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Birgitta Tomkinson - One of the best experts on this subject based on the ideXlab platform.
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Tripeptidyl Peptidase II update on an oldie that still counts
Biochimie, 2019Co-Authors: Birgitta TomkinsonAbstract:The huge exoPeptidase, Tripeptidyl-Peptidase II (TPP II), appears to be involved in a large number of important biological processes. It is present in the cytosol of most eukaryotic cells, where it ...
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Tripeptidyl Peptidase II mediates levels of nuclear phosphorylated erk1 and erk2
Molecular & Cellular Proteomics, 2015Co-Authors: Anne Wiemhoefer, Birgitta Tomkinson, Anita Stargardt, Wouter A Van Der Linden, Maria C Renner, Ronald E Van Kesteren, Jan Stap, Marcel Raspe, Helmut W Kessels, Huib OvaaAbstract:Tripeptidyl Peptidase II (TPP2) is a serine Peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a Peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function.
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Mitotic Infidelity and Centrosome Duplication Errors in Cells Overexpressing Tripeptidyl-Peptidase II
2013Co-Authors: Vaia Stavropoulou, Birgitta Tomkinson, Jianjun Xie, Marie Henriksson, Stefan Imreh, Maria G MasucciAbstract:The oligoPeptidase Tripeptidyl-Peptidase II (TPP II) is upregulated Burkitt’s lymphoma (BL) cells that overexpress the c-myc proto-oncogene and is required for their growth and survival. Here we show that overexpression of TPP II induces accelerated growth and resistance to apoptosis in human embryonic kidney 293 cells. This correlates with the appearance of multiple chromosomal aberrations, numerical and structural centrosome abnormalities, and multipolar cell divisions. Similar mitotic aberrations were also observed in a panel of BL lines and were suppressed, in parallel with TPP II down-regulation, upon reversion of BL-like characteristics in EBV-immortalized B lymphocytes carrying a tetracyclineregulated c-myc. Functional TPP II knockdown by small interfering RNA expression in BL cells caused the appearance of giant polynucleated cells that failed to complete cell division. Collectively, these data point to a role of TPP II in the regulation of centrosome homeostasis and mitotic fidelity suggesting that this enzyme may be a critical player in the induction and/or maintenance of genetic instability in malignant cells. (Cancer Res 2005; 65(4): 1361-8
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characterization of the endoPeptidase activity of Tripeptidyl Peptidase II
Biochemical and Biophysical Research Communications, 2012Co-Authors: Sandra Eklund, Jakob Dogan, Per Jemth, Hubert Kalbacher, Birgitta TomkinsonAbstract:Abstract Tripeptidyl-Peptidase II (TPP II) is a giant cytosolic Peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exoPeptidase activity, TPP II also has an endoPeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef69–87 and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endoPeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endoPeptidase activity could be determined to be 5 orders of magnitude lower than for the exoPeptidase activity.
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Exploring the active site of Tripeptidyl-Peptidase II through studies of pH dependence of reaction kinetics.
Biochimica et biophysica acta, 2012Co-Authors: Sandra Eklund, Ann-christin Lindås, Emil Hamnevik, Mikael Widersten, Birgitta TomkinsonAbstract:Abstract Tripeptidyl-Peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of kcatapp/KM probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the KM and kcatapp are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of kcatapp, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine Peptidases.
Margarita Del Val - One of the best experts on this subject based on the ideXlab platform.
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accumulation of polyubiquitylated proteins in response to ala ala phe chloromethylketone is independent of the inhibition of Tripeptidyl Peptidase II
Biochimica et Biophysica Acta, 2010Co-Authors: Eugenia M Villasevil, Sara Guil, Margarita Del Val, Lorena Lopezferreras, Carlos Sanchez, Luis C AntonAbstract:In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl Peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis.
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need for Tripeptidyl Peptidase II in major histocompatibility complex class i viral antigen processing when proteasomes are detrimental
Journal of Biological Chemistry, 2006Co-Authors: Sara Guil, Eugenia M Villasevil, Marta Rodriguezcastro, Francisco Aguilar, Luis C Anton, Margarita Del ValAbstract:CD8(+) T lymphocytes recognize infected cells that display virus-derived antigenic peptides complexed with major histocompatibility complex class I molecules. Peptides are mainly byproducts of cellular protein turnover by cytosolic proteasomes. Cytosolic Tripeptidyl-Peptidase II (TPPII) also participates in protein degradation. Several peptidic epitopes unexpectedly do not require proteasomes, but it is unclear which proteases generate them. We studied antigen processing of influenza virus nucleoprotein epitope NP(147-155), an archetype epitope that is even destroyed by a proteasome-mediated mechanism. TPPII, with the assistance of endoplasmic reticulum trimming metallo-aminoPeptidases, probably ERAAP (endoplasmic reticulum aminoPeptidase associated with antigen processing), was crucial for nucleoprotein epitope generation both in the presence of functional proteasomes and when blocked by lactacystin, as shown with specific chemical inhibitors and gene silencing. Different protein contexts and subcellular targeting all allowed epitope processing by TPPII as well as trimming. The results show the plasticity of the cell's assortment of proteases for providing ligands for recognition by antiviral CD8(+) T cells. Our observations identify for the first time a set of proteases competent for antigen processing of an epitope that is susceptible to destruction by proteasomes.
Peter Walter Maria Roevens - One of the best experts on this subject based on the ideXlab platform.
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Tripeptidyl Peptidase II tpp II inhibitory activity of s 2 3 dihydro 2 1h imidazol 2 yl 1h indoles a systematic sar evaluation part 2
ChemInform, 2004Co-Authors: Henry J Breslin, Hans De Winter, Tamara A Miskowski, Michael Joseph Kukla, Maria V F Somers, Peter Walter Maria Roevens, Robert W KavashAbstract:Abstract We have systematically explored the structure–activity relationship (SAR) for a series of compounds 2 as inhibitors of Tripeptidyl-Peptidase II (TPP II), a serine protease responsible for the degradation of cholecystokinin-8 (CCK-8). This SAR evaluation of the core structure 2 suggest a fairly restrictive pharmacophore for such related structures, but has yielded a limited set of compounds (2b, 2c, 2d, 2s, and 2t) with potent TPP II inhibitory activity (IC50 4-11 nM).
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design synthesis and Tripeptidyl Peptidase II inhibitory activity of a novel series of s 2 3 dihydro 2 4 alkyl 1h imidazol 2 yl 1h indoles
Journal of Medicinal Chemistry, 2002Co-Authors: Henry J Breslin, Hans De Winter, Tamara A Miskowski, Michael Joseph Kukla, Maria V F Somers, William Leister, Diane A Gauthier, Danielle Peeters, Peter Walter Maria RoevensAbstract:Butabindide, 1, was previously reported as a potent inhibitor (IC50 = 7 nM) of the serine protease enzyme Tripeptidyl Peptidase II (TPPII), an endogenous protease that degrades cholecystokinin-8 (CCK-8). We found that 1 has some inherent chemical instability, yielding diketopiperazine 2 fairly readily under mimicked physiological conditions. We therefore prepared imidazoles 3, which are void of 1's inherent instability, and have found that our novel analogues maintained comparable TPPII inhibitory activity (e.g.,for 3c, IC50 = 4 nM) as 1.
Eugenia M Villasevil - One of the best experts on this subject based on the ideXlab platform.
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Third Intracellular Proteolysis Meeting 839 Is there an alternative to the proteasome in cytosolic protein degradation?
2016Co-Authors: Luis C. Antón, Eugenia M VillasevilAbstract:While it is clear that the proteasome is the major player in degradative proteolysis in the nucleus and cytosol, there is a lack of complete agreement on whether there are alternative proteolytic pathways or activities responsible for a significant degradation of cytosolic/nuclear substrates. Particularly relevant is the case of the aminoPeptidase TPPII (Tripeptidyl Peptidase II), which has been suggested to be able to perform some of the proteasome functions. However, the current evidence seems to support only a limited role for these cytosolic alternatives. On the other hand, there is evidence of an alternative, autophagy, a pathway involving the delivery of cytosolic substrates to the lysosome for degradation
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accumulation of polyubiquitylated proteins in response to ala ala phe chloromethylketone is independent of the inhibition of Tripeptidyl Peptidase II
Biochimica et Biophysica Acta, 2010Co-Authors: Eugenia M Villasevil, Sara Guil, Margarita Del Val, Lorena Lopezferreras, Carlos Sanchez, Luis C AntonAbstract:In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl Peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis.
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Tripeptidyl Peptidase II is dispensable for the generation of both proteasome dependent and proteasome independent ligands of hla b27 and other class i molecules
European Journal of Immunology, 2008Co-Authors: Miguel Marcilla, Eugenia M Villasevil, Jose Lopez A De CastroAbstract:A significant fraction of the HLA-B27-bound peptide repertoire is resistant to proteasome inhibitors. The possible implication of Tripeptidyl Peptidase II (TPPII) in generating this subset was analyzed by quantifying the surface re-expression of HLA-B*2705 after acid stripping in the presence of two TPPII inhibitors, butabindide and Ala-Ala-Phe-chloromethylketone. Neither decreased HLA-B27 re-expression under conditions in which TPPII activity was largely inhibited. This was in contrast to a significant effect of the proteasome inhibitor epoxomicin. The failure of TPPII inhibition to decrease surface re-expression was not limited to HLA-B27, since it was also observed in several HLA-B27-negative cell lines, including Mel JuSo. Actually, HLA class I re-expression in Mel JuSo cells increased as a function of butabindide concentration, which is consistent with an involvement of TPPII in destroying HLA class I ligands. Inhibition of TPPII with small interfering RNA also failed to decrease the surface expression of HLA class I molecules on 143B cells. Our results indicate that TPPII is dispensable for the generation of proteasome-dependent HLA class I ligands and, without excluding its role in producing some individual epitopes, this enzyme is not involved to any quantitatively significant extent, in generating the proteasome-independent HLA-B27-bound peptide repertoire.
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need for Tripeptidyl Peptidase II in major histocompatibility complex class i viral antigen processing when proteasomes are detrimental
Journal of Biological Chemistry, 2006Co-Authors: Sara Guil, Eugenia M Villasevil, Marta Rodriguezcastro, Francisco Aguilar, Luis C Anton, Margarita Del ValAbstract:CD8(+) T lymphocytes recognize infected cells that display virus-derived antigenic peptides complexed with major histocompatibility complex class I molecules. Peptides are mainly byproducts of cellular protein turnover by cytosolic proteasomes. Cytosolic Tripeptidyl-Peptidase II (TPPII) also participates in protein degradation. Several peptidic epitopes unexpectedly do not require proteasomes, but it is unclear which proteases generate them. We studied antigen processing of influenza virus nucleoprotein epitope NP(147-155), an archetype epitope that is even destroyed by a proteasome-mediated mechanism. TPPII, with the assistance of endoplasmic reticulum trimming metallo-aminoPeptidases, probably ERAAP (endoplasmic reticulum aminoPeptidase associated with antigen processing), was crucial for nucleoprotein epitope generation both in the presence of functional proteasomes and when blocked by lactacystin, as shown with specific chemical inhibitors and gene silencing. Different protein contexts and subcellular targeting all allowed epitope processing by TPPII as well as trimming. The results show the plasticity of the cell's assortment of proteases for providing ligands for recognition by antiviral CD8(+) T cells. Our observations identify for the first time a set of proteases competent for antigen processing of an epitope that is susceptible to destruction by proteasomes.
Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.
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in situ structural studies of Tripeptidyl Peptidase II tppII reveal spatial association with proteasomes
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Yoshiyuki Fukuda, Florian Beck, Jurgen M Plitzko, Wolfgang BaumeisterAbstract:Tripeptidyl Peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies in vitro have revealed the basic architecture of TPPII, a two-stranded linear polymer that assembles to form a spindle-shaped complex of ∼6 MDa. Dependent on protein concentration, TPPII has a distinct tendency for polymorphism. Therefore, its structure in vivo has remained unclear. To resolve this issue, we have scrutinized cryo-electron tomograms of rat hippocampal neurons for the occurrence and spatial distribution of TPPII by template matching. The quality of the tomograms recorded with the Volta phase plate enabled a detailed structural analysis of TPPII despite its low abundance. Two different assembly states (36-mers and 32-mers) coexist as well as occasional extended forms with longer strands. A distance analysis of the relative locations of TPPII and 26S proteasomes confirmed the visual impression that these two complexes spatially associate in agreement with TPPII's role in postproteasomal degradation.
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the structure of human Tripeptidyl Peptidase II as determined by a hybrid approach
Structure, 2012Co-Authors: Anne Marie Schonegge, Jurgen Peters, Wolfgang Baumeister, Elizabeth Villa, Friedrich Forster, Reiner Hegerl, Beate RockelAbstract:Tripeptidyl-Peptidase II (TPPII) is a high molecular mass (∼5 MDa) serine protease, which is thought to act downstream of the 26S proteasome, cleaving peptides released by the latter. Here, the structure of human TPPII (HsTPPII) has been determined to subnanometer resolution by cryoelectron microscopy and single-particle analysis. The complex is built from two strands forming a quasihelical structure harboring a complex system of inner cavities. HsTPPII particles exhibit some polymorphism resulting in complexes consisting of nine or of eight dimers per strand. To obtain deeper insights into the architecture and function of HsTPPII, we have created a pseudoatomic structure of the HsTPPII spindle using a comparative model of HsTPPII dimers and molecular dynamics flexible fitting. Analyses of the resulting hybrid structure of the HsTPPII holocomplex provide new insights into the mechanism of maturation and activation.
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structure and function of Tripeptidyl Peptidase II a giant cytosolic protease
Biochimica et Biophysica Acta, 2012Co-Authors: Beate Rockel, Klaus O Kopec, Andrei N Lupas, Wolfgang BaumeisterAbstract:Tripeptidyl Peptidase II is the largest known eukaryotic Peptidase. It has been described as a multi-purpose Peptidase, which, in addition to its house-keeping function in intracellular protein degradation, plays a role in several vital cellular processes such as antigen processing, apoptosis, or cell division, and is involved in diseases like muscle wasting, obesity, and in cancer. Biochemical studies and bioinformatics have identified TPPII as a subtilase, but its structure is very unusual: it forms a large homooligomeric complex (6 MDa) with a spindle-like shape. Recently, the high-resolution structure of TPPII homodimers (300 kDa) was solved and a hybrid structure of the holocomplex built of 20 dimers was obtained by docking it into the EM-density. Here, we summarize our current knowledge about TPPII with a focus on structural aspects. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
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hybrid molecular structure of the giant protease Tripeptidyl Peptidase II
Nature Structural & Molecular Biology, 2010Co-Authors: Crystal K Chuang, Beate Rockel, Gonul Seyit, Peter J Walian, Anne Marie Schonegge, Jurgen Peters, Petrus H Zwart, Wolfgang Baumeister, Bing K JapAbstract:Tripeptidyl Peptidase II is an eukaryotic serine protease that forms huge, spindle-shaped homopolymeric complexes, whose building block is an enzymatically inactive dimer. Now a combination of cryo-EM and crystal structure analysis of Drosophila TPPII allows a view on dimer organization and polymer architecture assembly, as well as insight into the mechanism of activation.
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molecular architecture and assembly mechanism of drosophila Tripeptidyl Peptidase II
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Beate Rockel, Robert M Glaeser, Gonul Seyit, Jurgen Peters, Reiner Hegerl, Shirley A Muller, Philippe Ringler, Wolfgang BaumeisterAbstract:In eukaryotes, Tripeptidyl Peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino Peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.
