Number Of Post-translational Modifications Research Articles

Gap Junctions in Ctenophora.

Gap junction proteins form specialized intercellular communication channels, including electrical synapses, that regulate cellular metabolism and signaling. We present a molecular inventory of the gap junction proteins-innexins (INX-like) in ctenophores, focusing on two reference species, Pleurobrachia bachei and Mnemiopsis leidyi. Innexins were identified in more than 15 ctenophore species, including such genera as Euplokamis, Pukia, Hormiphora, Bolinopsis, Cestum, Ocyropsis, Dryodora, Beroe, benthic ctenophores, Coeloplana and Vallicula, and undescribed species of Mertensiidae. The observed diversity of innexins resulted from the independent expansion of this family from the common ancestor of ctenophores. Innexins show the conserved topology with four transmembrane domains connected by two extracellular loops, which bridge intracellular gaps. However, INX-like genes have highly diverse exon organization and low percentage identity for their amino acid sequences within the same species and between ctenophore species. Such a broad scope of molecular diversity differs from innexins in other phyla. We predicted posttranslational modifications in innexins: 249 and 188 for M. leidyi and P. bachei, respectively. Neither their number nor their locations were conserved within or between species. When the number of posttranslational modifications is factored into the innexins' radiation, the potential for molecular and physiological diversity within gap junctions of ctenophores is almost unfathomable. RNA-seq and in situ hybridization data revealed that innexins are expressed across embryogenesis, including early cleavage stages and gastrulation. They are abundant in all adult tissues, with the highest expression level in the aboral organ (the major integrative center and the gravity sensor in ctenophores), followed by tentacles and comb plates. Nevertheless, each organ and tissue has a unique combination of innexins, suggesting their involvement in complex integrative functions and behaviors of ctenophores.

Comparing extraction method efficiency for high-throughput palaeoproteomic bone species identification

High-throughput proteomic analysis of archaeological skeletal remains provides information about past fauna community compositions and species dispersals in time and space. Archaeological skeletal remains are a finite resource, however, and therefore it becomes relevant to optimize methods of skeletal proteome extraction. Ancient proteins in bone specimens can be highly degraded and consequently, extraction methods for well-preserved or modern bone might be unsuitable for the processing of highly degraded skeletal proteomes. In this study, we compared six proteomic extraction methods on Late Pleistocene remains with variable levels of proteome preservation. We tested the accuracy of species identification, protein sequence coverage, deamidation, and the number of post-translational modifications per method. We find striking differences in obtained proteome complexity and sequence coverage, highlighting that simple acid-insoluble proteome extraction methods perform better in highly degraded contexts. For well-preserved specimens, the approach using EDTA demineralization and protease-mix proteolysis yielded a higher number of identified peptides. The protocols presented here allowed protein extraction from ancient bone with a minimum number of working steps and equipment and yielded protein extracts within three working days. We expect further development along this route to benefit large-scale screening applications of relevance to archaeological and human evolution research.

Deubiquitinase OTUD3: a double-edged sword in immunity and disease.

Deubiquitination is an important form of post-translational modification that regulates protein homeostasis. Ovarian tumor domain-containing proteins (OTUDs) subfamily member OTUD3 was identified as a deubiquitinating enzyme involved in the regulation of various physiological processes such as immunity and inflammation. Disturbances in these physiological processes trigger diseases in humans and animals, such as cancer, neurodegenerative diseases, diabetes, mastitis, etc. OTUD3 is aberrantly expressed in tumors and is a double-edged sword, exerting tumor-promoting or anti-tumor effects in different types of tumors affecting cancer cell proliferation, metastasis, and metabolism. OTUD3 is regulated at the transcriptional level by a number of MicroRNAs, such as miR-520h, miR-32, and miR101-3p. In addition, OTUD3 is regulated by a number of post-translational modifications, such as acetylation and ubiquitination. Therefore, understanding the regulatory mechanisms of OTUD3 expression can help provide insight into its function in human immunity and disease, offering the possibility of its use as a therapeutic target to diagnose or treat disease.

A cellular metabolite directly and reversibly opens the ATP release pannexin channels.

Pannexins (Panx1-3) are a broadly expressed family of ion channels which mediate the cellular release of signaling molecules such as ATP. There are many established roles of pannexins in cell signaling and pathophysiology, particularly in the immune, cardiovascular, and nervous systems. Notably, Panx1 opens irreversibly following cleavage of its C-terminus by caspases in apoptotic cells, releasing ATP to recruit nearby phagocytes. However, there are many signaling roles of pannexin in non-apoptotic cells, which dictates that a reversible mode of activation must exist. To date, a number of post-translational modifications have been proposed as such candidates. Nevertheless, these findings typically relied on cell-based experiments and the simultaneous use of large, supraphysiological voltages to assess changes in channel function. Thus, it is unclear whether they act themselves as a channel gating stimulus or act as modulators. Based on our previous work, we postulated that small molecules may act as a natural gating stimulus for pannexins. An unbiased screen of cellular metabolites obtained from mouse tissues revealed a candidate pannexin agonist. Electrophysiological, cell-based, and in vitro assays confirmed that these metabolites act directly on pannexins to reversibly open the channel and trigger ATP release at resting membrane potential. These data establish this molecule class as the first bona fide pannexin agonists, and elucidate a novel biochemical pathway leading to ATP release through pannexins.

Post-Translation Modifications and Mutations of Human Linker Histone Subtypes: Their Manifestation in Disease.

Linker histones (LH) are a critical component of chromatin in addition to the canonical histones (H2A, H2B, H3, and H4). In humans, 11 subtypes (7 somatic and 4 germinal) of linker histones have been identified, and their diverse cellular functions in chromatin structure, DNA replication, DNA repair, transcription, and apoptosis have been explored, especially for the somatic subtypes. Delineating the unique role of human linker histone (hLH) and their subtypes is highly tedious given their high homology and overlapping expression patterns. However, recent advancements in mass spectrometry combined with HPLC have helped in identifying the post-translational modifications (PTMs) found on the different LH subtypes. However, while a number of PTMs have been identified and their potential nuclear and non-nuclear functions explored in cellular processes, there are very few studies delineating the direct relevance of these PTMs in diseases. In addition, recent whole-genome sequencing of clinical samples from cancer patients and individuals afflicted with Rahman syndrome have identified high-frequency mutations and therefore broadened the perspective of the linker histone mutations in diseases. In this review, we compile the identified PTMs of hLH subtypes, current knowledge of the relevance of hLH PTMs in human diseases, and the correlation of PTMs coinciding with mutations mapped in diseases.

Cross-Talk between Wnt Signaling and Src Tyrosine Kinase.

Src, a non-receptor tyrosine kinase, was first discovered as a prototype oncogene and has been shown to critical for cancer progression for a variety of tissues. Src activity is regulated by a number of post-translational modifications in response to various stimuli. Phosphorylations of Src Tyr419 (human; 416 in chicken) and Src Tyr530 (human; 527 in chicken) have been known to be critical for activation and inactivation of Src, respectively. Wnt signaling regulates a variety of cellular functions including for development and cell proliferation, and has a role in certain diseases such as cancer. Wnt signaling is carried out through two pathways: β-catenin-dependent canonical and β-catenin-independent non-canonical pathways as Wnt ligands bind to their receptors, Frizzled, LRP5/6, and ROR1/2. In addition, many signaling components including Axin, APC, Damm, Dishevelled, JNK kinase and Rho GTPases contribute to these canonical and non-canonical Wnt pathways. However, the communication between Wnt signaling and Src tyrosine kinase has not been well reviewed as Src regulates Wnt signaling through LRP6 tyrosine phosphorylation. GSK-3β phosphorylated by Wnt also regulates Src activity. As Wnt signaling and Src mutually regulate each other, it is noted that aberrant regulation of these components give rise to various diseases including typically cancer, and as such, merit a closer look.

Citrullination of glucokinase is linked to autoimmune diabetes

Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications of self-proteins. A number of post-translational modifications have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes. Here we show the citrullination of pancreatic glucokinase as a result of inflammation, triggering autoimmunity and affecting glucokinase biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4+ T cells to glucokinase epitopes in the circulation of Type 1 diabetes patients and NOD mice. Finally, citrullination alters glucokinase biologic activity and suppresses glucose-stimulated insulin secretion. Our study define glucokinase as a Type 1 diabetes biomarker, providing new insights of how inflammation drives post-translational modifications to create both neoautoantigens and affect beta cell metabolism.

The Thermal Stability of the Collagen Triple Helix Is Tuned According to the Environmental Temperature.

Triple helix formation of procollagen occurs in the endoplasmic reticulum (ER) where the single-stranded α-chains of procollagen undergo extensive post-translational modifications. The modifications include prolyl 4- and 3-hydroxylations, lysyl hydroxylation, and following glycosylations. The modifications, especially prolyl 4-hydroxylation, enhance the thermal stability of the procollagen triple helix. Procollagen molecules are transported to the Golgi and secreted from the cell, after the triple helix is formed in the ER. In this study, we investigated the relationship between the thermal stability of the collagen triple helix and environmental temperature. We analyzed the number of collagen post-translational modifications and thermal melting temperature and α-chain composition of secreted type I collagen in zebrafish embryonic fibroblasts (ZF4) cultured at various temperatures (18, 23, 28, and 33 °C). The results revealed that thermal stability and other properties of collagen were almost constant when ZF4 cells were cultured below 28 °C. By contrast, at a higher temperature (33 °C), an increase in the number of post-translational modifications and a change in α-chain composition of type I collagen were observed; hence, the collagen acquired higher thermal stability. The results indicate that the thermal stability of collagen could be autonomously tuned according to the environmental temperature in poikilotherms.

Abstract IA09: Control of PIP3 levels by PI3Kα and PTEN

Abstract Phosphatidylinositol 3,4,5-triphosphate (PIP3), a lipid second messenger, signals the initiation of cascades that result in, among other processes, cell proliferation, migration, and survival. PIP3 is generated by the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-3-kinase-α (PI3Kα) in response to activation by receptor tyrosine kinases (RTKs) or their substrates. PI3Kα has a residual constitutive activity that is counteracted by the action of PTEN, a phosphatase that is subjected to a large number of post-translational modifications. Thus, levels of PIP3 are controlled by a balance between the opposite actions of PI3Kα, a kinase, and PTEN, a phosphatase. Both enzymes are highly mutated in tumors. Oncogenic mutations have opposite effects on the activities of the two proteins: mutations activate PI3Kα but suppress the activity of PTEN. Both of these effects result in increased levels of PIP3 and provide selective advantage to the tumor cells carrying the mutations. In the physiologic activation of PI3Kα, the increase in activity is caused by the phosphotyrosine-containing region of the RTK dislodging the nSH2, a PI3Kα inhibitory domain from the regulatory subunit p85α, from its inhibitory interaction with the catalytic subunit p110α. A similar mechanism is used by some oncogenic mutations: they activate the enzyme by reducing the inhibitory action of the nSH2. Importantly, this effect is not a consequence of a transition to a new structure that can be identified as a new ground state. Instead, the mutations affect the dynamics of the protein in such a way that the new landscape favors catalytically competent conformations. Analysis of the interdomain interactions between the p110α and the p85α of the wild-type and the mutants suggests that the tumor-associated mutations effectively weaken the interactions between p110α and p85α by disrupting key stabilizing interactions. These findings support the notion that oncogenic mutations increase the enzymatic activity by enhancing the dynamics of the protein. Other oncogenic PI3Kα mutations increase the kinase activity by increasing the interaction of the enzyme with the membrane, and therefore augmenting accessibility to the substrate. In the case of PTEN, activity is controlled, at least in part, by the phosphorylation of four Ser/Thr residues (380, 382, 383, 385) in its 52-residue long C-terminal tail. This multiple phosphorylation results in an inhibition of the membrane-associated phosphatase activity. Using a variety of biochemical and biophysical techniques, we determined structural and physical chemical details of the mechanism controlling this inhibition. Briefly, in contrast to the unphosphorylated form, the tetra phosphorylated PTEN adopts a more compact conformation in which its carboxy terminal tail interacts with the C2 domain in a fashion that reduces its affinity for the cell membrane and diminishes its catalytic activity. Citation Format: Ignacia Echeverria, Yunlong Liu, Sweta Maheshwari, Mayukh Chakrabarti, Michelle Miller, David Bolduc, Philip Cole, Sandra B. Gabelli, L. Mario Amzel. Control of PIP3 levels by PI3Kα and PTEN [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA09.

Understanding the Limit of Open Search in the Identification of Peptides With Post-translational Modifications - A Simulation-Based Study.

Peptide identification from tandem mass spectrometry data is a fundamental task in computational proteomics. Traditional algorithms perform well when facing unmodified peptides. However, when peptides have post-translational modifications (PTMs), these methods cannot provide satisfactory results. Recently, open search methods have been proposed to identify peptides with PTMs. While the performance of these new methods is promising, the identification results vary greatly with respect to the quality of tandem mass spectra and the number of PTMs in peptides. This motivates us to systematically study the relationship between the performance of open search methods and the quality parameters of tandem mass spectrometry data as well as the number of PTMs in peptides. In this paper, we have proposed an analytical model derived from simulated data to describe the relationship between the probability of obtaining correct results and the spectrum quality as well as the number of PTMs. The proposed model is verified using 1,464,146 real experimental spectra. The consistent trend observed in both simulated data and real data reveals the necessary conditions to effectively apply open search methods. Source code of our study is available at http://bioinformatics.ust.hk/PST.html.

Bigram-PGK: phosphoglycerylation prediction using the technique of bigram probabilities of position specific scoring matrix

BackgroundThe biological process known as post-translational modification (PTM) is a condition whereby proteomes are modified that affects normal cell biology, and hence the pathogenesis. A number of PTMs have been discovered in the recent years and lysine phosphoglycerylation is one of the fairly recent developments. Even with a large number of proteins being sequenced in the post-genomic era, the identification of phosphoglycerylation remains a big challenge due to factors such as cost, time consumption and inefficiency involved in the experimental efforts. To overcome this issue, computational techniques have emerged to accurately identify phosphoglycerylated lysine residues. However, the computational techniques proposed so far hold limitations to correctly predict this covalent modification.ResultsWe propose a new predictor in this paper called Bigram-PGK which uses evolutionary information of amino acids to try and predict phosphoglycerylated sites. The benchmark dataset which contains experimentally labelled sites is employed for this purpose and profile bigram occurrences is calculated from position specific scoring matrices of amino acids in the protein sequences. The statistical measures of this work, such as sensitivity, specificity, precision, accuracy, Mathews correlation coefficient and area under ROC curve have been reported to be 0.9642, 0.8973, 0.8253, 0.9193, 0.8330, 0.9306, respectively.ConclusionsThe proposed predictor, based on the feature of evolutionary information and support vector machine classifier, has shown great potential to effectively predict phosphoglycerylated and non-phosphoglycerylated lysine residues when compared against the existing predictors. The data and software of this work can be acquired from https://github.com/abelavit/Bigram-PGK.

An optimized method using peptide arrays for the identification of in vitro substrates of lysine methyltransferase enzymes

While a number of post-translational modifications (PTM), such as phosphorylation and ubiquitination, have been extensively studied, lysine methylation is emerging as an important PTM with implications in a growing number of diverse cellular processes. To date, there are approximately 5000 identified methylation sites on non-histone proteins, and as the methyllysine proteome expands it becomes important to identify the lysine methyltransferase enzymes responsible for each methylation event. The use of peptide SPOT methylation assay has proven to be a useful in the identification and validation of novel substrates for lysine methyltransferase enzymes as it uses a weak beta emitter coupled with fluorography to detect methylation events. The method described in this paper provides improvements to the typical protocol for this assay, as a highly sensitive tritium assay can be developed with less radioactivity than previously described. This protocol provides an inexpensive alternative to weak beta signal enhancer sprays and washes for use in lysine methylation peptide SPOT arrays, and a simple open-source method for array quantification.

Regulation of FOXO Factors in Mammalian Cells.

Forkhead box O (FOXO) transcription factors are central regulators of cellular homeostasis. FOXOs respond to a wide range of external stimuli, including growth factor signaling, oxidative stress, genotoxic stress, and nutrient deprivation. These signaling inputs regulate FOXOs through a number of posttranslational modifications, including phosphorylation, acetylation, ubiquitination, and methylation. Covalent modifications can affect localization, DNA binding, and interactions with other cofactors in the cell. FOXOs integrate the various modifications to regulate cell type-specific gene expression programs that are essential for metabolic homeostasis, redox balance, and the stress response. Together, these functions are critical for coordinating a response to environmental fluctuations in order to maintain cellular homeostasis during development and to support healthy aging.

Determination of Posttranslational Modifications by 2D PAGE: Applications to Polyamines.

Polyamines not only affect transcription and translation but also may induce a number of posttranslational modifications. The identification of polyamine-induced posttranslational modifications can be performed by 2D PAGE analyses. Here, we provide a protocol for 2D-gel electrophoresis that has been optimized for plants. The combined use of this protocol with epitope-tagged proteins expressed in plants enables the detailed analysis of posttranslational modifications induced by different polyamines in vivo.

Post-translational modifications of FDA-approved plasma biomarkers in glioblastoma samples.

Liquid chromatography-tandem mass spectrometry was used to analyze plasma proteins of volunteers (control) and patients with glioblastoma multiform (GBM). A database search was pre-set with a variable post-translational modification (PTM): phosphorylation, acetylation or ubiquitination. There were no significant differences between the control and the GBM groups regarding the number of protein identifications, sequence coverage or number of PTMs. However, in GBM plasma, we unambiguously observed a decreased fraction in post-translationally modified peptides identified with high quality. The disease-specific PTM patterns were extracted and mapped to the set of FDA-approved plasma protein markers. Decreases of 46% and 24% in the number of acetylated and ubiquitinated peptides, respectively, were observed in the GBM samples. Significance of capturing disease-associated patterns of protein modifications was envisaged.

Characterization of histone-related chemical modifications in formalin-fixed paraffin-embedded and fresh-frozen human pancreatic cancer xenografts using LC-MS/MS

Post-translational modifications (PTMs) of histones including acetylation, methylation, and ubiquitination are known to be involved in the epigenetic regulation of gene expression and thus can have an important role in tumorigenesis. A number of PTMs have been linked to pancreatic cancer and are frequently studied as potential targets for cancer therapy or diagnosis. The availability of biobank-stored, formalin-fixed, paraffin-embedded (FFPE) materials and advanced proteomic analytical tools make it possible to detect histone-related PTMs using predicted mass shifts caused by specific modification. It is, however, important to take into account the fact that formaldehyde (FA) present in the FFPE material is chemically reactive and may undergo condensation reactions, for example, with terminal amino groups and active CH functionalities of the studied proteins. As supported by the results of this study, the possibility to misinterpret such protein condensation product as endogenous PTMs should be taken into consideration in all proteomic analytical work involving FFPE materials. In this study, we used liquid chromatography-tandem mass spectrometry to assess preassumed modification of the lysine residues of histone proteins in FFPE or fresh-frozen (FF) tumor xenografts, derived from the human pancreatic cancer cell line, Capan-1. Here we report modifications with a defined mass shift of +14.016, +28.031, +42.011, or +114.043 Da, corresponding to apparent methylation, dimethylation, acetylation, or ubiquitination that were differentially distributed between the groups. The identified modifications were significantly more frequent in FFPE samples as compared with FF samples. Our results indicate that FFPE tissue processing may result in persistent chemical modifications of histones, which correspond in mass shift of important PTMs. Herein, we highlight the importance to investigate and report FA-formed modifications in FFPE-treated tissues, as well as the necessity of careful manual examination of observed modifications to eliminate false-positive PTMs.

Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances.

Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation.

Modulation of post-translational modifications in β-catenin and LRP6 inhibits Wnt signaling pathway in pancreatic cancer

β-Catenin/Wnt signaling pathway is critically regulated in a normal cell by a number of post-translational modifications. In pancreatic cancer however, aberrant activation of this pathway plays a significant role in tumor progression and metastasis. Though a number of studies have focused on understanding Wnt signaling pathway in pancreatic cancer, there has been no systematic study to evaluate molecules that may be affecting this pathway. In the current study, we used a diterpene triepoxide, triptolide, to inhibit post-translational modifications in Wnt pathway and evaluated how this compound may be affecting the intricate signaling that regulates cell proliferation in pancreatic cancer.Our results showed that triptolide inhibits the activation of WNT1, FZD1, and disheveled (DSH) in pancreatic cancer cell lines MIA PaCa-2 and S2-VP10 by inhibiting the phosphorylation of LRP6 and simultaneously blocked translocation of β-catenin to the nucleus by inhibiting its glycosylation.Additionally, inhibition of post-translational modification of the Wnt-signaling pathway also demonstrated regression of tumor growth in a Syngenic Tumor Implantation Model (STIM). Interestingly, these findings suggest Wnt signaling is a vital molecular pathway in pancreatic cancer and may be amenable to targeted drug therapy.

Reactive Oxygen Species Regulate Myocardial Mitochondria through Post-Translational Modification

Post-translational modifications (PTMs) generally refer to covalent and enzymatic modifications of proteins during or after protein biosynthesis, as well as during cleavage or degradation of proteins. These modification processes govern not only protein homeostasis but also new cellular protein functions. As an essential part of complex cellular signal sensing and transduction networks, PTMs respond to various reactive oxygen species (ROS) and contribute to multi-organ injuries although the precise underlying mechanisms of action remain poorly understood. Up to date, a number of PTMs have been postulated to participate in the regulation of mitochondrial function through free radical species and other second messengers. Mitochondrion is believed to serve as a main ROS generator, and at the same time, also an ROS receptor. Modification of mitochondrial proteins may result in detrimental biological consequences through oxidative injury and mitochondrial dysfunction. In this mini-review we will recapitulate some aspects of PTMs in the control of mitochondrial integrity in the heart. The major forms of PTM related to mitochondrial regulation will be discussed here, which include phosphorylation, ubiquitination, acetylation, and oxidative and nitrosative modification.

A study of bias and increasing organismal complexity from their post-translational modifications and reaction site interplays.

Post-translational modifications (PTMs) are important steps in the biosynthesis of proteins. Aside from their integral contributions to protein development, i.e. perform specialized proteolytic cleavage of regulatory subunits, the covalent addition of functional groups of proteins or the degradation of entire proteins, PTMs are also involved in enabling proteins to withstand and recover from temporary environmental stresses (heat shock, microgravity and many others). The literature supports evidence of thousands of recently discovered PTMs, many of which may likely contribute similarly (perhaps, even, interchangeably) to protein stress response. Although there are many PTM actors on the biological stage, our study determines that these PTMs are generally cast into organism-specific, preferential roles. In this work, we study the PTM compositions across the mitochondrial (Mt) and non-Mt proteomes of 11 diverse organisms to illustrate that each organism appears to have a unique list of PTMs, and an equally unique list of PTM-associated residue reaction sites (RSs), where PTMs interact with protein. Despite the present limitation of available PTM data across different species, we apply existing and current protein data to illustrate particular organismal biases. We explore the relative frequencies of observed PTMs, the RSs and general amino-acid compositions of Mt and non-Mt proteomes. We apply these data to create networks and heatmaps to illustrate the evidence of bias. We show that the number of PTMs and RSs appears to grow along with organismal complexity, which may imply that environmental stress could play a role in this bias.