Post-translational modifications (PTMs) of proteins are a major tool that the cell uses to monitor events and initiate appropriate responses to internal and external signals. While a protein is defined by its backbone of amino acid sequence, its function is often determined by...
Post-translational modifications (PTMs) of proteins are a major tool that the cell uses to monitor events and initiate appropriate responses to internal and external signals. While a protein is defined by its backbone of amino acid sequence, its function is often determined by PTMs, which specify stability, activity, or cellular localization. More than 200 types of PTMs are known to date, dramatically increasing the complexity of our genome and proteome1. Yet, although much is known about the chemistry of PTMs, we still lack understanding of their dynamics and regulation. Even a single modification may have a profound effect on cellular state1. How then, are multiple PTM signals integrated to achieve specific spatial and temporal control of the proteome at the molecular, cellular and organism level? How does PTM activity change with cellular differentiation and specialization, for example of immune cells? How does PTM activity change in diseases such as cancer? Deciphering currently unknown molecular mechanisms governing PTM regulation bears critical importance in understanding their role in homeostasis and disease.
For this ERC project we propose to use our state-of-the-art PTM profiling system combined with MS analysis, high-throughput imaging, molecular and biochemical approaches to tackle fundamental questions in ubiquitin and Ubl biology and tumor growth, including: Can a Ubl signature of the proteome report on its physiological state? Can the Ubl profile teach us about the molecular pathways that are misregulated in disease? What are the regulatory principles governing substrate specificity and recognition? How is the ubiquitin system involved in mediating host-tumor interactions? Can we reshape the cellular environment by controlling the enzymatic machinery? To address these challenging questions we will identify, validate and study the enzymatic machinery of the ubiquitin system as well as regulatory substrates affecting tumor growth, by following these aims:
Aim 1 â€“ Mapping the ubiquitin and ubiquitin-like modification landscape in cancer
Aim 2 â€“ Deciphering determinants regulating recognition and specificity of human E3 ligases
Aim 3 â€“ Elucidating ubiquitin-dependent mechanisms regulating tumor-microenvironment interactions
The three aims can each be carried out independently and in parallel, and each has the capacity to generate novel insights about the complex regulation of PTMs. However, the real power of our approach will be the combination of all three aims, which should endow us with a comprehensive view of the important and intricate regulatory principles involved in protein modifications and in the ubiquitin family in particular. It will shed new light on the interactions between Ubl proteins, the modifying enzymes and how these affect the downstream cellular network (Figure 2). Uncovering the PTM landscape in cancer should afford a new paradigm in understanding the molecular basis of the disease and identifying putative therapeutic targets
AIM 1 - Mapping the ubiquitin and ubiquitin-like modification landscape in cancer
Over the past decade high-throughput analyses have profoundly broadened our understanding of the processes underlying cancer development and progression but these have been primarily focused on the genetic and genomic levels. Changes in the proteome and the protein post-translational modification (PTMs) landscape remained relatively untouched. Partly this is because despite the clinical importance of PTMs in general, and ubiquitin-like modifications specifically, there are few analytical tools to analyze them. Our research focused on analyzing the PTM landscape in cancer where major focus has been given to Non-Small Cell Lung Cancer.
To enable unbiased study of PTMs in solid tumors extracted from patients we spent time and efforts to calibrate the PTM profiling protocol and the corresponding analysis pipeline. This calibration and optimization included isolating cells from tumorous tissues under conditions that preserve enzymatic activity and the ability to probe these reactivates by modification of absorbed proteins on the microarray. This development and improvements involved six months of work and were covered by my ERC grant. We then used this set up to study the changes in the ubiquitin and FAT10 modification landscape of human lung cancer cell lines and patients.
In the frame of my ERC grant we characterized the changes that occur in NSCLC. Using PTM profiling we found and were successful in identifying distinct PTM signatures in certain patients carrying mutations in KRAS and EGFR genes. Among the targets were components of E3-ligases and key regulators of cell cycle regulation and other relevant pathways which may be involved in regulating tumor growth. Specific targets will be followed up by biochemical and molecular analysis. Importantly, we provide a proof-of-concept for the ability to identify PTM-based signatures that are associated with unique clinical manifestations (PMID: 28809000; PMID:27229346).
Aim 2 â€“ Deciphering determinants regulating recognition and specificity of human E3
We hypothesize that the generating a library of catalytically-dead HECT E3 enzymes would allow us to compare the ubiquitination of cells expressing wildtype and mutant forms. We have started to set up the mutant library in order to generate a cassette for each member of the family (28 proteins). However, due to high copy number of chromosome in several cancer cell lines we did not manage to generate an endogenous tag or mutation. We are now preparing an exogenous library that would allow us to examine these mutant by overexpression in cancer cell lines. In parallel, we have set up a novel system to identify degradation products of cellular proteasomes from human cells. This system was recently published in Nature Biotech (PMID: 30346940) and all the relevant analysis pipeline was already set up. Our method examines the cellular profile based on observing proteins that were targeted for degradation in the â€˜garbage canâ€™ of cells, which are called â€˜proteasomesâ€™. Importantly, like in regular detective work, â€˜dumpster-divingâ€™ and examining the content of the garbage cans can teach us a lot about the state of cells and their regulation. Most proteomic techniques generate lists of all the proteins expressed in cells, but do not produce data about how or when these proteins are used. By focusing on degraded proteins, on the other hand, we are able to identify which proteins were recently used and which may be damaged and therefore sheds light on the major activities in the cell at a particular time. Our results show that proteasomal profiling is more sensitive than regular standard proteomic and identify relatively small changes in the cellular proteostasis. This system would allow us to determine the functional consequences of mutant HECT E3 in a very robust manner.
Aim 3 â€“Elucidating ubiquitin-dependent mechanisms regula
We have already accomplished several milestones in this project which would enable advancing the PTM profiling technology and developing it further for biomarker discovery.
In additon, we are elucidating novel insight into the role of the FAT10 in cancer-related inflammation, which may have therapeutic implications, due to its role in tumor mmicroenvironment
In addition, we have developed a novel methodology which allows us to examine degradation products in cells and tissues. Major achievemnts already reached are:
1. Establishment of an activity-based assay for PTM profiling in tumor samples and blood samples.
2. Developing a novel technology for Mass-Spectrometry analysis of Proteolytic Peptides (Nat Bio, 2018).
3. Licensing of FAT10 insight and know-how to an incubator interested in finding an inhibitor for FAT10 pathway for cancer therapy, based on our results.