein Signaling (RGS) proteins.

Using SAPH-ire, we identified PTMs with elevated function potential in superoxide dismutase proteins. One of these ranking in the top 10 (K122 acylation) was tested and found to have an important function in regulating mitochondrial respiration.

Here we describe the impact of PTM features on function potential prediction by SAPH-ire.

Here we demonstrate a neural network machine learning approach for prediction of functional PTMs.

A review of heterotrimeric G protein regulation by ubiquitination including a comparison of ubiquitin site topologies between large and small G proteins.

Here we characterized the proteolytic cleavage site preference of an intramembrane aspartyl protease by mass spectrometry.

The inaugural proof-of-principle test for SAPH-ire, showing that integration of PTM features serves to improve prediction of functional PTMs. Using the approach, we discovered novel PTM regulatory elements in heterotrimeric G protein systems.

This manuscript highlights several examples of combinatorial PTMs in proteins, and describes recent technological developments, which are driving our ability to understand how PTM patterns may "code" for biological outcomes.