The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, low concentrations of interleukin 1 ß.

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 ß cells, as this might facilitate autoantigen presentation by ß cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in ß 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 ß5i 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-1ß stimulating proinsulin biosynthesis. These findings suggest that the ß cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S.

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.

Determination of differentially regulated proteins upon proteasome inhibition in AML cell lines by the combination of large-scale and targeted quantitative proteomics.

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. In-depth 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.

Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance.

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 cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1-directed pharmacological compounds represents an anti-stromal-targeted therapy with promising chemosensitization potential.

Deciphering preferential interactions within supramolecular protein complexes: the proteasome case.

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. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.

Label-free quantitative proteomics reveals the dynamics of proteasome complexes composition and stoichiometry in a wide range of human cell lines.

The proteasome is the main proteolytic system involved in intracellular proteins homeostasis in eukaryotes. Although the structure of proteasome complexes has been well characterized, the distribution of its activators and associated proteins are less studied. Here, we determine the composition and the stoichiometry of proteasome complexes and their associated proteins in a wide range of human cell lines using a one-step affinity purification method and a label-free quantitative proteomic approach. We show that proteasome complexes are highly dynamic protein assemblies, the activity of which being regulated at different levels by variations in the stoichiometry of bound regulators, in the composition of catalytic subunits and associated proteins, and in the rate of the 20S catalytic core complex assembly.

Control of Paip1-eukayrotic translation initiation factor 3 interaction by amino acids through S6 kinase.

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.

Subcellular distribution and dynamics of active proteasome complexes unraveled by a workflow combining in vivo complex cross-linking and quantitative proteomics.

Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins-which modulate proteasome activity, stability, localization, or substrate uptake-rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.

Analysis of the processing of seven human tumor antigens by intermediate proteasomes.

We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (ß5i) or two (ß1i and ß5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3(114-122), MAGE-C2(42-50), MAGE-C2(336-344)) or the standard proteasome (Melan-A(26-35), tyrosinase(369-377), gp100(209-217)). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2(336-344) was only produced by intermediate proteasome ß1i-ß5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2(191-200) is produced only by intermediate proteasome ß1i-ß5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.

Proteomics to study the diversity and dynamics of proteasome complexes: from fundamentals to the clinic.

This article covers the latest contributions of proteomics to the structural and functional characterization of proteasomes and their associated proteins, but also to the detection of proteasomes as clinical biomarkers in diseases. Proteasomes are highly heterogenous supramolecular complexes and constitute important cellular proteases controlling the pool of proteins involved in key cellular functions. The comprehension of the structure/function relationship of proteasomes is therefore of major interest in biology. Numerous biochemical methods have been employed to purify proteasomes, and have led to the identification of complexes of various compositions - depending on the experimental conditions and the type of strategy used. In association with protein separation and enrichment techniques, modern mass spectrometry instruments and mass spectrometry-based quantitative methods, they have led to unprecedented breakthroughs in the in-depth analysis of the diversity and dynamics of proteasome composition and localization under various stimuli or pathological contexts. Proteasome inhibitors are now used in clinics for the treatment of cancer, and recent studies propose that the proteasome should be considered as a predictive biomarker for various pathologies.

Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules.

Most antigenic peptides presented by MHC class I molecules result from the degradation of intracellular proteins by the proteasome. In lymphoid tissues and cells exposed to IFNγ, the standard proteasome is replaced by the immunoproteasome, in which all of the standard catalytic subunits ß1, ß2, and ß5 are replaced by their inducible counterparts ß1i, ß2i, and ß5i, which have different cleavage specificities. The immunoproteasome thereby shapes the repertoire of antigenic peptides. The existence of additional forms of proteasomes bearing a mixed assortment of standard and inducible catalytic subunits has been suggested. Using a new set of unique subunit-specific antibodies, we have now isolated, quantified, and characterized human proteasomes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (ß5i) or two (ß1i and ß5i) of the three inducible catalytic subunits of the immunoproteasome. These intermediate proteasomes represent between one-third and one-half of the proteasome content of human liver, colon, small intestine, and kidney. They are also present in human tumor cells and dendritic cells. We identified two tumor antigens of clinical interest that are processed exclusively either by intermediate proteasomes ß5i (MAGE-A3(271-279)) or by intermediate proteasomes ß1i-ß5i (MAGE-A10(254-262)). The existence of these intermediate proteasomes broadens the repertoire of antigens presented to CD8 T cells and implies that the antigens presented by a given cell depend on their proteasome content.

Proteasome inhibitor-induced apoptosis in acute myeloid leukemia: a correlation with the proteasome status.

The proteasome plays a critical role in the regulation of many cellular processes, including the cell cycle and tumor growth. The proteasome inhibitor bortezomib has recently been approved for the treatment of relapsed and refractory multiple myeloma. In this study, we investigated the induction of apoptosis by proteasome inhibitors in several human acute myeloid leukemia (AML) cell lines and in primary cells from patients. We demonstrate that these drugs induce a high level of apoptosis in the KG1a cell line, in which the therapeutic drug daunorubicin is poorly active, compared to other AML cell lines. In parallel, we found that significantly different levels of apoptosis were induced in primary cells from patients depending on the FAB-based differentiation status of these cells. Moreover, the level of 20S proteasome in KG1a cells was also high compared to other AML cell lines, suggesting a relationship between the high sensitivity to proteasome inhibitors and an elevated amount of 20S proteasome. In good accordance, we identified two groups of patient cells expressing high and low levels of 20S proteasome, with respective high and low sensitivity to proteasome inhibitors. Further comparison of the proteasome status in KG1a and U937 cells also suggests that a high proportion of the 19S regulatory complex in U937 cells compared to the 20S core complex may explain an increased proteasome activity. Altogether, our results suggest that various AML subtypes may present different responses to proteasome inhibitors, that these molecules can be potentially considered as interesting therapeutic alternatives for these pathologies, and that the amount of 20S proteasome in AML cells may be predictive of the cellular response to these inhibitors.

Chronic ethanol feeding affects proteasome-interacting proteins.

Studies on alcoholic liver injury mechanisms show a significant inhibition of the proteasome activity. To investigate this phenomenon, we isolated proteasome complexes from the liver of rats fed ethanol chronically, and from the liver of their pair-fed controls, using a non-denaturing multiple centrifugations procedure to preserve proteasome-interacting proteins (PIPs). ICAT and MS/MS spectral counting, further confirmed by Western blot, showed that the levels of several PIPs were significantly decreased in the isolated ethanol proteasome fractions. This was the case of PA28alpha/beta proteasome activator subunits, and of three proteasome-associated deubiquitinases, Rpn11, ubiquitin C-terminal hydrolase 14, and ubiquitin carboxyl-terminal hydrolase L5. Interestingly, Rpn13 C-terminal end was missing in the ethanol proteasome fraction, which probably altered the linking of ubiquitin carboxyl-terminal hydrolase L5 to the proteasome. 20S proteasome and most 19S subunits were however not changed but Ecm29, a protein known to stabilize the interactions between the 20S and its activators, was decreased in the isolated ethanol proteasome fractions. It is proposed that ethanol metabolism causes proteasome inhibition by several mechanisms, including by altering PIPs and proteasome regulatory complexes binding to the proteasome.

Affinity purification strategy to capture human endogenous proteasome complexes diversity and to identify proteasome-interacting proteins.

An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ/tissue and in different cell physiological states.

Toward a full characterization of the human 20S proteasome subunits and their isoforms by a combination of proteomic approaches.

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.

Scaled-down purification protocol to access proteomic analysis of 20S proteasome from human tissue samples: comparison of normal and tumor colorectal cells.

The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the cytoplasmic 20S proteasome of adjacent normal and tumor colorectal cells arising from tissue samples of several patients. Proteomic analyses based on two-dimensional gel electrophoresis (2DE) and mass spectrometry showed that the subunit composition of 20S proteasomes from these normal and tumor cells were not significantly different. The proteasome activity was also assessed in the cytoplasmic extracts and was similar or higher in tumor colorectal than in the corresponding normal cells. The scaled-down 20S proteasome purification protocol developed here can be applied to any human clinical tissue samples and is compatible with further proteomic analyses.

Purification and proteomic analysis of 20S proteasomes from human cells.

The 20S proteasome is a multicatalytic protein complex present in all eukaryotic cells. Associated to regulatory complexes, it plays a major role in cellular protein degradation and in the generation of Major Histocompatibility Complex (MHC) class I antigenic peptides. In mammalian cells, this symmetrical cylindrical complex is composed of two copies of 14 distinct subunits, three of which possess a proteolytic activity. The catalytic standard subunits can be replaced by immunosubunits to form the immunoproteasome, which possesses different proteolytic efficiencies. Both types of 20S proteasomes can be present in cells in varying distributions. The heterogeneity of 20S proteasome complexes in cells leads to different protein degradation patterns. The characterization of the subunit composition of 20S proteasomes in cells thus represents an important step in the understanding of the effect of the heterogeneity of proteasome complexes on their activity. This chapter describes the use of proteomic approaches to study the subunit composition of 20S proteasome complexes purified from human cells. An immunoaffinity purification method is presented. The separation of all 20S proteasome subunits by 2D gel electrophoresis and the subunit identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis and database search are then described. These methods are discussed with the study of 20S proteasomes purified from two human cancer cell lines.

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity.

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters.

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity (a(w)). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a(w) variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.

Water plays a different role on activation thermodynamic parameters of alcoholysis reaction catalyzed by lipase in gaseous and organic media.

The effect of water on the alcoholysis of methyl propionate and n-propanol catalyzed by immobilized Candida antarctica lipase B (CALB) has been compared in a continuous solid-gas reactor and in an organic liquid medium. The enthalpic and entropic contributions of water to the Gibbs free energy of activation in the gas phase were different from the ones in the organic phase, the inverse trends being observed for the variation of both DeltaH* and DeltaS* with water activity. Different phenomena were identified for their influence on the thermodynamic parameters. When increasing a(w), the enhanced flexibility of the enzyme was predominant in the gas phase whereas substrate-solvent interactions due to an increased polarity of the solvent affected mainly the thermodynamic parameters in the organic phase. The observed variations of DeltaG* with water activity were in accordance with kinetics results previously obtained in both reaction media.