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Project Hyperion: Revolutionizing Disease Detection through Kinome Hyperphosphorylation Patterns

The Project Hyperion embarks on a visionary quest rooted in understanding kinome hyperphosphorylation patterns to redefine the landscape of early disease detection. At its core, this project delves into the intricate mechanisms of protein regulation and their implications in health and disease. The theoretical underpinnings of Project Hyperion are anchored in three key pillars: the significance of kinome hyperphosphorylation patterns, the fundamental workings of hyperphosphorylation on proteins, and the central role played by the kinome in orchestrating cell fates.

I. Kinome Hyperphosphorylation Patterns:

Kinome, a portmanteau of "kinase" and "genome," encompasses the entire complement of protein kinases within an organism. Protein kinases are vital cellular components that catalyze the phosphorylation of specific amino acid residues, primarily serine, threonine, and tyrosine, on target proteins. The intricate and context-specific patterns of these phosphorylation events are referred to as kinome hyperphosphorylation patterns. These patterns serve as dynamic molecular signatures that orchestrate intricate cellular processes, including signal transduction, cellular proliferation, differentiation, and apoptosis. Dysregulation of kinome phosphorylation patterns is increasingly recognized as a hallmark of various diseases, particularly cancer.

II. Hyperphosphorylation Mechanisms:

Hyperphosphorylation entails an excess of phosphorylation events on target proteins. Protein kinases add phosphate groups to target proteins, modulating their structure, function, and interactions. This dynamic post-translational modification can induce conformational changes, alter enzymatic activities, and dictate protein-protein interactions. Importantly, hyperphosphorylation can lead to the activation or inactivation of crucial signaling pathways, providing an intricate level of control over cellular behavior.

III. Kinome Regulation of Cell Fates:

The kinome plays a pivotal role in modulating cell fates by serving as a master regulator of cellular processes. Through phosphorylation of downstream effectors, kinases govern critical decisions in cell life, including proliferation, differentiation, and programmed cell death. Aberrant kinome activity, often stemming from hyperphosphorylation patterns, contributes to pathogenic cellular behaviors, such as uncontrolled growth, resistance to apoptosis, and metastasis. Thus, an in-depth understanding of kinome regulation offers profound insights into cellular physiology, disease etiology, and potential therapeutic avenues.

IV. Antibody-immobilization technologies applied to phospho-proteins profiling:

• As mentioned above, the entire set of proteins existing in a given cell of any kind of organism (bacteria, vegetal and animal) is called Proteome.
• Proteins can be classified in families based on their functions. There are structural proteins that maintain the cell-shape and functional proteins (enzymes) that regulate cell-division, proliferation, differentiation, and apoptosis.
• In order to work, proteins such as enzymes undergo modifications after synthesis (post-translational modification). Phosphorylation, the attachment of one or more phosphate groups to a protein chain, is one of the most important modifications and is executed by a class of functional proteins (enzymes) called kinases.
• The human cell has 518 different kinases. This sub-set of all functional proteins that are able to attach to the phosphate group (kinases) is generally referred to as Kinome.
• Phosphorylation is not permanent and the phosphate(s) group(s) can be removed by a class of enzymes named phosphatases.
• Since kinases are proteins (enzymes) their activity is also controlled by phosphorylation. In other words, for example, they can be phosphorylated (active) and in turn they phosphorylate additional proteins.
• For example, in a simple signaling pathway, an activation/inhibition signal from a biologically active molecule (hormone, neurotransmitter) or a stress signal is mediated via a cascade of consecutive phosphorylation. Once the first kinase, of a specific pathway, is active as a result of phosphorylation, it is able to phosphorylate other downstream members of the pathway. In this way, the signal is transmitted through the cell rapidly.
• Phosphorylation can regulate almost every property of a protein (including enzymes) and is involved in all fundamental cellular processes.
• Deregulation of the kinome activity usually leads to pathologic conditions and, in some cases, to cancer.
• Anti-cancer drug treatments targeting kinases or general chemotherapy can be initially highly effective in eliciting a clinical response, but progression to resistance ultimately occurs.
• This adaptive response involves reprogramming of the kinome to effectively bypass inhibition (or stress conditions) of the targeted kinases or proteins.
• It is imperative to delineate the complex network of phosphorylation-based kinase signaling for therapeutically applicable understanding of the functioning of the cell in a disease state.
• A promising approach to get a "snapshot" of the network of phosphorylation-based kinase signaling in normal and pathological conditions is to evaluate the phosphorylation status of the entire kinome (518 proteins) in both situations.
• The available technologies for kinome profiling of clinical specimens range from those based on arrays of phospho-specific antibodies (antibodies that can bind a specific protein only if it is phosphorylated) to approaches in which phospho-proteins are separated on gels or by chromatography, followed by mass spectrometry (MS).
• An alternative to these deficiencies could be an array of normal antibodies (not phospho-specific, to avoid cross-reactivity) for the entire kinome (518 antibodies already available) immobilized in single wells.
• The array of 518 antibodies is then incubated with all cell-proteins for a relatively short time, and the interaction between antibody and kinase is detected using an interdigital capacitor.
• A wash step will remove the unbound proteins from each single well.
• Then, to evaluate the phosphorylation status, an enzymatic reaction is used. These enzymes (phosphatases) can remove phosphate molecules from any kind of substrate, including proteins.
• The molecules of free phosphate will remain trapped in every single well, thanks to the fact that the reaction takes place in isolated wells in parallel.
• Then, the free phosphate molecules will be detected with an optical system because a reagent that will change the color of the solution (or alternatively will release UV or visible light) will be added.
• Steps 3 and 4 can also be done simultaneously.

V. Methods for Detecting Protein Phosphorylation:

• Kinase Activity Assays: Kinase activity within a biological sample is commonly measured in vitro by incubating the immunoprecipitated kinase with an exogenous substrate in the presence of ATP.

• Phospho-Specific Antibody: Kinase activity within a biological sample is commonly measured in vitro by incubating the immunoprecipitated kinase with an exogenous substrate in the presence of ATP.

• Enzyme-Linked Immunosorbent Assay (ELISA): The target protein, either purified or as a component in a complex heterogeneous sample such as a cell lysate, is bound to the antibody-coated plate. A detection antibody specific for the phosphorylation site to be analyzed is then added.

• Intracellular Flow Cytometry: Flow cytometry uses a laser to excite the fluorochrome used for antibody detection. It allows for rapid, quantitative, single cell analysis. Labeled phospho-antibodies are required.

• Immunohistochemistry (IHC): IHC refers to protein detection in intact tissue sections. Like flow cytometry, these techniques allow for the assessment of multiple proteins within a cell or tissue provided that adequate attention is given to avoid overlapping fluorescence spectra or color. Few available colors and labeled phospho-antibodies are required.

• Mass Spectrometry: Large-scale phospho-protein analysis in complex protein mixtures involves identification of phospho-proteins and phosphopeptides and sequencing of the phosphorylated residues. No antibodies are required but a large amount of samples is required.

• Phospho-Protein Multiplex Assays: In general, these involve the use of phospho-specific antibodies and include microplate-based and membrane-based detection formats. Cross-reactions problems are present, and a low number of proteins detected simultaneously.

VI. Technology developed by our group to immobilize proteins on synthetic surfaces

A laser is used to realize wells on top of a surface with specific size. Subsequently proteins are immobilized inside each well.

Test proteins (not phosphorylated) are revealed with fluorescent light. 1p, 2p and 5 indicate well size realized with laser technology.

VII. Overview of the system to construct and use the biochip

Two main machineries need to be realized:

• Laser system to realize microwells and immobilize antibodies
• System for sample hybridization and biochip reading

References:

1. Manning, G., Whyte, D. B., Martinez, R., Hunter, T., & Sudarsanam, S. (2002). The protein kinase complement of the human genome. Science, 298(5600), 1912-1934.
2. Pearce, L. R., Komander, D., & Alessi, D. R. (2010). The nuts and bolts of AGC protein kinases. Nature Reviews Molecular Cell Biology, 11(1), 9-22.
3. Johnson, L. N., & Lewis, R. J. (2001). Structural basis for control by phosphorylation. Chemical Reviews, 101(8), 2209-2242.
4. Manning, B. D., & Cantley, L. C. (2007). AKT/PKB signaling: navigating downstream. Cell, 129(7), 1261-1274.
5. Ubersax, J., Ferrell Jr, J. Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8, 530–541 (2007). https://doi.org/10.1038/nrm2203
6. E. Sarantopoulou et al. Protein immobilization and detection on laser processed polystyrene surfaces. Journal of Applied Physicsvol. 110 issue 6(2011)pp: 064309
7. Citation: Georgas, A.; Nestoras, L.; Kanaris, A.I.; Angelopoulos, S.; Ferraro, A.; Hristoforou, E. Packaging and Optimization of a Capacitive Biosensor and Its Readout Circuit. Sensors 2023, 23, 765. https://doi.org/10.3390/s23020765