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Papers by Marie-pierre Bousquet-dubouch

Embo Molecular Medicine, Apr 1, 2015

HAL (Le Centre pour la Communication Scientifique Directe), 2005

HAL (Le Centre pour la Communication Scientifique Directe), Sep 5, 2001

HAL (Le Centre pour la Communication Scientifique Directe), Jan 14, 2003

Alcoholysis catalysed by Candida antarctica lipase B in

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their ... more During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>...

Molecular & Cellular Proteomics, 2012

Molecular systems biology, 2015

In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin-proteas... more In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin-proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly 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 subtypes, 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....

PLOS ONE

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type... more A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic β cells, as this might facilitate autoantigen presentation by β cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in β cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the β5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by low concentrations of IL-1β stimulating proinsulin biosynthesis. These findings suggest that the β cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type... more A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic β cells, as this might facilitate autoantigen presentation by β cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in β cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rode...

Cellular and molecular life sciences : CMLS, 2018

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assembli... more The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) ≤ 30 nM and activating at (IDE) ≥ 30 nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S.

PROTEOMICS, 2016

The ubiquitin-proteasome pathway (UPP) plays a critical role in the degradation of proteins impli... more The ubiquitin-proteasome pathway (UPP) plays a critical role in the degradation of proteins implicated in cell cycle control, signal transduction, DNA damage response, apoptosis and immune response. Proteasome inhibitors can inhibit the growth of a broad spectrum of human cancer cells by altering the balance of intracellular proteins. However, the targets of these compounds in acute myeloid leukemia (AML) cells have not been fully characterized. Herein, we combined large-scale quantitative analysis by SILAC-MS and targeted quantitative proteomic analysis in order to identify proteins regulated upon proteasome inhibition in two AML cell lines displaying different stages of maturation: immature KG1a cells and mature U937 cells. Indepth data analysis enabled accurate quantification of more than 7000 proteins in these two cell lines. Several candidates were validated by selected reaction monitoring (SRM) measurements in a large number of samples. Despite the broad range of proteins known to be affected by proteasome inhibition, such as heat shock (HSP) and cell cycle proteins, our analysis identified new differentially regulated proteins, including IL-32, MORF family mortality factors and apoptosis inducing factor SIVA, a target of p53. It could explain why proteasome inhibitors induce stronger apoptotic responses in immature AML cells.

The kinetics of alcoholysis of methylpropionate and n-propanol catalyzed by Candida antarctica li... more The kinetics of alcoholysis of methylpropionate and n-propanol catalyzed by Candida antarctica lipase B supported onto silanized Chromosorb P was studied in a continuous solid/gas reactor. In this system the solid phase is composed of a packed enzymatic sample and is percolated by nitrogen as carrier gas, which simultaneously carries substrates to the enzyme while removing reaction products. In this reactor the thermodynamic activity of substrates and effectors can be perfectly adjusted allowing kinetics studies to be performed under different operating conditions. The kinetics obtained for alcoholysis were suggested to fit a Ping Pong Bi Bi mechanism with dead-end inhibition by the alcohol. The values of all apparent kinetic parameters were calculated and the apparent dissociation constant of enzyme for gaseous ester was found very low compared with the one obtained for liquid ester in organic medium, certainly due to the more efficient diffusion in the gaseous phase. The effect of water thermodynamic activity was also investigated. Water was found to act as a competitive inhibitor, with a higher inhibition constant than n-propanol. Thus alcoholysis of gaseous methylpropionate and n-propanol catalyzed by of Candida antarctica lipase B was found to obey the same kinetics mechanism than in other non conventional media such as organic liquid media and supercritical carbon dioxide, but with much higher affinity for the substrates.

Journal of Proteome Research, 2014

Molecular and Cellular Biology, 2014

The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initia... more The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3= poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway.

Journal of Proteome …, 2008

A 20S proteasome immuno-purification protocol was optimized for the analysis of samples containin... more A 20S proteasome immuno-purification protocol was optimized for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the 20S proteasome of adjacent normal and tumor colorectal cells arising ...

EMBO molecular medicine, 2015

Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (... more Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome-triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E-BP1 regulatory pathway which we found highly activated in primary cultures of α-SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E-BP1 pathway and the resultant synthesis of secreted proteins including IL-6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine-induced ...

Methods in Molecular Biology, 2008

The 20S proteasome is a multicatalytic protein complex, present in all eukaryotic cells, that pla... more The 20S proteasome is a multicatalytic protein complex, present in all eukaryotic cells, that plays a major role in intracellular protein degradation. In mammalian cells, this symmetrical cylindrical complex is composed of two copies each of seven different alpha and beta subunits arranged into four stacked rings (alpha(7)beta(7)beta(7)alpha(7)). Separation by two-dimensional (2D) gel electrophoresis of the human erythrocytes 20S proteasome subunits and mass spectrometry (MS) identification of all the observed spots reveal the presence of multiple isoforms for most of the subunits. These isoforms could correspond to protein variants and/or posttranslational modifications that may influence the 20S proteasome proteolytic activity. Their characterization is therefore important to establish the rules governing structure/activity relationships of the human 20S proteasome. This chapter describes the use of a combination of proteomic approaches to characterize the human 20S proteasome subunit isoforms separated by 2D gel electrophoresis. A "top-down" strategy was developed to determine by electrospray MS the molecular mass of the intact protein after its passive elution from the gel. Comparison of the experimental molecular mass to the theoretical one can reveal the presence of possible modifications. "Bottom-up" proteomic approaches are then performed and, after protein digestion, tandem MS analyses of the modified peptides allow the characterization and location of the modification. These methods are discussed for the study of the human erythrocytes 20S proteasome subunit isoforms.

EuPA Open Proteomics, 2014

Embo Molecular Medicine, Apr 1, 2015

HAL (Le Centre pour la Communication Scientifique Directe), 2005

HAL (Le Centre pour la Communication Scientifique Directe), Sep 5, 2001

HAL (Le Centre pour la Communication Scientifique Directe), Jan 14, 2003

Alcoholysis catalysed by Candida antarctica lipase B in

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their ... more During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>...

Molecular & Cellular Proteomics, 2012

Molecular systems biology, 2015

In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin-proteas... more In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin-proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly 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 subtypes, 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....

PLOS ONE

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type... more A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic β cells, as this might facilitate autoantigen presentation by β cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in β cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the β5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by low concentrations of IL-1β stimulating proinsulin biosynthesis. These findings suggest that the β cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type... more A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic β cells, as this might facilitate autoantigen presentation by β cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in β cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rode...

Cellular and molecular life sciences : CMLS, 2018

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assembli... more The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) ≤ 30 nM and activating at (IDE) ≥ 30 nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S.

PROTEOMICS, 2016

The ubiquitin-proteasome pathway (UPP) plays a critical role in the degradation of proteins impli... more The ubiquitin-proteasome pathway (UPP) plays a critical role in the degradation of proteins implicated in cell cycle control, signal transduction, DNA damage response, apoptosis and immune response. Proteasome inhibitors can inhibit the growth of a broad spectrum of human cancer cells by altering the balance of intracellular proteins. However, the targets of these compounds in acute myeloid leukemia (AML) cells have not been fully characterized. Herein, we combined large-scale quantitative analysis by SILAC-MS and targeted quantitative proteomic analysis in order to identify proteins regulated upon proteasome inhibition in two AML cell lines displaying different stages of maturation: immature KG1a cells and mature U937 cells. Indepth data analysis enabled accurate quantification of more than 7000 proteins in these two cell lines. Several candidates were validated by selected reaction monitoring (SRM) measurements in a large number of samples. Despite the broad range of proteins known to be affected by proteasome inhibition, such as heat shock (HSP) and cell cycle proteins, our analysis identified new differentially regulated proteins, including IL-32, MORF family mortality factors and apoptosis inducing factor SIVA, a target of p53. It could explain why proteasome inhibitors induce stronger apoptotic responses in immature AML cells.

The kinetics of alcoholysis of methylpropionate and n-propanol catalyzed by Candida antarctica li... more The kinetics of alcoholysis of methylpropionate and n-propanol catalyzed by Candida antarctica lipase B supported onto silanized Chromosorb P was studied in a continuous solid/gas reactor. In this system the solid phase is composed of a packed enzymatic sample and is percolated by nitrogen as carrier gas, which simultaneously carries substrates to the enzyme while removing reaction products. In this reactor the thermodynamic activity of substrates and effectors can be perfectly adjusted allowing kinetics studies to be performed under different operating conditions. The kinetics obtained for alcoholysis were suggested to fit a Ping Pong Bi Bi mechanism with dead-end inhibition by the alcohol. The values of all apparent kinetic parameters were calculated and the apparent dissociation constant of enzyme for gaseous ester was found very low compared with the one obtained for liquid ester in organic medium, certainly due to the more efficient diffusion in the gaseous phase. The effect of water thermodynamic activity was also investigated. Water was found to act as a competitive inhibitor, with a higher inhibition constant than n-propanol. Thus alcoholysis of gaseous methylpropionate and n-propanol catalyzed by of Candida antarctica lipase B was found to obey the same kinetics mechanism than in other non conventional media such as organic liquid media and supercritical carbon dioxide, but with much higher affinity for the substrates.

Journal of Proteome Research, 2014

Molecular and Cellular Biology, 2014

The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initia... more The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3= poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway.

Journal of Proteome …, 2008

A 20S proteasome immuno-purification protocol was optimized for the analysis of samples containin... more A 20S proteasome immuno-purification protocol was optimized for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the 20S proteasome of adjacent normal and tumor colorectal cells arising ...

EMBO molecular medicine, 2015

Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (... more Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome-triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E-BP1 regulatory pathway which we found highly activated in primary cultures of α-SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E-BP1 pathway and the resultant synthesis of secreted proteins including IL-6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine-induced ...

Methods in Molecular Biology, 2008

The 20S proteasome is a multicatalytic protein complex, present in all eukaryotic cells, that pla... more The 20S proteasome is a multicatalytic protein complex, present in all eukaryotic cells, that plays a major role in intracellular protein degradation. In mammalian cells, this symmetrical cylindrical complex is composed of two copies each of seven different alpha and beta subunits arranged into four stacked rings (alpha(7)beta(7)beta(7)alpha(7)). Separation by two-dimensional (2D) gel electrophoresis of the human erythrocytes 20S proteasome subunits and mass spectrometry (MS) identification of all the observed spots reveal the presence of multiple isoforms for most of the subunits. These isoforms could correspond to protein variants and/or posttranslational modifications that may influence the 20S proteasome proteolytic activity. Their characterization is therefore important to establish the rules governing structure/activity relationships of the human 20S proteasome. This chapter describes the use of a combination of proteomic approaches to characterize the human 20S proteasome subunit isoforms separated by 2D gel electrophoresis. A "top-down" strategy was developed to determine by electrospray MS the molecular mass of the intact protein after its passive elution from the gel. Comparison of the experimental molecular mass to the theoretical one can reveal the presence of possible modifications. "Bottom-up" proteomic approaches are then performed and, after protein digestion, tandem MS analyses of the modified peptides allow the characterization and location of the modification. These methods are discussed for the study of the human erythrocytes 20S proteasome subunit isoforms.

EuPA Open Proteomics, 2014