Discovering protein degradation through bioinformatics

29th June 2022
Posted by: Breige McBride
Category: Uncategorised

The degradation of proteins is a crucial regulatory mechanism affecting the availability of structural proteins, enzymes, and signalling factors in all living organisms. While the production of proteins has been extensively studied by both academia and industry, protein degradation is currently emerging as the other half of this equation. In addition, the targeted degradation of specific proteins offers a new paradigm in therapy. Omics technologies from RNA-sequencing to proteomics, supplemented with specific methods like PROTAC are increasingly employed to shed light on this field. Analysis of such complex data sets brings new challenges to bioinformatics, and at Fios Genomics we offer cutting-edge solutions to these.

What is protein degradation?

Protein degradation is the biological process of breaking down proteins and cells can achieve it via multiple, independent mechanisms. Proteolytic enzymes cleave proteins at specific sites to create peptides, while peptidases break these chunks down to even smaller components as observed during digestion. This can occur both in the extracellular environment such as the gastrointestinal tract of animals, or within cells inside lysosomes. Cells also operate the proteasome, a complex machinery that degrades specific proteins that target it using ubiquitin marks. Ubiquitin mediated proteasomal degradation is a key process regulating the level of most proteins in a cell, and is an important target for drug discovery.

The role of ubiquitin in proteasomal function

For the proteasome to accept a target protein for degradation, it needs to be marked. Ubiquitin is a peptide the cell can attach to proteins, to act as such a mark. The ubiquitin monomer is a small peptide, but enzyme complexes often add it multiple times to a protein. This creates long, branching polyubiquitin chains that carry complex signalling information on the planned removal of their target. Ubiquitinylated proteins are transported to the proteasome where the complex chops them up to short peptide fragments. Peptidases then digest these peptides in the cytosol to create amino acids ready to act as building blocks for new proteins.

Picking targets for protein degradation

A cell may target a protein for degradation for multiple reasons. It often labels misfolded proteins for degradation directly after synthesis. It may reduce enzyme numbers dynamically to maintain the desired steady state of key metabolites. The cell can also dismantle structural proteins to free up building blocks for other proteins to be made. Most crucially, signalling factors are under tight expressional control to manage the delicate balance of pathway activities in the cell.

Proteins in key points of signalling pathways often exist in a steady state of constant production and degradation to dynamically control their levels and thus pathway activation. An example of this is beta-catenin, the key factor controlling the Wnt signalling pathway. A resting cell constantly produces beta-catenin. However, at the same time it also constantly degrades it, leaving a low concentration of this factor in the cell. Once Wnt is activated, the cell no longer targets beta-catenin for degradation while its synthesis remains unchanged. This leads to the rapid accumulation of the factor, launching a chain of events throughout the cell.

E3 ligases: the gatekeepers of the proteasome

The attachment of ubiquitin onto targets is a multi-step process. First, the E1 ubiquitin activating enzyme attaches to the E2 ubiquitin conjugating factor. This complex can transfer a ubiquitin molecule to a target, but is not able to select a target. E3 ligases are the specific targeting proteins that direct the E1-E2 complex to the appropriate protein it needs to mark for removal. This difference in roles is evident in the diversity of the members of this protein group; while we only know one major E1 enzyme in humans, an estimated 1000 different E3 ligases are necessary for targeting. This means that a notable proportion of the total human proteome is E3 ligases.

These factors represent a huge and largely untapped resource for therapeutic approaches. By fine tuning the activity of specific E3 ligases we can reprogram cells to remove harmful factors, change their metabolism, or reset malfunctioning pathways. Therefore, novel therapies may achieve their aims using the naturally available, targetable degradation machinery using ubiquitin ligases and the proteasome.

OMICS approaches for understanding protein degradation

The advent of high throughput technologies paved the way for the cell-wide mapping of protein degradation systems. Researchers are using DNA sequencing to map potential genes involved in the process. Today, databases that collect mutations in E3 ligases are available and we can use them to explain various malignancies. Meanwhile, RNA sequencing elucidates expression levels of members of ubiquitinylation pathways. Researchers use this approach for everything from cancer research to developing novel wheat and strawberry varieties.

However, the most accurate representation of protein degradation comes from proteomics experiments. Here, not only the speed of synthesis and availability, but the speed of removal can also be directly quantified. Using different incubation times, pulse-chase labelling, or SILAC experiments, protein-level omics data has been instrumental in understanding degradation mechanisms.

Going a step further, multi-omics approaches capture protein turnover on various levels of cellular function. Researchers are using such a combined approach to uncover how the mutant p53 radically upsets proteasomal function. They found that it leads to a cell-wide rebalancing protein homeostasis, culminating in the emergence of cancer.

Harnessing protein degradation for cancer therapy

The latest advancements in the field are methods to not only trace, but to actively control protein degradation pathways. The first protein degrader drug was an inadvertent discovery that led to a long and tragic clinical history. Researchers found Thalidomide to attach specific proteins to E3 ligases thus targeting them for proteasomal degradation. Once they uncovered the mechanism of action, the logic behind it was used to create a novel class of therapeutic agents that expanded the druggable proteome. Quantitative clinical proteomics coupled with pharmacodynamic monitoring led to the development and reuse of drugs like lenalidomide in the treatment of multiple myeloma. Other factors previously thought undruggable are now active therapeutic targets via the built-in ubiquitin-mediated specific degradation pathway existing in every cell.

PROTAC: engineering adapters to degrade proteins of interest

While a rich assortment of E3 ligases target cellular factors for removal, many proteins are not conveniently targetable. A novel technique aims to adapt ubiquitin mediated proteasomal degradation for hard to access targets. The proteolysis targeting chimeric (PROTAC) technology mimics the function of E3 ligases and selects specific factors for cellular decomposition.

The technique works by creating adapters that bind the target protein on one end, and an active E3 ligase on the other end, thus pinpointing the target protein for proteasomal degradation. Contrary to chemo- or radiotherapy, this method specifically removes only a protein of interest, thus providing much needed specificity in cancer treatment. As a recent study demonstrated, a single warhead (foretinib) was sufficient to create multiple, isoform specific degraders in a human setting. As a readout, quantitative proteomics is not only able to measure the effectiveness of the removal, but also monitor the specificity of the process by confirming other proteins are not affected.

Bioinformatic analysis unlocks therapeutic potential

The techniques outlined here all rely on rigorous wet-lab experimenting followed by cutting-edge bioinformatic data analysis. At Fios, we have over a decade of experience in interrogating clinical data and uncovering key features for biomarker selection, method validation, or exploratory data analysis. We tailor our bioinformatics services to adapt to the latest trends to help you get the most out of large biomedical datasets. Contact us today to discuss bioinformatics analysis of protein degradation data, or any other biological data sets.

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Author: Mate Ravasz, Bioinformatician, Fios Genomics