Our team focuses on the crosstalk between Ub and UbL proteins in the proteotoxic and genotoxic stress responses. Nevertheless, we collaborate with many public and private laboratories working with distinct biological models or diseases, given the importance of protein modification by distinct members of the ubiquitin family in multiple physiologic or pathologic events.

Our current research lines aim at …

Studying the regulation of the ubiquitin-code and in particular the role of ubiquitin enzymes

Distinct ubiquitin chains are important to a large diversity of cellular events. Having the correct ubiquitin chains at a given moment would be crucial to respond to a particular situation. Our projects tackle one main question: can we correct a process that is affected by the absence of particular chain types? There are several ways to develop a phenotype that do not contain the correct chain types to link with a process. Generally speaking, we can consider that it is the consequence of the excessive action of one or a group of de-ubiquitylating enzymes, or the lack or excess of activity of particular E3s. How can these enzymes sense situations where they have to stop or accelerate their activity? These simplistic hypotheses cannot ignore that regulation at the level of transcription is also possible. To explore these questions, we used MCL cells that are resistant (ZBR) or sensitive (Z138) to BTZ. In the BZT-resistant phenotype, we know that there are both a reduction of chains that are required to drive proteasome degradation and an increase of chains driving autophagy-mediated proteolysis. We are performing deep MS analysis using new tools and strategies in order to figure out how chain remodelling can be regulated. Identifying enzymes, substrates and cofactors implicated in chain remodelling will provide information on mechanistic aspects of regulation of chain remodelling under proteotoxic stress conditions.

We have started this project by exploring the role of TRIM and UBR enzymes in ubiquitin chain remodelling using multiple strategies including chemical inhibition through proteolysis targeting chimeras (PROTACs) or siRNA. In addition to the effects on chain composition, we are also analysing their role on proteaphagy and cell survival in various BTZ resistant MCL and MM cell lines. This work is performed in collaboration with Gael Roué, Brigitte Sola and Cedric-Olivier Turrin.

My project aims to explore ubiquitin chain composition and remodelling in BTZ-resistant and sensitive MCL cells. For this purpose, different molecular tools (TUBES and nanobodies) have been optimized to be used in mass spectrometry (MS) analysis. Based on previous MS analyses we identified ubiquitin ligases (E3) and deubiquitylating enzymes (DUBs) which could potentially be implicated in the development of the BTZ-resistant phenotype. I am also exploring the role of these enzymes in determining ubiquitin chain architecture, in activating proteaphagy and in the acquisition of BTZ-resistant phenotype. E3 and DUBs will be targeted using pharmacological approaches and different silencing strategies to observe the impact of these enzymes on these molecular and clinically relevant events. We expect to bring insights into the molecular basis of this disease. 

Maria Gonzalez Santamarta

UbiCODE PhD student

Exploring the UPS-ALS crosstalk under genotoxic and proteotoxic stress conditions

We use several treatments that activate the genotoxic and proteotoxic stress response using various biologic models in collaboration with distinct public and private laboratories in order to explore the UPS-ALS crosstalk. Some of the stimuli we study in our laboratory block processes by clinically relevant chemical inhibitors.  This is the case of the proteasome inhibitors that are used for the treatment of Mantle Cell Lymphoma (MCL) or Multiple Myeloma (MM) and generate proteotoxic stress.

Mantle Cell Lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma that accounts for 5-10% of all lymphomas. This disease is associated with poor prognosis, with a median survival of 5-7 years, and no standard treatment. The therapeutic options being limited, new approaches for the treatment of this lymphoma are urgently needed. The reversible proteasome inhibitor Bortezomib (BTZ) is an excellent example of this new class of agents, where in vitro studies have been immediately applied to the clinical practice. BTZ was first reported to induce a high response rate in multiple myeloma (MM) patients, leading to its approval by the Food and Drug Administration in 2003. BTZ was later approved for the treatment of MCL patients who had received at least one prior therapy. However, some patients are still developing resistance to BTZ, possibly due to alterations in a subset of the many signalling pathways affected by this drug. In this scenario, the ex vivo evaluation of chemosensitivity may be useful for the prediction of clinical response. In the last years, it has been reported for the first time that BTZ was active in preclinical models of MCL cells as single agent, and in combination with other chemotherapeutic treatments. Despite promising results of these BTZ-based combinations in various Phase II clinical trial, none of them has been introduced in Phase III trial so far, highlighting a lack of efficacy or toxicity at long term for most of them, as well as the gap existing between preclinical and clinical efficacy. Thus, the identification of response/resistance mechanisms to this drug and the design of new combination therapies are vital to optimize the therapeutic effect of BTZ.

The inhibition of the proteasome activity by BTZ modulates the degradation of poly-ubiquitylated proteins involved in signalling pathways essential for tumour progression, such as the control of programmed cell death, cell cycle, inflammation and immune surveillance. BTZ induces proteotoxic stress leading to the accumulation (due to the inability to be degraded by the proteasome) of many intracellular factors potentially implicated in the response or resistance to this drug. To decipher the cellular functions affected in cells resistant to BTZ Team 1 isolated the ubiquitin proteome (ubiquitome) associated to BTZ-resistant (ZBR) vs.BTZ-sensitive (Z138) MCL cells using a ubiquitin-traps (TUBEs)-mass spectrometry (MS) approach (unpublished). BTZ-resistant MCL cell lines were generated by our collaborator Gael Roué by exposing cells to increasing doses of BTZ going from 10 to 30 nM during 5 months until they become resistant. These models of BTZ-acquired resistance were selected to compare the proteome from parental versus BTZ resistant cells and better identify differences. Gene ontology analysis of enriched proteins (263) showed distinct functional categories affected in both celllines. In the top of the list, components of the Ubiquitin-Proteasome System (UPS) are dramatically reduced in ZBR cells while cellular factors implicated in the Autophagy-Lysosome System (ALS) were increased. These observations were also confirmed in other BTZ resistant cell lines such as JBR which were derived from the bortezomib-sensitive, TP53mutant cell line Jeko-1. The use of distinct inhibitors of autophagy and biochemical/cellular approaches allowed us to demonstrate that in ZBR, JBR and other BTZ resistant cells, proteasome subunits are continuously degraded by autophagy by a mechanism named proteaphagy underling important mechanisms of crosstalk between these two proteolytic systems.

I am currently in the last year of my PhD and my project aims at understanding the role of protein degradation in the chemoresistance using Mantle Cell Lymphoma as a main model. The proteasome inhibitor bortezomib is efficient but resistance is observed in patients. Some patients also show natural resistance, and the biologic mechanisms underlying this resistance are not fully understood. Over these three years of investigation, we have accumulated evidence indicating that string exchanges between the Ubiquitin Proteasome System and Autophagy are generated, in particular proteaphagy where the proteasome is permanently degraded by autophagy in bortezomib resistant Mantle Cell Lymphoma cells. Pharmacological inhibition of proteaphagy has revealed promising to sensitize bortezomib resistant cells to treatment. As part of my PhD, I have also developed new molecular traps to explore the complex regulation of selective autophagy events. This technology enables to isolate and purify key proteins in an endogenous manner, without artefacts.

Grégoire Quinet

Phd student