Decoding Protein Complexity: The Fundamental Role of Post-Translational Modifications in Cellular Function and Disease

In recent decades, our understanding of PTMs has grown immensely, identifying over 500 distinct types to date. The most studied PTMs, representing nearly 90 % of reported PTM sites, include phosphorylation, acetylation, ubiquitination, succinylation, and methylation. Excitingly, new discoveries revealed additional modifications such as crotonylation, monoaminylation, and lactylation. 

PTMs play a critical role in cellular responsiveness and adaptation. Their predominantly reversible nature allows a precise spatial and temporal control over protein chemistry and function, enabling cells to quickly adapt to environmental changes. For instance, phosphorylation, one of the best studied PTMs, activates enzymes like protein kinases, triggering distinct signaling pathways. Further, PTMs regulate gene expression, protein homeostasis, protein functions, protein-protein recognition, interactions, as well as their spatial localization. With this, PTMs significantly enhance the structural and functional diversity of proteins, impacting nearly every aspect of cellular functions. However, the complete impact of many identified PTMs remains to be fully elucidated.
PTM Proteomics Infograph Functional Diversification

PTMs play a critical role in cellular responsiveness and adaptation. Their predominantly reversible nature allows a precise spatial and temporal control over protein chemistry and function, enabling cells to quickly adapt to environmental changes. 

Dysregulation of PTMs is linked to a wide spectrum of diseases, from cancer and cardiovascular conditions to neurological disorders, neurodegenerative diseases, and infectious diseases. Recent research shows that diseases are primarily associated with phosphorylation (81%), methylation (9%), and ubiquitination (4%) among PTM types.  However, it is important to note that these distributions may reflect the current research focus rather than the definitive role of PTM types in disease pathology. These associations are frequently observed in neurological disorders, breast diseases, and blood-related conditions. Exploring PTMs in the context of disease remains a dynamic area of investigation, shedding light on their nuanced roles in various health conditions.

Comprehensive proteome-wide analysis methods enable systematic exploration of PTMs in health and disease. Understanding the role of PTMs in disease onset and progression is complex and requires a diverse toolset of techniques. Nowadays, most techniques require pre-processing of the specimen, such as immunoaffinity enrichment or protease digestion. Mass spectrometry (MS) is the most widely used approach for PTM site discovery, offering comprehensive qualitative and quantitative analyses. However, reliance on protein digestion to generate analyzable peptides presents certain limitations and challenges.

A less well-known but very successful approach is the use of antibody microarrays for a combined protein and PTM analysis. scioDiscover, a microarray-based platform by Sciomics, enables the simultaneous analysis of more than 1400 proteins, providing comprehensive insights into protein changes – without prior protein digestion, fractionation or protein-depletion and using only small sample amounts. This protein abundance profiling can be combined with a simultaneous profiling of phosphorylation status, covering serine, tyrosine, and threonine phosphorylation of all profiled proteins. The combined protein and phosphorylation-status analysis platform scioPhospho yields robust data and allows direct comparison of protein abundance and PTM-status.

Phospho-proteome analysis has already proven its scientific value in multiple studies. Recently, scioPhospho was applied in finding a solution for cyclosporine-A (CsA)-induced anemia and kidney toxicity. CsA is an immunosuppressant and standard of care in organ transplantation, acting through inhibition of the phosphatase calcineurin. Even though CsA’s benefits are substantial, CsA may exert short- and long-term adverse effects on the kidney. Long-term CsA treatment is known to cause renal vascular constriction, occlusive afferent arteriolopathy, nephron ischemia, and striped tubular-interstitial fibrosis. Therefore, loss of kidney function may outweigh the benefits of CsA. The pathophysiology of CsA-induced kidney injury is not completely understood, but hypoxia of nephron segments likely is involved. In their study ‘Daprodustat prevents cyclosporine-A-mediated anemia and peritubular capillary loss’, Labes et al. investigated the effect of CsA on the phospho-proteome in the kidney and identified a promising combinatorial approach for alleviating both CsA-induced anemia and kidney toxicity. 

Daprodustat, a selective inhibitor of the prolyl-hydroxylase domain, has emerged as an erythropoiesis-stimulating agent, based on its ability to upregulate hypoxia-inducible factor (HIF), and thus, erythropoietin (EPO). In the study, mice were treated with CsA, Daprodustat, or in combination for up to eight weeks.  CsA-induced kidney injury was assessed in the mouse model by plasma creatinine, routine histology, and kidney injury markers. Daprodustat or Daprodustat/CsA had no impact on plasma creatinine. However, following CsA treatment, focal tubular atrophy and interstitial fibrosis were observed. The histological findings, indicating CsA-induced kidney injury, were supported by multiple molecular markers, including KIM-1, NGAL, dickkopf-3 and vimentin. 

Since CsA acts as phosphatase inhibitor, alterations in protein phosphorylation levels are supposed to be involved in CsA-induced kidney injury. For in-depth analysis of the phospho-proteome, the scioPhospho platform was used to analyze proteins involved in tissue injury and repair, including T-cell activation, cell cycle regulation, oxidative stress response, cell adhesion, apoptosis, and angiogenesis. CsA treatment changed the expression of 79 and the phosphorylation of 86 proteins. Interestingly, CsA downregulated both expression and phosphorylation of mTOR. Daprodustat alone had a limited effect, with altered phosphorylation levels of only two proteins. In combination, Daprodustat abolished the effect of CsA on protein phosphorylation, as only six of previously 86 proteins were differentially phosphorylated. Daprodustat had no significant effect on CsA-induced phospho-mTOR depression, and therefore, it appears unlikely that Daprodustat restored angiogenesis through mTOR. However, Daprodustat reversed the effect of CsA on phospho-PRKAR2A, another key regulator of angiogenesis. In conclusion, microarray-based phospho-proteomics revealed phospho-PRKAR2A as a potential prognostic tissue marker in CsA toxicity. Daprodustat abolished the changes in phosphorylation induced by CsA and this suppression of broad phosphorylation changes was involved in the protective action of Daprodustat after CsA treatment. Thus, by leveraging scioPhospho, the potential of Daprodustat as a promissing intervention to counteract CsA-induced kidney injury could be revealed.

Beyond phosphorylation, the antibody microarray technique provides a flexible platform which can be adapted to analyze a range of PTM types. With the scioUbi platform, Sciomics offers protein profiling alongside ubiquitination analysis. In recent years, Sciomics has dedicated significant efforts to developing and optimizing platforms for combined protein and PTM discovery, including acetylation and methylation analysis, with a focus on asymmetric and symmetric dimethylation of arginine. This fast and cost-effective analysis of various PTMs, coupled with protein level data, facilitates the profiling and identification of new PTM signatures in health and disease. Such insights into underlying biological mechanisms might not only identify potential PTM protein biomarkers but also reveal promising therapeutic targets.

Labes, R., Brinkmann, L., Kulow, V. A., Roegner, K., Mathia, S., Balcerek, B., Persson, P. B., Rosenberger, C., & Fähling, M. (2022). Daprodustat prevents cyclosporine-A-mediated anemia and peritubular capillary loss. Kidney international, 102(4), 750–765. https://doi.org/10.1016/j.kint.2022.04.025

Published: 19 June 2024

 

Publications using scioPhospho

Article | Prevention of anemia and peritubular capillary loss

| October 2022 | Dapr. protects against induced kidney toxicity and anemia by preventing protein phosphorylation changes, preserving microcirculation, and maintaining hemoglobin levels.
Sciomics contributed to this study by analyzing mouse kidney samples using the scioPhospho proteomic assay.

Labes, R., Brinkmann, L. , Kulow, V.A., et al. Dapr. prevents cyclosp.-A-mediated anemia and peritubular capillary loss. Kidney Int. 2022;102(4):750-765.

Article | Protection from ischemia reperfusion injury-induced inflammation and fibrosis in C5aR2-deficient mice

| June 2018 | Complement component 5a receptor 2 (C5AR2) deficient mice are protected from inflammation and fibrosis in response to ischemia reperfusion injury. Our scioPhospho platform supported scientists at the Medical School Hannover to elucidate the underlying mechanism. 

Thorenz, A., Derlin, K., Schröder, C., et al. 2018 Enhanced activation of interleukin-10, heme oxygenase-1, and AKT in C5aR2-deficient mice is associated with protection from ischemia reperfusion injury–induced inflammation and fibrosis. Kidney International 94, 741–755.

Testimonials phosphorylation analysis

Dr. Bettina Grötsch

Department of Medicine 3 - Rheumatology and Immunology, University Clinics Erlangen, Erlangen, Germany

"Our lab is working with human osteoclast that were isolated and differentiated from human peripheral blood. Therefore, we were quite limited in cell numbers and protein content for our planned Phosphoprotein analyses. Nonetheless, Sciomics and especially Camille Lowy helped us a lot to test and improve our protein isolation techniques to achieve some interesting phosphoproteome data in human osteoclasts. We were really impressed by the detailed data report and their willingness to discuss and reanalyze the data repeatedly until we got the best results out of our measurements. Even several month after their first report, we could ask for help and suggestions with an immediate reply.  The phosphoproteome analyses completed our preliminary project data and are now published in Frontiers Immunology.  Altogether, we were very happy with the service of Sciomics and special thanks to Camille Lowy and Christoph Schröder who guided us through the whole process."

Product: scioPhospho
Publication: Grötsch B. et al. 2022 Front. Immunol. 13:958974 

Dr. Leonardo Guasti

Centre for Endocrinology, William Harvey Research Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom

"We were satisfied with the service provided by Sciomics back in 2017, so when it came to study a newly discovered secreted protein, for which little biology is known, we decided to engage again with them. Indeed, the phosphorylation and protein expression level data provided by Sciomics was of great help in assessing modulation of pathways initiated by adrenal cortex cells treated with this protein. Crucially, the Sciomics team actively engaged with us during both the planning of the experiments and subsequently in data analysis. This comprehensive data set has truly put us in the right path to dig into the role of a newly discovered protein in adrenocortical pathobiology."

Product: scioPhospho

 

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