Matthew P. Torres

Deciphering post-translational modification codes.

Post‐translational modifications (PTMs) occur on nearly all proteins. Many domains within proteins are modified on multiple amino acid sidechains by diverse enzymes to create a myriad of possible protein species. How these combinations of PTMs lead to distinct biological outcomes is only beginning to be understood. 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.

Structural Analysis of PTM Hotspots (SAPH-ire)--A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families.

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.

Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease

Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable body of potential regulatory elements that impact hundreds of different biological processes important in eukaryotic biology and human health.

Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Background: The yeast Gα protein contains a unique domain that is monoubiquitinated, leading to vacuolar degradation. Results: A gene deletion screen reveals ubiquitin binding domain proteins necessary for Gα trafficking. Loss of the ubiquitination domain impedes cellular morphogenesis and mating. Conclusion: Proper endocytosis of Gα is required for sustained morphogenesis and efficient mating. Significance: Gα endocytosis promotes signaling. Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.

Systematic analysis of non-structural protein features for the prediction of PTM function potential by artificial neural networks

Post-translational modifications (PTMs) provide an extensible framework for regulation of protein behavior beyond the diversity represented within the genome alone. While the rate of identification of PTMs has rapidly increased in recent years, our knowledge of PTM functionality encompasses less than 5% of this data. We previously developed SAPH-ire (Structural Analysis of PTM Hotspots) for the prioritization of eukaryotic PTMs based on function potential of discrete modified alignment positions (MAPs) in a set of 8 protein families. A proteome-wide expansion of the dataset to all families of PTM-bearing, eukaryotic proteins with a representational crystal structure and the application of artificial neural network (ANN) models demonstrated the broader applicability of this approach. Although structural features of proteins have been repeatedly demonstrated to be predictive of PTM functionality, the availability of adequately resolved 3D structures in the Protein Data Bank (PDB) limits the scope of these methods. In order to bridge this gap and capture the larger set of PTM-bearing proteins without an available, homologous structure, we explored all available MAP features as ANN inputs to identify predictive models that do not rely on 3D protein structural data. This systematic, algorithmic approach explores 8 available input features in exhaustive combinations (247 models; size 2–8). To control for potential bias in random sampling for holdback in training sets, we iterated each model across 100 randomized, sample training and testing sets—yielding 24,700 individual ANNs. The size of the analyzed dataset and iterative generation of ANNs represents the largest and most thorough investigation of predictive models for PTM functionality to date. Comparison of input layer combinations allows us to quantify ANN performance with a high degree of confidence and subsequently select a top-ranked, robust fit model which highlights 3,687 MAPs, including 10,933 PTMs with a high probability of biological impact but without a currently known functional role.

Predicted Functional Implications of Phosphorylation of Regulator of G Protein Signaling Protein in Plants

Heterotrimeric G proteins function in development, biotic, and abiotic stress responses, hormone signaling as well as sugar sensing. We previously proposed that discrimination of these various external signals in the G protein pathway is accomplished in plants by membrane-localized receptor-like kinases (RLKs) rather than G-protein-coupled receptors. Arabidopsis thaliana Regulator of G Signaling protein 1 (AtRGS1) modulates G protein activation and is phosphorylated by several RLKs and by WITH-NO-LYSINE kinases (WNKs). Here, a combination of in vitro kinase assays, mass spectrometry, and computational bioinformatics identified and functionally prioritized phosphorylation sites in AtRGS1. Phosphosites for two more RLKs (BRL3 and PEPR1) were identified and added to the AtRGS1 phosphorylation profile. Bioinformatics analyses revealed that RLKs and WNK kinases phosphorylate plant RGS proteins within regions that are conserved across eukaryotes and at a high frequency. Four phospho-sites among 14 identified are proximal to equivalent mammalian phosphosites that impact RGS function, including: pS437 and pT267 in GmRGS2, and pS339 and pS436 in AtRGS1. Based on these analyses, we propose that pS437 and pS436 regulate GmRGS2 and AtRGS1 protein interactions and/or localization, whereas pT267 is important for modulation of GmRGS2 GAP activity and localization. Moreover, pS339 most likely affects AtRGS1 activation.

Heme bioavailability and signaling in response to stress in yeast cells

Protoheme (hereafter referred to as heme) is an essential cellular cofactor and signaling molecule that is also potentially cytotoxic. To mitigate heme toxicity, heme synthesis and degradation are tightly coupled to heme utilization in order to limit the intracellular concentration of “free” heme. Such a model, however, would suggest that a readily accessible steady-state, bioavailable labile heme (LH) pool is not required for supporting heme-dependent processes. Using the yeast Saccharomyces cerevisiae as a model and fluorescent heme sensors, site-specific heme chelators, and molecular genetic approaches, we found here that 1) yeast cells preferentially use LH in heme-depleted conditions; 2) sequestration of cytosolic LH suppresses heme signaling; and 3) lead (Pb2+) stress contributes to a decrease in total heme, but an increase in LH, which correlates with increased heme signaling. We also observed that the proteasome is involved in the regulation of the LH pool and that loss of proteasomal activity sensitizes cells to Pb2+ effects on heme homeostasis. Overall, these findings suggest an important role for LH in supporting heme-dependent functions in yeast physiology.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His–Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr–Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala–Thr) and the other to the non-pathogenic Aβ48 (Thr–Leu). For the former site, we observed more favorable kinetics in lipid bilayer–mimicking bicelles than in detergent solution, indicating that substrate–lipid and substrate–enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Tyrosine phosphorylation switching of a G protein substrate

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein–coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr166-dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr166 forms a salt bridge in this interface and potentially affects the RGS protein–accelerated GTPase cycle. Using a Tyr166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism “substrate phosphoswitching.”