Cymoxanil disrupts RNA synthesis through inhibiting the activity of dihydrofolate reductase.

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.

Hydrogen peroxide sensitivity connects the activity of COX5A and NPR3 to the regulation of YAP1 expression.

Reactive oxygen species (ROS) are among the most severe types of cellular stressors with the ability to damage essential cellular biomolecules. Excess levels of ROS are correlated with multiple pathophysiological conditions including neurodegeneration, diabetes, atherosclerosis, and cancer. Failure to regulate the severely imbalanced levels of ROS can ultimately lead to cell death, highlighting the importance of investigating the molecular mechanisms involved in the detoxification procedures that counteract the effects of these compounds in living organisms. One of the most abundant forms of ROS is H2 O2 , mainly produced by the electron transport chain in the mitochondria. Numerous genes have been identified as essential to the process of cellular detoxification. Yeast YAP1, which is homologous to mammalian AP-1 type transcriptional factors, has a key role in oxidative detoxification by upregulating the expression of antioxidant genes in yeast. The current study reveals novel functions for COX5A and NPR3 in H2 O2 -induced stress by demonstrating that their deletions result in a sensitive phenotype. Our follow-up investigations indicate that COX5A and NPR3 regulate the expression of YAP1 through an alternative mode of translation initiation. These novel gene functions expand our understanding of the regulation of gene expression and defense mechanism of yeast against oxidative stress.

Semi-automated water sampling module for repeated sampling and concentration of Bacillus cereus group spores.

Monitoring the presence of pathogenic Bacillus spores is important for industrial applications, as well as necessary for ensuring human health. Bacillus thuringiensis is used as a biopesticide against several insect pests. Bacillus cereus spores are a significant cause of food poisoning, and Bacillus anthracis is a recognized biosecurity threat. Laboratory-based methods, such as polymerase chain reaction, enzyme-linked immunosorbent assay, or matrix-assisted laser desorption ionization spectroscopy provide sensitive detection of bacteria and spores, but the application of those methods for quasi-continuous environmental monitoring presents a significant challenge requiring frequent human intervention. To address this challenge, we developed a workstation for quasi-autonomous monitoring of water reservoirs for the presence of bacteria and spores, and designed and validated the functionality of a microprocessor-controlled module capable of repetitive collection and pre-concentration of spores in liquid samples tested with fiberglass (FG), polyether sulfone and polyvinylidene fluoride filters. The best results were obtained with FG filters delivering a 20× concentration of B. thuringiensis and B. cereus spores from saline suspensions. The successful 20× pre-concentration of Bacillus spores demonstrated with FG filters could be repeated up to 3 times when bleach decontamination is applied between filtrations. Taken together, our results demonstrate an attractive instrument suitable for semi-automated, quasi-continuous sampling and pre-processing of water samples for biosensing of bacterial spores originating from a complex environment.

DBP7 and YRF1-6 Are Involved in Cell Sensitivity to LiCl by Regulating the Translation of PGM2 mRNA.

Lithium chloride (LiCl) has been widely researched and utilized as a therapeutic option for bipolar disorder (BD). Several pathways, including cell signaling and signal transduction pathways in mammalian cells, are shown to be regulated by LiCl. LiCl can negatively control the expression and activity of PGM2, a phosphoglucomutase that influences sugar metabolism in yeast. In the presence of galactose, when yeast cells are challenged by LiCl, the phosphoglucomutase activity of PGM2p is decreased, causing an increase in the concentration of toxic galactose metabolism intermediates that result in cell sensitivity. Here, we report that the null yeast mutant strains DBP7∆ and YRF1-6∆ exhibit increased LiCl sensitivity on galactose-containing media. Additionally, we demonstrate that DBP7 and YRF1-6 modulate the translational level of PGM2 mRNA, and the observed alteration in translation seems to be associated with the 5'-untranslated region (UTR) of PGM2 mRNA. Furthermore, we observe that DBP7 and YRF1-6 influence, to varying degrees, the translation of other mRNAs that carry different 5'-UTR secondary structures.

Rapid, Sensitive, and Selective Quantification of Bacillus cereus Spores Using xMAP Technology.

Bacillus cereus is a spore-forming ubiquitous bacterium notable as a food poisoning agent. Detection of B. cereus spores using selective media is laborious and non-specific. Herein, the quantitative detection of B. cereus spores was investigated with commercial antibodies and published aptamer sequences. Several detection reagents were screened for affinity to Bacillus collagen-like protein A (BclA), an abundant exosporium glycoprotein. Sensitivity and selectivity toward B. cereus spores were tested using immunoassays and multi-analyte profiling (xMAP). A recombinant antibody developed in llama against BclA protein showed B. cereus spore selectivity and sensitivity between 102 and 105 spores/mL using xMAP. DNA aptamer sequences demonstrated sensitivity from 103 to 107 spores/mL and no cross-reaction to B. megaterium and B. subtilis. Selectivity for B. cereus spores was also demonstrated in a mixture of several diverse microorganisms and within a food sample with no compromise of sensitivity. As proof of concept for multiplexed measurement of human pathogens, B. cereus and three other microorganisms, E. coli, P. aeruginosa, and S. cerevisiae, were simultaneously detected using xMAP. These data support the development of a rapid, sensitive, and selective system for quantitation of B. cereus spores and multiplexed monitoring of human pathogens in complex matrices.

Lithium chloride sensitivity connects the activity of PEX11 and RIM20 to the translation of PGM2 and other mRNAs with structured 5'-UTRs.

Lithium chloride (LiCl) is a widely used and extensively researched drug for the treatment of bipolar disorder (BD). As a result, LiCl has been the subject of research studying its toxicity, mode of action, and downstream cellular responses. LiCl has been shown to influence cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3 in mammalian cells. LiCl's significant downstream effects on the translational pathway necessitate further investigation. In yeast, LiCl is found to lower the activity and alter the expression of PGM2, a gene encoding a sugar-metabolism enzyme phosphoglucomutase. When phosphoglucomutase activity is reduced in the presence of galactose, intermediates of galactose metabolism aggregate, causing cell sensitivity to LiCl. In this study, we identified that deleting the genes PEX11 and RIM20 increases yeast LiCl sensitivity. We further show that PEX11 and RIM20 regulate the expression of PGM2 mRNA at the translation level. The observed alteration of translation seems to target the structured 5'-untranslated region (5'-UTR) of the PGM2 mRNA.

Discovery and identification of genes involved in DNA damage repair in yeast.

DNA repair defects are common in tumour cells and can lead to misrepair of double-strand breaks (DSBs), posing a significant challenge to cellular integrity. The overall mechanisms of DSB have been known for decades. However, the list of the genes that affect the efficiency of DSB repair continues to grow. Additional factors that play a role in DSB repair pathways have yet to be identified. In this study, we present a computational approach to identify novel gene functions that are involved in DNA damage repair in Saccharomyces cerevisiae. Among the primary candidates, GAL7, YMR130W, and YHI9 were selected for further analysis since they had not previously been identified as being active in DNA repair pathways. Originally, GAL7 was linked to galactose metabolism. YHI9 and YMR130W encode proteins of unknown functions. Laboratory testing of deletion strains gal7Δ, ymr130wΔ, and yhi9Δ implicated all 3 genes in Homologous Recombination (HR) and/or Non-Homologous End Joining (NHEJ) repair pathways, and enhanced sensitivity to DNA damage-inducing drugs suggested involvement in the broader DNA damage repair machinery. A subsequent genetic interaction analysis revealed interconnections of these three genes, most strikingly through SIR2, SIR3 and SIR4 that are involved in chromatin regulation and DNA damage repair network.

Strategies for capturing Bacillus thuringiensis spores on surfaces of (001) GaAs-based biosensors.

Bacillus thuringiensis (Bt) is used as a bioinsecticide since it effectively kills insect larvae. Bt is also genetically similar to Bacillus cereus (Bc), a well recognized foodborne human pathogen; they are both members of the Bacillus cereus group (BC group). Although approved Bt bioinsecticide products have been confirmed to be non-pathogenic to humans, close monitoring of Bt during dissemination is important for cost considerations and to limit impact on biodiversity towards nontarget organisms. As such, developing rapid, sensitive, and specific tools for quantitative detection of Bt spores during and following spray operations is highly desirable. The goals of this study were to investigate commercially available detection reagents for sensitivity and selectivity in detecting Bt spores, and then functionalize a surface of (001) GaAs used in photonic biosensing. To achieve these goals, we (1) screened commercial antibodies for their capacity to bind recombinant proteins from Bt spores, (2) screened antibodies and aptamers for their sensitivity and selectivity against Bt spores, and (3) tested the efficiency of selected antibodies and aptamers in capturing Bt spores on the surface of functionalized GaAs biochips. Seven genes encoding Bt spore proteins were cloned and expressed in Escherichia coli. The binding of each purified spore antigen was tested by commercially available polyclonal and monoclonal antibodies claimed to exclusively target spores. Of the seven targets, Bacillus collagen-like protein A, was the most abundant protein on Bt spores and demonstrated the strongest binding affinity to all test antibodies. The commercial antibodies (Abs) were also tested for specificity to BC Group versus non-BC Group spores. Three of six commercial antibodies showed selectivity to Bt spores, with recombinant Abs providing the most robust lower range of detection (102 to 6 × 103 spores/mL). The sensitivity and selectivity of three published DNA aptamer sequences demonstrated a wide range of detection sensitivity for Bt spores. Two of the three test aptamers also showed reasonable selectivity towards Bt spores while the third demonstrated reactivity to non-BC Group B. megaterium and B. subtilis. Of the reagents tested, a thiolated aptamer and llama recombinant Ab showed highest Bt spore capture efficiency as measured by spore coverage of the GaAs surface. These results confirm that the selected aptamer and llama rAb can be considered strong candidates for the development of GaAs-based biosensing devices.

Manganese-Induced Neurotoxicity through Impairment of Cross-Talk Pathways in Human Neuroblastoma Cell Line SH-SY5Y Differentiated with Retinoic Acid.

Manganese (Mn) is an important element; yet acute and/or chronic exposure to this metal has been linked to neurotoxicity and neurodegenerative illnesses such as Parkinson's disease and others via an unknown mechanism. To better understand it, we exposed a human neuroblastoma cell model (SH-SY5Y) to two Mn chemical species, MnCl2 and Citrate of Mn(II) (0-2000 µM), followed by a cell viability assay, transcriptomics, and bioinformatics. Even though these cells have been chemically and genetically modified, which may limit the significance of our findings, we discovered that by using RA-differentiated cells instead of undifferentiated SH-SY5Y cell line, both chemical species induce a similar toxicity, potentially governed by disruption of protein metabolism, with some differences. The MnCl2 altered amino acid metabolism, which affects RNA metabolism and protein synthesis. Citrate of Mn(II), however, inhibited the E3 ubiquitin ligases-target protein degradation pathway, which can lead to the buildup of damaged/unfolded proteins, consistent with histone modification. Finally, we discovered that Mn(II)-induced cytotoxicity in RA-SH-SY5Y cells shared 84 percent of the pathways involved in neurodegenerative diseases.

Manganese-induced neurotoxicity in cerebellar granule neurons due to perturbation of cell network pathways with potential implications for neurodegenerative disorders.

Manganese (Mn) is essential for living organisms, playing an important role in nervous system function. Nevertheless, chronic and/or acute exposure to this metal, especially during early life stages, can lead to neurotoxicity and dementia by unclear mechanisms. Thus, based on previous works of our group with yeast and zebrafish, we hypothesized that the mechanisms mediating manganese-induced neurotoxicity can be associated with the alteration of protein metabolism. These mechanisms may also depend on the chemical speciation of manganese. Therefore, the current study aimed at investigating the mechanisms mediating the toxic effects of manganese in primary cultures of cerebellar granule neurons (CGNs). By exposing cultured CGNs to different chemical species of manganese ([[2-[(dithiocarboxy)amino]ethyl]carbamodithioato]](2-)-kS,kS']manganese, named maneb (MB), and [[1,2-ethanediylbis[carbamodithioato]](2-)]manganese mixture with [[1,2-ethanediylbis[carbamodithioato]](2-)]zinc, named mancozeb (MZ), and manganese chloride (MnCl2)), and using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, we observed that both MB and MZ induced similar cytotoxicity (LC50∼ 7-9 µM), which was higher than that of MnCl2 (LC50∼ 27 µM). Subsequently, we applied systems biology approaches, including metallomics, proteomics, gene expression and bioinformatics, and revealed that independent of chemical speciation, for non-cytotoxic concentrations (0.3-3 µM), Mn-induced neurotoxicity in CGNs is associated with metal dyshomeostasis and impaired protein metabolism. In this way, we verified that MB induced more post-translational alterations than MnCl2, which can be a plausible explanation for cytotoxic differences between both chemical species. The metabolism of proteins is one of the most energy consuming cellular processes and its impairment appears to be a key event of some cellular stress processes reported separately in other studies such as cell cycle arrest, energy impairment, cell signaling, excitotoxicity, immune response, potential protein accumulation and apoptosis. Interestingly, we verified that Mn-induced neurotoxicity shares pathways associated with the development of Alzheimer's disease, Amyotrophic Lateral Sclerosis, Huntington's disease, and Parkinson's disease. This has been observed in baker's yeast and zebrafish suggesting that the mode of action of Mn may be evolutionarily conserved.

Lithium Chloride Sensitivity in Yeast and Regulation of Translation.

For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5'-Untranslated region (5'-UTR) of PGM2 mRNA in yeast.

Sensitivity of yeast to lithium chloride connects the activity of YTA6 and YPR096C to translation of structured mRNAs.

Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.

Manganese-induced cellular disturbance in the baker's yeast, Saccharomyces cerevisiae with putative implications in neuronal dysfunction.

Manganese (Mn) is an essential element, but in humans, chronic and/or acute exposure to this metal can lead to neurotoxicity and neurodegenerative disorders including Parkinsonism and Parkinson's Disease by unclear mechanisms. To better understand the effects that exposure to Mn2+ exert on eukaryotic cell biology, we exposed a non-essential deletion library of the yeast Saccharomyces cerevisiae to a sub-inhibitory concentration of Mn2+ followed by targeted functional analyses of the positive hits. This screen produced a set of 43 sensitive deletion mutants that were enriched for genes associated with protein biosynthesis. Our follow-up investigations demonstrated that Mn reduced total rRNA levels in a dose-dependent manner and decreased expression of a ß-galactosidase reporter gene. This was subsequently supported by analysis of ribosome profiles that suggested Mn-induced toxicity was associated with a reduction in formation of active ribosomes on the mRNAs. Altogether, these findings contribute to the current understanding of the mechanism of Mn-triggered cytotoxicity. Lastly, using the Comparative Toxicogenomic Database, we revealed that Mn shared certain similarities in toxicological mechanisms with neurodegenerative disorders including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's and Huntington's diseases.

In Silico Engineering of Synthetic Binding Proteins from Random Amino Acid Sequences.

Synthetic proteins with high affinity and selectivity for a protein target can be used as research tools, biomarkers, and pharmacological agents, but few methods exist to design such proteins de novo. To this end, the In-Silico Protein Synthesizer (InSiPS) was developed to design synthetic binding proteins (SBPs) that bind pre-determined targets while minimizing off-target interactions. InSiPS is a genetic algorithm that refines a pool of random sequences over hundreds of generations of mutation and selection to produce SBPs with pre-specified binding characteristics. As a proof of concept, we design SBPs against three yeast proteins and demonstrate binding and functional inhibition of two of three targets in vivo. Peptide SPOT arrays confirm binding sites, and a permutation array demonstrates target specificity. Our foundational approach will support the field of de novo design of small binding polypeptide motifs and has robust applicability while offering potential advantages over the limited number of techniques currently available.

Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast.

Heavy metal and metalloid contaminations are among the most concerning types of pollutant in the environment. Consequently, it is important to investigate the molecular mechanisms of cellular responses and detoxification pathways for these compounds in living organisms. To date, a number of genes have been linked to the detoxification process. The expression of these genes can be controlled at both transcriptional and translational levels. In baker's yeast, Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferases. Yeast URE2 encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. The URE2 expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes, ITT1, an inhibitor of translation termination, and RPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain for URE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affect URE2 mRNA expression at the level of translation.

A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis.

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.

Uncharacterized ORF HUR1 influences the efficiency of non-homologous end-joining repair in Saccharomyces cerevisiae.

Non-Homologous End Joining (NHEJ) is a highly conserved pathway that repairs Double-Strand Breaks (DSBs) within DNA. Here we show that the deletion of yeast uncharacterized ORF HUR1, Hydroxyurea Resistance1 affects the efficiency of NHEJ. Our findings are supported by Protein-Protein Interaction (PPI), genetic interaction and drug sensitivity analyses. To assess the activity of HUR1 in DSB repair, we deleted its non-overlapping region with PMR1, referred to as HUR1-A. We observed that similar to deletion of TPK1 and NEJ1, and unlike YKU70 (important for NHEJ of DNA with overhang and not blunt end), deletion of HUR1-A reduced the efficiency of NHEJ in both overhang and blunt end plasmid repair assays. Similarly, a chromosomal repair assay showed a reduction for repair efficiency when HUR1-A was deleted. In agreement with a functional connection for Hur1p with Tpk1p and NEJ1p, double mutant strains Δhur1-A/Δtpk1, and Δhur1-A/Δnej1 showed the same reduction in the efficiency of plasmid repair, compared to both single deletion strains. Also, using a Homologous Recombination (HR) specific plasmid-based DSB repair assay we observed that deletion of HUR1-A influenced the efficiency of HR repair, suggesting that HUR1 might also play additional roles in other DNA repair pathways.

Designing anti-Zika virus peptides derived from predicted human-Zika virus protein-protein interactions.

The production of anti-Zika virus (ZIKV) therapeutics has become increasingly important as the propagation of the devastating virus continues largely unchecked. Notably, a causal relationship between ZIKV infection and neurodevelopmental abnormalities has been widely reported, yet a specific mechanism underlying impaired neurological development has not been identified. Here, we report on the design of several synthetic competitive inhibitory peptides against key pathogenic ZIKV proteins through the prediction of protein-protein interactions (PPIs). Often, PPIs between host and viral proteins are crucial for infection and pathogenesis, making them attractive targets for therapeutics. Using two complementary sequence-based PPI prediction tools, we first produced a comprehensive map of predicted human-ZIKV PPIs (involving 209 human protein candidates). We then designed several peptides intended to disrupt the corresponding host-pathogen interactions thereby acting as anti-ZIKV therapeutics. The data generated in this study constitute a foundational resource to aid in the multi-disciplinary effort to combat ZIKV infection, including the design of additional synthetic proteins.

The sensitivity of the yeast, Saccharomyces cerevisiae, to acetic acid is influenced by DOM34 and RPL36A.

The presence of acetic acid during industrial alcohol fermentation reduces the yield of fermentation by imposing additional stress on the yeast cells. The biology of cellular responses to stress has been a subject of vigorous investigations. Although much has been learned, details of some of these responses remain poorly understood. Members of heat shock chaperone HSP proteins have been linked to acetic acid and heat shock stress responses in yeast. Both acetic acid and heat shock have been identified to trigger different cellular responses including reduction of global protein synthesis and induction of programmed cell death. Yeast HSC82 and HSP82 code for two important heat shock proteins that together account for 1-2% of total cellular proteins. Both proteins have been linked to responses to acetic acid and heat shock. In contrast to the overall rate of protein synthesis which is reduced, the expression of HSC82 and HSP82 is induced in response to acetic acid stress. In the current study we identified two yeast genes DOM34 and RPL36A that are linked to acetic acid and heat shock sensitivity. We investigated the influence of these genes on the expression of HSP proteins. Our observations suggest that Dom34 and RPL36A influence translation in a CAP-independent manner.

Evolution of protein-protein interaction networks in yeast.

Interest in the evolution of protein-protein and genetic interaction networks has been rising in recent years, but the lack of large-scale high quality comparative datasets has acted as a barrier. Here, we carried out a comparative analysis of computationally predicted protein-protein interaction (PPI) networks from five closely related yeast species. We used the Protein-protein Interaction Prediction Engine (PIPE), which uses a database of known interactions to make sequence-based PPI predictions, to generate high quality predicted interactomes. Simulated proteomes and corresponding PPI networks were used to provide null expectations for the extent and nature of PPI network evolution. We found strong evidence for conservation of PPIs, with lower than expected levels of change in PPIs for about a quarter of the proteome. Furthermore, we found that changes in predicted PPI networks are poorly predicted by sequence divergence. Our analyses identified a number of functional classes experiencing fewer PPI changes than expected, suggestive of purifying selection on PPIs. Our results demonstrate the added benefit of considering predicted PPI networks when studying the evolution of closely related organisms.