Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

International audienceIn Brief 20S proteasomes are very heterogeneous protein complexes involved in many cellular processes. In the present study, we combined an MRM-based assay with the production and purification of entire SILAC labelled pro-teasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte-derived stem cells (ADSCs) amplified under various conditions highlights an increased expression of immunoproteasome when this type of cell is primed with IFN␥ or amplified in a 20% O 2 environment. Graphical Abstract Highlights • Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. • Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. • Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O 2 levels might be causal for change in cells differentiation capacity. • The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity

FAN1 controls mismatch repair complex assembly via MLH1 retention to stabilize CAG repeat expansion in Huntington's disease.

CAG repeat expansion in the HTT gene drives Huntington's disease (HD) pathogenesis and is modulated by DNA damage repair pathways. In this context, the interaction between FAN1, a DNA-structure-specific nuclease, and MLH1, member of the DNA mismatch repair pathway (MMR), is not defined. Here, we identify a highly conserved SPYF motif at the N terminus of FAN1 that binds to MLH1. Our data support a model where FAN1 has two distinct functions to stabilize CAG repeats. On one hand, it binds MLH1 to restrict its recruitment by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG repeat expansion. On the other hand, it promotes accurate repair via its nuclease activity. These data highlight a potential avenue for HD therapeutics in attenuating somatic expansion

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus

Développement de méthodes de spectrométrie de masse pour l'étude de la dynamique des complexes de protéasome : application aux cellules stromales/souches mésenchymateuses humaines

Les protéines sont des biomolécules majeures dans l'ensemble des processus cellulaires. La régulation fine de leur concentration est donc essentielle dans la survie et le développement d'une cellule et peut avoir lieu en amont ou en aval de leur synthèse. Un des systèmes cellulaires capable de dégrader les protéines est le système Ubiquitine-Protéasome. Le protéasome 26S, élément central de ce système, est composé d'un cœur catalytique, le protéasome 20S et d'une particule activatrice, le régulateur 19S. Le protéasome 20S est un complexe multiprotéique présentant une très grande diversité structurale de par l'incorporation d'isoformes de certaines sous-unités. De plus, le protéasome 20S peut être retrouvé sous forme libre ou associé avec un ou deux régulateurs 19S dont la fonction est d'augmenter l'activité protéolytique mais aussi de recruter des substrats à dégrader via la reconnaissance de leur chaîne d'ubiquitine. D'autres activateurs du protéasome 20S ont été décrits, PA28aß, PA28ƴ et PA200, mais fonctionnent de manière ubiquitine-indépendante et leurs rôles cellulaires et leurs substrats sont moins étudiés que ceux du régulateur 19S. Le but de ce travail de thèse a été de développer des méthodes originales de spectrométrie de masse permettant une meilleure compréhension du fonctionnement de ces complexes et d'appliquer ces méthodes aux cellules souches/stromales mésenchymateuses du tissu adipeux humain (ADSC), actuellement très étudiées pour leurs potentiels thérapeutiques. Lors d'une première partie, nous avons développé une méthode de spectrométrie de masse ciblée permettant de déterminer de façon sensible et précise la quantité absolue de protéasome 20S total ainsi que la stœchiométrie des différentes formes de protéasome 20S actuellement décrits. Cette méthode a été appliquée à des cellules en culture, à des tissus humains, mais également à des cellules primaires de type ADSC. Cette nouvelle technologie nous a permis de montrer une modulation de la quantité de protéasome 20S en fonction des conditions de culture utilisées. Ces résultats suggèrent que le protéasome 20S pourrait être un indicateur à suivre lors de la production de ces cellules à des fins cliniques. Dans une deuxième partie de cette thèse, nous sommes intéressés à la caractérisation de FAM192A, une protéine de fonction jusqu'alors inconnue que nous avons identifiée dans l'interactome du protéasome 20S nucléaire, et plus particulièrement comme interagissant avec le régulateur PA28ƴ. Nos résultats ont montré que FAM192A est phosphorylée, module la localisation subcellulaire de PA28ƴ et son rôle dans les corps nucléaires de Cajal, et augmente la capacité d'association de PA28ƴ avec le protéasome 20S. Enfin, dans une dernière partie, nous avons mis au point une méthode basée sur une analyse par spectrométrie de masse quantitative multiplexée permettant l'identification de substrats dégradés par le protéasome 20S associé au régulateur PA28ƴ. Cette approche permet d'identifier des substrats sans apriori en une unique analyse, ce qui la distingue des études déjà publiées.Proteins are the main biomolecules controlling cellular biological processes. The precise control of their concentration is essential for the survival and development of cells. This regulation can be done upstream of their synthesis (modulation of gene expression), or through degradation by specific cellular pathways such as the Ubiquitin-Proteasome System. The 26S proteasome, which plays a central role in this pathway, is composed of a catalytic core, the 20S proteasome, and an associated regulator, the 19S regulator. The 20S proteasome is a multi-protein complex presenting a high structural diversity resulting from the combination of specific subunits in proportions that vary in different cell types. Other regulators are described as modulator of proteasomal activity, such as PA28aß, PA28ƴ and PA200, but their substrate recognition is ubiquitin-independent and their mechanism are less described compared to the 19S regulator. The aim of this thesis was to develop original mass spectrometry methods to characterize the proteasome subunit associations and apply these methods to the study of human adipose-derived mesenchymal stromal/stem cells (ADSC), currently appreciated and studied for their therapeutic potentials. In the first part of this thesis, we developed a mass spectrometry-based strategy to determine absolute quantity of total 20S proteasome and the different described 20S proteasome forms stoichiometry. With this approach, we were able to detect all the different forms of the 20S proteasome described to date. This method was then applied to several human tissues and to ADSC. This method permitted us to measure the quantity and proportion variation of the different 20S proteasome forms depending on the cell culture conditions. This results suggest that 20S proteasome could be a potential marker to measure during cell production for clinical purposes. In a second part, we focused on FAM192A, a functionally undescribed protein identified in nuclear 20S proteasome interactome and more specifically with the PA28ƴ. Our results showed that FAM192A is phosphorylated, modulates PA28ƴ subcellular localization and its role in Cajal bodies but also increase its capacity to interact with the 20S proteasome. Finally, in the last part, we developed a method to identify the proteasome substrates that are degraded by 20S proteasome associated with the PA28ƴ regulator. In contrast to classical approaches used to identify proteasome substrates, this multiplexed mass spectrometry analysis allowed us to determine which proteins are degraded in a regulator-dependent fashion without a priori

Mass spectrometry methods development to study proteasome complexes dynamic : application to human mesenchymal stromal/stem cells

Les protéines sont des biomolécules majeures dans l'ensemble des processus cellulaires. La régulation fine de leur concentration est donc essentielle dans la survie et le développement d'une cellule et peut avoir lieu en amont ou en aval de leur synthèse. Un des systèmes cellulaires capable de dégrader les protéines est le système Ubiquitine-Protéasome. Le protéasome 26S, élément central de ce système, est composé d'un cœur catalytique, le protéasome 20S et d'une particule activatrice, le régulateur 19S. Le protéasome 20S est un complexe multiprotéique présentant une très grande diversité structurale de par l'incorporation d'isoformes de certaines sous-unités. De plus, le protéasome 20S peut être retrouvé sous forme libre ou associé avec un ou deux régulateurs 19S dont la fonction est d'augmenter l'activité protéolytique mais aussi de recruter des substrats à dégrader via la reconnaissance de leur chaîne d'ubiquitine. D'autres activateurs du protéasome 20S ont été décrits, PA28aß, PA28ƴ et PA200, mais fonctionnent de manière ubiquitine-indépendante et leurs rôles cellulaires et leurs substrats sont moins étudiés que ceux du régulateur 19S. Le but de ce travail de thèse a été de développer des méthodes originales de spectrométrie de masse permettant une meilleure compréhension du fonctionnement de ces complexes et d'appliquer ces méthodes aux cellules souches/stromales mésenchymateuses du tissu adipeux humain (ADSC), actuellement très étudiées pour leurs potentiels thérapeutiques. Lors d'une première partie, nous avons développé une méthode de spectrométrie de masse ciblée permettant de déterminer de façon sensible et précise la quantité absolue de protéasome 20S total ainsi que la stœchiométrie des différentes formes de protéasome 20S actuellement décrits. Cette méthode a été appliquée à des cellules en culture, à des tissus humains, mais également à des cellules primaires de type ADSC. [...]Proteins are the main biomolecules controlling cellular biological processes. The precise control of their concentration is essential for the survival and development of cells. This regulation can be done upstream of their synthesis (modulation of gene expression), or through degradation by specific cellular pathways such as the Ubiquitin-Proteasome System. The 26S proteasome, which plays a central role in this pathway, is composed of a catalytic core, the 20S proteasome, and an associated regulator, the 19S regulator. The 20S proteasome is a multi-protein complex presenting a high structural diversity resulting from the combination of specific subunits in proportions that vary in different cell types. Other regulators are described as modulator of proteasomal activity, such as PA28aß, PA28ƴ and PA200, but their substrate recognition is ubiquitin-independent and their mechanism are less described compared to the 19S regulator. The aim of this thesis was to develop original mass spectrometry methods to characterize the proteasome subunit associations and apply these methods to the study of human adipose-derived mesenchymal stromal/stem cells (ADSC), currently appreciated and studied for their therapeutic potentials. In the first part of this thesis, we developed a mass spectrometry-based strategy to determine absolute quantity of total 20S proteasome and the different described 20S proteasome forms stoichiometry. With this approach, we were able to detect all the different forms of the 20S proteasome described to date. This method was then applied to several human tissues and to ADSC. This method permitted us to measure the quantity and proportion variation of the different 20S proteasome forms depending on the cell culture conditions. This results suggest that 20S proteasome could be a potential marker to measure during cell production for clinical purposes. In a second part, we focused on FAM192A, a functionally undescribed protein identified in nuclear 20S proteasome interactome and more specifically with the PA28ƴ. Our results showed that FAM192A is phosphorylated, modulates PA28ƴ subcellular localization and its role in Cajal bodies but also increase its capacity to interact with the 20S proteasome.[...

Comparison of label-free quantification methods for the determination of protein complexes subunits stoichiometry

Protein complexes are the main molecular machines that support all major cellular pathways and their in-depth characterization are essential to understand their functions. Determining the stoichiometry of the different subunits of a protein complex still remains challenging. Recently, many label-free quantitative proteomic approaches have been developed to study the composition of protein complexes. It is therefore of great interest to evaluate these different methods in a stoichiometry oriented objective. Here we compare the ability of four absolute quantitative label-free methods currently used in proteomic studies to determine the stoichiometry of a well-characterized protein complex, the 26S proteasome

Dynamics of cell and tissue growth acquired by means of 25 mm² to 10 cm² lensfree imaging

International audienceIn this paper, we discuss a new methodology based on lens-free imaging to perform wound healing assay with unprecedented statistics. Our video lens-free microscopy setup is a simple optical system featuring only a CMOS sensor and a semi coherent illumination system. Yet it is a powerful means for the real-time monitoring of cultivated cells. It presents several key advantages, e.g., integration into standard incubator, compatibility with standard cell culture protocol, simplicity and ease of use. It can perform the follow-up in a large field of view (25 mm2) of several crucial parameters during the culture of cells i.e. their motility, their proliferation rate or their death. Consequently the setup can gather large statistics both in space and time. But in the case of tissue growth experiments, the field of view of 25 mm2 remains not sufficient and results can be biased depending on the position of the device with respect to the recipient of the cell culture. Hence, to conduct exhaustive wound healing assay, here we propose to enlarge the field of view up to 10 cm2 through two different approaches. The first method consists in performing a scan of the cell culture by moving the source/sensor couple and then stitch the stack of images. The second is to make an acquisition by scanning with a line scan camera. The two approaches are compared in term of resolution, complexity and acquisition time. Next we have performed acquisitions of wound healing assay (keratinocytes HaCaT) both in real-time (25 mm2) and in final point (10 cm2) to assess the combination of these two complementary modalities. In the future, we aim at combining directly super wide field of view acquisitions (>10 cm2) with real time ability inside the incubator

Tailoring nanostructured lipid carriers for the delivery of protein antigens Physicochemical properties versus immunogenicity studies

International audienceNew vaccine formulations are still highly anticipated in the near-future to face incoming health challenges, such as emergence or reemergence of severe infectious diseases, immunosenescence associated with elderly or the spread of pathogens resistant to antibiotics. In particular, new nanoparticle-based adjuvants are promising for sub-unit vaccines in order to elicit potent and long lasting immune responses with a better control on their safety. In this context, an innovative delivery system of protein antigens has been designed based on the chemical grafting of the antigen onto the shell of Nanostructured Lipid Carriers (NLC). By using the well-known ovalbumin (OVA) as model of protein antigen, we have compared the immunogenicity properties in mice of different formulations of NLC grafted with OVA, by studying the influence of two main parameters the size (80 nm versus 120 nm) and the surface charge (anionic versus cationic). We have shown that all mice immunized with OVA delivered through NLC produced much higher antibody titers for all tested formulations as compared to that immunized with OVA or OVA formulated in Complete Freund Adjuvant (CFA, positive control). More interestingly, the 80 nm anionic lipid particles were the most efficient antigen carrier for eliciting higher humoral immune response, as well as cellular immune response characterized by a strong secretion of gamma interferon (IFN-gamma). These results associated with the demonstrated non-immunogenicity of the NLC carrier by itself open new avenues for the design of smart sub-unit vaccines containing properly engineered lipid nanoparticles which could stimulate or orient the immune system in a specific way

Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependent inter-ring crosstalks

International audienceHydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) is now common practice in structural biology. However, it is most of the time applied to rather small oligomeric complexes. Here, we report on the use of HDX-MS to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28αβ or PA28γ activators. Their solvent accessibility is analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirm the existence of allosteric differences between the std20S and i20S at the surface of the α-ring triggered from inside the catalytic β-ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modify solvent accessibility due to conformational changes of the β-rings. This work is not only a proof-of-concept that HDX-MS can be used to get structural insights on large multi-protein complexes in solution, it also demonstrates that the binding of the std20S or i20S subtype to any of its PA28 activator triggers allosteric changes that are specific to this 20S/PA28 pair

Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

Intracellular protein breakdown is mainly performed by the Ubiquitin-Proteasome System in eukaryotic cells. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. They thus exhibit a very high heterogeneity whose function still needs to be understood. Here, using a new developed method based on the combination of affinity purification and protein correlation profiling associated with high resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome sub-types, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be widened to unravel the dynamic and heterogeneous nature of many other biologically relevant molecular systems