The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
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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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.
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development evaluation and application of Tripeptidyl Peptidase ii sequence signatures
Archives of Biochemistry and Biophysics, 2009Co-Authors: Sandra Eriksson, Omar A Gutierrez, Pernilla Bjerling, Birgitta TomkinsonAbstract:Tripeptidyl-Peptidase II (TPP II) is a cytosolic Peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.
Elizabeth Kida - One of the best experts on this subject based on the ideXlab platform.
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Functional consequences and rescue potential of pathogenic missense mutations in Tripeptidyl Peptidase I.
Human mutation, 2010Co-Authors: Marius Walus, Elizabeth Kida, Adam A. GolabekAbstract:There are 35 missense mutations among 68 different mutations in the TPP1 gene, which encodes Tripeptidyl Peptidase I (TPPI), a lysosomal aminoPeptidase associated with classic late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). To elucidate the molecular mechanisms underlying TPPI deficiency in patients carrying missense mutations and to test the amenability of mutant proteins to chemical chaperones and permissive temperature treatment, we introduced individually 14 disease-associated missense mutations into human TPP1 cDNA and analyzed the cell biology of these TPPI variants expressed in Chinese hamster ovary cells. Most TPPI variants displayed obstructed transport to the lysosomes, prolonged half-life of the proenzyme, and residual or no enzymatic activity, indicating folding abnormalities. Protein misfolding was produced by mutations located in both the prosegment (p.Gly77Arg) and throughout the length of the mature enzyme. However, the routes of removal of misfolded proteins by the cells varied, ranging from their efficient degradation by the ubiquitin/proteasome system to abundant secretion. Two TPPI variants demonstrated enhanced processing in response to folding improvement treatment, and the activity of one of them, p.Arg447His, showed a fivefold increase under permissive temperature conditions, which suggests that folding improvement strategies may ameliorate the function of some misfolding TPPI mutant proteins. Hum Mutat 31:1–12, 2010. © 2010 Wiley-Liss, Inc.
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Prosegment of Tripeptidyl Peptidase I is a potent, slow-binding inhibitor of its cognate enzyme.
The Journal of biological chemistry, 2008Co-Authors: Adam A. Golabek, Marius Walus, Krystyna E. Wisniewski, Natalia Dolzhanskaya, Elizabeth KidaAbstract:Abstract Tripeptidyl Peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.
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Tripeptidyl-Peptidase I in health and disease
Biological chemistry, 2006Co-Authors: Adam A. Golabek, Elizabeth KidaAbstract:The lysosomal lumen contains numerous acidic hydrolases involved in the degradation of carbohydrates, lipids, proteins, and nucleic acids, which are basic cell components that turn over continuously within the cell and/or are ingested from outside of the cell. Deficiency in almost any of these hydrolases causes accumulation of the undigested material in secondary lysosomes, which manifests itself as a form of lysosomal storage disorder (LSD). Mutations in Tripeptidyl-Peptidase I (TPP I) underlie the classic late-infantile form of neuronal ceroid lipofuscinoses (CLN2), the most common neurodegenerative disorders of childhood. TPP I is an aminoPeptidase with minor endoPeptidase activity and Ser475 serving as an active-site nucleophile. The enzyme is synthesized as a highly glycosylated precursor transported by mannose-6-phosphate receptors to lysosomes, where it undergoes proteolytic maturation. This review summarizes recent progress in understanding of TPP I biology and molecular pathology of the CLN2 disease process, including distribution of the enzyme, its biosynthesis, glycosylation, transport and activation, as well as catalytic mechanisms and their potential implications for pathogenesis and treatment of the underlying disease. Promising data from gene and stem cell therapy in laboratory animals raise hope that CLN2 will be the first neurodegenerative LSD for which causative treatment will become available for humans.
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Ser475, Glu272, Asp276, Asp327, and Asp360 are involved in catalytic activity of human Tripeptidyl-Peptidase I
FEBS letters, 2005Co-Authors: Marius Walus, Elizabeth Kida, Krystyna E. Wisniewski, Adam A. GolabekAbstract:Tripeptidyl-Peptidase I (TPP I) is a lysosomal aminoPeptidase that sequentially removes tripeptides from small polypeptides and also shows a minor endoprotease activity. Mutations in TPP I are associated with a fatal lysosomal storage disorder – the classic late-infantile form of neuronal ceroid lipofuscinoses. In the present study, we analyzed the catalytic mechanism of the human enzyme by using a site-directed mutagenesis. We demonstrate that apart from previously identified Ser475 and Asp360, also Glu272, Asp276, and Asp327 are important for catalytic activity of the enzyme. Involvement of serine, glutamic acid, and aspartic acid in the catalytic reaction validates the idea, formulated on the basis of significant amino acid sequence homology and inhibition studies, that TPP I is the first mammalian representative of a growing family of serine-carboxyl Peptidases.
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Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-Peptidase I in Vitro and in Vivo
The Journal of biological chemistry, 2004Co-Authors: Adam A. Golabek, Marius Walus, Krystyna E. Wisniewski, Elizabeth KidaAbstract:Abstract Tripeptidyl-Peptidase I (TPP I, CLN2 protein) is a lysosomal exoPeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t½ of mature enzyme at pH 7.4 increased by ∼8-fold in the presence of heparin) and in vivo (∼2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.
Adam A. Golabek - One of the best experts on this subject based on the ideXlab platform.
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Functional consequences and rescue potential of pathogenic missense mutations in Tripeptidyl Peptidase I.
Human mutation, 2010Co-Authors: Marius Walus, Elizabeth Kida, Adam A. GolabekAbstract:There are 35 missense mutations among 68 different mutations in the TPP1 gene, which encodes Tripeptidyl Peptidase I (TPPI), a lysosomal aminoPeptidase associated with classic late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). To elucidate the molecular mechanisms underlying TPPI deficiency in patients carrying missense mutations and to test the amenability of mutant proteins to chemical chaperones and permissive temperature treatment, we introduced individually 14 disease-associated missense mutations into human TPP1 cDNA and analyzed the cell biology of these TPPI variants expressed in Chinese hamster ovary cells. Most TPPI variants displayed obstructed transport to the lysosomes, prolonged half-life of the proenzyme, and residual or no enzymatic activity, indicating folding abnormalities. Protein misfolding was produced by mutations located in both the prosegment (p.Gly77Arg) and throughout the length of the mature enzyme. However, the routes of removal of misfolded proteins by the cells varied, ranging from their efficient degradation by the ubiquitin/proteasome system to abundant secretion. Two TPPI variants demonstrated enhanced processing in response to folding improvement treatment, and the activity of one of them, p.Arg447His, showed a fivefold increase under permissive temperature conditions, which suggests that folding improvement strategies may ameliorate the function of some misfolding TPPI mutant proteins. Hum Mutat 31:1–12, 2010. © 2010 Wiley-Liss, Inc.
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Prosegment of Tripeptidyl Peptidase I is a potent, slow-binding inhibitor of its cognate enzyme.
The Journal of biological chemistry, 2008Co-Authors: Adam A. Golabek, Marius Walus, Krystyna E. Wisniewski, Natalia Dolzhanskaya, Elizabeth KidaAbstract:Abstract Tripeptidyl Peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.
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Tripeptidyl-Peptidase I in health and disease
Biological chemistry, 2006Co-Authors: Adam A. Golabek, Elizabeth KidaAbstract:The lysosomal lumen contains numerous acidic hydrolases involved in the degradation of carbohydrates, lipids, proteins, and nucleic acids, which are basic cell components that turn over continuously within the cell and/or are ingested from outside of the cell. Deficiency in almost any of these hydrolases causes accumulation of the undigested material in secondary lysosomes, which manifests itself as a form of lysosomal storage disorder (LSD). Mutations in Tripeptidyl-Peptidase I (TPP I) underlie the classic late-infantile form of neuronal ceroid lipofuscinoses (CLN2), the most common neurodegenerative disorders of childhood. TPP I is an aminoPeptidase with minor endoPeptidase activity and Ser475 serving as an active-site nucleophile. The enzyme is synthesized as a highly glycosylated precursor transported by mannose-6-phosphate receptors to lysosomes, where it undergoes proteolytic maturation. This review summarizes recent progress in understanding of TPP I biology and molecular pathology of the CLN2 disease process, including distribution of the enzyme, its biosynthesis, glycosylation, transport and activation, as well as catalytic mechanisms and their potential implications for pathogenesis and treatment of the underlying disease. Promising data from gene and stem cell therapy in laboratory animals raise hope that CLN2 will be the first neurodegenerative LSD for which causative treatment will become available for humans.
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Ser475, Glu272, Asp276, Asp327, and Asp360 are involved in catalytic activity of human Tripeptidyl-Peptidase I
FEBS letters, 2005Co-Authors: Marius Walus, Elizabeth Kida, Krystyna E. Wisniewski, Adam A. GolabekAbstract:Tripeptidyl-Peptidase I (TPP I) is a lysosomal aminoPeptidase that sequentially removes tripeptides from small polypeptides and also shows a minor endoprotease activity. Mutations in TPP I are associated with a fatal lysosomal storage disorder – the classic late-infantile form of neuronal ceroid lipofuscinoses. In the present study, we analyzed the catalytic mechanism of the human enzyme by using a site-directed mutagenesis. We demonstrate that apart from previously identified Ser475 and Asp360, also Glu272, Asp276, and Asp327 are important for catalytic activity of the enzyme. Involvement of serine, glutamic acid, and aspartic acid in the catalytic reaction validates the idea, formulated on the basis of significant amino acid sequence homology and inhibition studies, that TPP I is the first mammalian representative of a growing family of serine-carboxyl Peptidases.
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Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-Peptidase I in Vitro and in Vivo
The Journal of biological chemistry, 2004Co-Authors: Adam A. Golabek, Marius Walus, Krystyna E. Wisniewski, Elizabeth KidaAbstract:Abstract Tripeptidyl-Peptidase I (TPP I, CLN2 protein) is a lysosomal exoPeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t½ of mature enzyme at pH 7.4 increased by ∼8-fold in the presence of heparin) and in vivo (∼2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.
Peter Lobel - One of the best experts on this subject based on the ideXlab platform.
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protein thermal stability does not correlate with cellular half life global observations and a case study of Tripeptidyl Peptidase 1
bioRxiv, 2019Co-Authors: Aaron M Collier, David E. Sleat, Yuliya Nemtsova, Narendra Kuber, Whitney Banachpetrosky, Anurag Modak, Vikas Nanda, Peter LobelAbstract:Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease Tripeptidyl-Peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6{degrees}C to 64.4{degrees}C and increased its enzymatic half-life at 60{degrees}C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL-patient derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.
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lysosomal enzyme Tripeptidyl Peptidase 1 plays a role in degradation of beta amyloid fibrils
bioRxiv, 2019Co-Authors: Dana Cruz, David E. Sleat, Peter Lobel, Mukarram Elbanna, Amitabha Majumdar, Michelle Muldowney, Frederick R MaxfieldAbstract:Alzheimers disease (AD) is characterized by the accumulation of amyloid plaques surrounded by microglia. In cell culture, microglia internalize fibrillar {beta}-amyloid but do not degrade it efficiently. Unactivated microglia have a relatively high lysosomal pH, which impairs the activity of lysosomal proteases. Previous studies showed that activation of microglia with macrophage colony stimulating factor decreases lysosomal pH and enhances fibrillar {beta}-amyloid degradation. We investigated the role of the lysosomal protease Tripeptidyl Peptidase 1 (TPP1) in cell culture and in a mouse model of Alzheimers disease. Increased levels of TPP1 in unactivated microglia enhanced fibrillar {beta}-amyloid degradation. Conversely, reduction of TPP1 led to decreased fibrillar {beta}-amyloid degradation in activated microglia, macrophages, and other cells that degrade fibrillar {beta}-amyloid efficiently. Reduction of TPP1 in an AD model mouse using a gene-targeted hypomorphic Tpp1 allele increased plaque burden. These results suggest that decreased TPP1 potentiates AD pathogenesis and that strategies to increase TPP1 activity may have therapeutic value.nnHighlightsO_LIIn microglia, TPP1 is important for the degradation of fibrillar {beta}-amyloid.nC_LIO_LIIncreased TPP1 in microglia results in enhanced fibrillar {beta}-amyloid degradation.nC_LIO_LIIn an AD mouse model, reduction of TPP1 led to increased amyloid plaque deposition.nC_LI
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lysosomal enzyme Tripeptidyl Peptidase 1 destabilizes fibrillar aβ by multiple endoproteolytic cleavages within the β sheet domain
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Santiago Soledomenech, Peter Lobel, Ana V Rojas, Gia G Maisuradze, Harold A Scheraga, Frederick R MaxfieldAbstract:Accumulation of amyloid-beta (Aβ), which is associated with Alzheimer's disease, can be caused by excess production or insufficient clearance. Because of its β-sheet structure, fibrillar Aβ is resistant to proteolysis, which would contribute to slow degradation of Aβ plaques in vivo. Fibrillar Aβ can be internalized by microglia, which are the scavenger cells of the brain, but the fibrils are degraded only slowly in microglial lysosomes. Cathepsin B is a lysosomal protease that has been shown to proteolyze fibrillar Aβ. Tripeptidyl Peptidase 1 (TPP1), a lysosomal serine protease, possesses endoPeptidase activity and has been shown to cleave peptides between hydrophobic residues. Herein, we demonstrate that TPP1 is able to proteolyze fibrillar Aβ efficiently. Mass spectrometry analysis of peptides released from fibrillar Aβ digested with TPP1 reveals several endoproteolytic cleavages including some within β-sheet regions that are important for fibril formation. Using molecular dynamics simulations, we demonstrate that these cleavages destabilize fibrillar β-sheet structure. The demonstration that TPP1 can degrade fibrillar forms of Aβ provides insight into the turnover of fibrillar Aβ and may lead to new therapeutic methods to increase degradation of Aβ plaques.
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Inducible transgenic expression of Tripeptidyl Peptidase 1 in a mouse model of late-infantile neuronal ceroid lipofuscinosis
2018Co-Authors: Yuliya Nemtsova, Mukarram El-banna, Peter Lobel, Jennifer A. Wiseman, David E. SleatAbstract:Late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease of children caused by mutations resulting in loss of activity of the lysosomal protease, Tripeptidyl Peptidase 1 (TPP1). While Tpp1-targeted mouse models of LINCL exist, the goal of this study was to create a transgenic mouse with inducible TPP1 to benchmark treatment approaches, evaluate efficacy of treatment at different stages of disease, and to provide insights into the pathobiology of disease. A construct containing a loxP-flanked stop cassette inserted between the chicken-actin promoter and a sequence encoding murine TPP1 (TgLSL-TPP1) was integrated into the ROSA26 locus in mice by homologous recombination. Tested in both transfected CHO cells and in transgenic mice, the TgLSL-TPP1 did not express TPP1 until cre-mediated removal of the LSL cassette, which resulted in supraphysiological levels of TPP1 activity. We tested four cre/ERT2 transgenes to allow tamoxifen-inducible removal of the LSL cassette and subsequent TPP1 expression at any stage of disease. However, two of the cre/ERT2 driver transgenes had significant cre activity in the absence of tamoxifen, while cre-mediated recombination could not be induced by tamoxifen by two others. These results highlight potential problems with the use of cre/ERT2 transgenes in applications that are sensitive to low levels of basal cre expression. However, the germline-recombined mouse transgenic that constitutively overexpresses TPP1 will allow long-term evaluation of overexposure to the enzyme and in cell culture, the inducible transgene may be a useful tool in biomarker discovery projects.
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systemic administration of Tripeptidyl Peptidase i in a mouse model of late infantile neuronal ceroid lipofuscinosis effect of glycan modification
PLOS ONE, 2012Co-Authors: Yu Meng, David E. Sleat, Peter Lobel, István Sohár, Lingling WangAbstract:Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a recessive genetic disease of childhood caused by deficiencies in the lysosomal protease Tripeptidyl Peptidase I (TPP1). Disease is characterized by progressive and extensive neuronal death. One hurdle towards development of enzyme replacement therapy is delivery of TPP1 to the brain. In this study, we evaluated the effect of modifying N-linked glycans on recombinant human TPP1 on its pharmacokinetic properties after administration via tail vein injection to a mouse model of LINCL. Unmodified TPP1 exhibited a dose-dependent serum half-life of 12 min (0.12 mg) to 45 min (2 mg). Deglycosylation or modification using sodium metaperiodate oxidation and reduction with sodium borohydride increased the circulatory half-life but did not improve targeting to the brain compared to unmodified TPP1. Analysis of liver, brain, spleen, kidney and lung demonstrated that for all preparations, >95% of the recovered activity was in the liver. Interestingly, administration of a single 2 mg dose (80 mg/kg) of unmodified TPP1 resulted in ∼10% of wild-type activity in brain. This suggests that systemic administration of unmodified recombinant enzyme merits further exploration as a potential therapy for LINCL.
Krystyna E. Wisniewski - One of the best experts on this subject based on the ideXlab platform.
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Prosegment of Tripeptidyl Peptidase I is a potent, slow-binding inhibitor of its cognate enzyme.
The Journal of biological chemistry, 2008Co-Authors: Adam A. Golabek, Marius Walus, Krystyna E. Wisniewski, Natalia Dolzhanskaya, Elizabeth KidaAbstract:Abstract Tripeptidyl Peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.
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Ser475, Glu272, Asp276, Asp327, and Asp360 are involved in catalytic activity of human Tripeptidyl-Peptidase I
FEBS letters, 2005Co-Authors: Marius Walus, Elizabeth Kida, Krystyna E. Wisniewski, Adam A. GolabekAbstract:Tripeptidyl-Peptidase I (TPP I) is a lysosomal aminoPeptidase that sequentially removes tripeptides from small polypeptides and also shows a minor endoprotease activity. Mutations in TPP I are associated with a fatal lysosomal storage disorder – the classic late-infantile form of neuronal ceroid lipofuscinoses. In the present study, we analyzed the catalytic mechanism of the human enzyme by using a site-directed mutagenesis. We demonstrate that apart from previously identified Ser475 and Asp360, also Glu272, Asp276, and Asp327 are important for catalytic activity of the enzyme. Involvement of serine, glutamic acid, and aspartic acid in the catalytic reaction validates the idea, formulated on the basis of significant amino acid sequence homology and inhibition studies, that TPP I is the first mammalian representative of a growing family of serine-carboxyl Peptidases.
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Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-Peptidase I in Vitro and in Vivo
The Journal of biological chemistry, 2004Co-Authors: Adam A. Golabek, Marius Walus, Krystyna E. Wisniewski, Elizabeth KidaAbstract:Abstract Tripeptidyl-Peptidase I (TPP I, CLN2 protein) is a lysosomal exoPeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t½ of mature enzyme at pH 7.4 increased by ∼8-fold in the presence of heparin) and in vivo (∼2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.
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Maturation of Human Tripeptidyl-Peptidase I in Vitro
The Journal of biological chemistry, 2004Co-Authors: Adam A. Golabek, Peter Wujek, Marius Walus, Sylvain Bieler, Claudio Soto, Krystyna E. Wisniewski, Elizabeth KidaAbstract:Abstract Tripeptidyl-Peptidase I (TPP I, CLN2 protein) is a lysosomal aminoPeptidase that cleaves off tripeptides from the free N termini of oligopeptides and also shows minor endoPeptidase activity. TPP I is synthesized as a preproenzyme. Its proenzyme autoactivates under acidic conditions in vitro, resulting in a rapid conversion into the mature form. In this study, we examined the process of maturation in vitro of recombinant latent human TPP I purified to homogeneity from secretions of Chinese hamster ovary cells overexpressing TPP I cDNA. Autoprocessing of TPP I proenzyme was carried out at a wide pH range, from ∼2.0 to 6.0, albeit with different efficiencies depending on the pH and the type of buffer. However, the acquisition of enzymatic activity in the same buffer took place in a narrower pH “window,” usually in the range of 3.6–4.2. N-terminal sequencing revealed that mature, inactive enzyme generated during autoactivation at higher pH contained N-terminal extensions (starting at 6 and 14 amino acid residues upstream of the prosegment/mature enzyme junction), which could contribute to the lack of activity of TPP I generated in this manner. Autoprocessing was not associated with any major changes of the secondary structure of the proenzyme, as revealed by CD spectroscopy. Both the activation and proteolytic processing of the recombinant TPP I precursor were primarily concentration-independent. The addition of the mature enzyme did not accelerate the processing of the proenzyme. In addition, the maturation of the proenzyme was not affected by the presence of glycerol. Finally, the proenzyme with the active site mutated (S475L) was not processed in the presence of the wild-type enzyme. All of these findings indicate a primarily intramolecular (unimolecular) mechanism of TPP I activation and autoprocessing and suggest that in vivo mature enzyme does not significantly participate in its own generation from the precursor.
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N-Glycosylation Is Crucial for Folding, Trafficking, and Stability of Human Tripeptidyl-Peptidase I
The Journal of biological chemistry, 2003Co-Authors: Peter Wujek, Elizabeth Kida, Marius Walus, Krystyna E. Wisniewski, Adam A. GolabekAbstract:Tripeptidyl-Peptidase I (TPP I) is a lysosomal serine-carboxyl Peptidase that sequentially removes tripeptides from polypeptides. Naturally occurring mutations in TPP I are associated with the classic late infantile neuronal ceroid lipofuscinosis. Human TPP I has five potential N-glycosylation sites at Asn residues 210, 222, 286, 313, and 443. To analyze the role of N-glycosylation in the function of the enzyme, we obliterated each N- glycosylation consensus sequence by substituting Gln for Asn, either individually or in combinations, and expressed mutated cDNAs in Chinese hamster ovary and human embryonic kidney 293 cells. Here, we demonstrate that human TPP I in vivo utilizes all five N-glycosylation sites. Elimination of one of these sites, at Asn-286, dramatically affected the folding of the enzyme. However, in contrast to other misfolded proteins that are retained in the endoplasmic reticulum, only a fraction of misfolded TPP I mutant expressed in Chinese hamster ovary cells, but not in human embryonic kidney 293 cells, was arrested in the ER, whereas its major portion was secreted. Secreted proenzyme formed non-native, interchain disulfide bridges and displayed only residual TPP I activity upon acidification. A small portion of TPP I missing Asn-286-linked glycan reached the lysosome and was processed to an active species; however, it showed low thermal and pH stability. N-Glycans at Asn-210, Asn-222, Asn-313, and Asn-443 contributed slightly to the specific activity of the enzyme and its resistance to alkaline pH-induced inactivation. Phospholabeling experiments revealed that N-glycans at Asn-210 and Asn-286 of TPP I preferentially accept a phosphomannose marker. Thus, a dual role of oligosaccharide at Asn-286 in folding and lysosomal targeting could contribute to the unusual, but cell type-dependent, fate of misfolded TPP I conformer and represent the molecular basis of the disease process in subjects with naturally occurring missense mutation at Asn-286.
