The Experts below are selected from a list of 138 Experts worldwide ranked by ideXlab platform
Brigitte T Huber - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and activity of a potent, specific azabicyclo [3.3.0] octane-based DPP II inhibitor
Bioorganic & medicinal chemistry letters, 2006Co-Authors: Olga V. Danilova, Brigitte T Huber, A. Katrin Szardenings, Jonathan S RosenblumAbstract:A cell permeable DPP II [also known as DPP2, DPP7, and quiescent cell Proline Dipeptidase (QPP)] inhibitor has been synthesized. The azabicyclo[3.3.0]octane-based inhibitor is potent and selective and elicits very similar quiescent lymphocyte death to previously characterized inhibitors that are not as selective.
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vesicular localization and characterization of a novel post Proline cleaving aminoDipeptidase quiescent cell Proline Dipeptidase
Journal of Immunology, 2000Co-Authors: Murali Chiravuri, Henry Lee, Suzanne Mathieu, Fernando A Agarraberes, Brigitte T HuberAbstract:A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell Proline Dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized with a propeptide and undergoes N -glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of post-translational regulation.
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homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell Proline Dipeptidase
Journal of Biological Chemistry, 2000Co-Authors: Murali Chiravuri, Henry Lee, Suzanne Mathieu, Brigitte T HuberAbstract:Quiescent cell Proline Dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.
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sequence purification and cloning of an intracellular serine protease quiescent cell Proline Dipeptidase
Journal of Biological Chemistry, 1999Co-Authors: Robert Underwood, Murali Chiravuri, Henry Lee, Tracy Schmitz, Alisa K Kabcenell, Kurt Yardley, Brigitte T HuberAbstract:We recently observed that specific inhibitors of post-Proline cleaving aminoDipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell Proline Dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-Proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-Proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.
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a novel apoptotic pathway in quiescent lymphocytes identified by inhibition of a post Proline cleaving aminoDipeptidase a candidate target protease quiescent cell Proline Dipeptidase
Journal of Immunology, 1999Co-Authors: Murali Chiravuri, Robert Underwood, Tracy Schmitz, Kurt Yardley, Yogeshwar Dayal, Brigitte T HuberAbstract:The vast majority of lymphocytes in vivo persist in a quiescent state. These resting lymphocytes are maintained through a cellular program that suppresses apoptosis. We show here that quiescent PBMC, but not activated PBMC or transformed lymphocytes, die in the presence of highly specific post-Proline aminoDipeptidase inhibitors. This form of death has the hallmarks of apoptosis, such as phosphatidylserine externalization and loss of mitochondrial transmembrane potential. However, it differs from apoptosis induced by gamma irradiation in the same cells or by Fas ligation in transformed lymphocytes in terms of caspase involvement. In addition, the aminoDipeptidase inhibitor-induced cell death, but not gamma-irradiation-mediated apoptosis, can be prevented by inhibition of the proteasome complex. The target of these inhibitors is not CD26/DPPIV, but probably a novel serine protease, quiescent cell Proline Dipeptidase, that we have recently isolated and cloned. These studies will yield a better understanding of the requirements and the mechanisms that mediate quiescent lymphocyte homeostasis in vivo.
Barbara Leiting - One of the best experts on this subject based on the ideXlab platform.
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dipeptidyl peptidase iv inhibition for the treatment of type 2 diabetes potential importance of selectivity over dipeptidyl peptidases 8 and 9
Diabetes, 2005Co-Authors: George R Lankas, Ranabir Sinha Roy, George J Eiermann, Maria G Beconi, Chichung Chan, Scott D Edmondson, William P Feeney, Tesfaye Biftu, Barbara Leiting, Huaibing HeAbstract:Dipeptidyl peptidase (DPP)-IV inhibitors are a new approach to the treatment of type 2 diabetes. DPP-IV is a member of a family of serine peptidases that includes quiescent cell Proline Dipeptidase (QPP), DPP8, and DPP9; DPP-IV is a key regulator of incretin hormones, but the functions of other family members are unknown. To determine the importance of selective DPP-IV inhibition for the treatment of diabetes, we tested selective inhibitors of DPP-IV, DPP8/DPP9, or QPP in 2-week rat toxicity studies and in acute dog tolerability studies. In rats, the DPP8/9 inhibitor produced alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes, and mortality. In dogs, the DPP8/9 inhibitor produced gastrointestinal toxicity. The QPP inhibitor produced reticulocytopenia in rats only, and no toxicities were noted in either species for the selective DPP-IV inhibitor. The DPP8/9 inhibitor was also shown to attenuate T-cell activation in human in vitro models; a selective DPP-IV inhibitor was inactive in these assays. Moreover, we found DPP-IV inhibitors that were previously reported to be active in models of immune function to be more potent inhibitors of DPP8/9. These results suggest that assessment of selectivity of potential clinical candidates may be important to an optimal safety profile for this new class of antihyperglycemic agents.
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Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell Proline Dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7).
The Biochemical journal, 2005Co-Authors: Marie-berthe Maes, Kristel Senten, Pieter Van Der Veken, Koen Augustyns, Kambiz Gilany, Barbara Leiting, Anne-marie Lambeir, Simon Scharpé, Ingrid De MeesterAbstract:The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K(i) approximately 0.9 microM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell Proline Dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/K(m) clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing Proline at the P1 position and lysine at P2. The importance of the P1' group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/K(m)). Lys-Pro-pNA (k(cat)/K(m) 4.1x10(6) s(-1) x M(-1)) and Ala-Pro-pNA (kcat/K(m) 2.6x10(6) s(-1) x M(-1)) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/K(m) 0.4x10(6) s(-1) x M(-1)).
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Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell Proline Dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7)
Biochemical Journal, 2005Co-Authors: Marie-berthe Maes, Kristel Senten, Pieter Van Der Veken, Koen Augustyns, Kambiz Gilany, Barbara Leiting, Anne-marie Lambeir, Simon Scharpé, Ingrid De MeesterAbstract:The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (Ki~0.9 μM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell Proline Dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/Km clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing Proline at the P1 position and lysine at P2. The importance of the P1′ group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/Km). Lys-Pro-pNA (kcat/Km 4.1×106 s−1·M−1) and Ala-Pro-pNA (kcat/Km 2.6×106 s−1· M−1) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/Km 0.4×106 s−1·M−1).
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Fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors.
Bioorganic & medicinal chemistry letters, 2004Co-Authors: Charles G. Caldwell, George J Eiermann, Barbara Leiting, Frank Marsilio, Reshma A. Patel, Ping Chen, Emma R. Parmee, Aleksandr PetrovAbstract:Amides derived from fluorinated pyrrolidines and 4-substituted cyclohexylglycine analogues have been prepared and evaluated as inhibitors of dipeptidyl Dipeptidase IV (DP-IV). Analogues which incorporated (S)-3-fluoropyrrolidine showed good selectivity for DP-IV over quiescent cell Proline Dipeptidase (QPP). Compound 48 had good pharmacokinetic properties and was orally active in an oral glucose tolerance test in lean mice.
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Catalytic properties and inhibition of Proline-specific dipeptidyl peptidases II, IV and VII
Biochemical Journal, 2003Co-Authors: Barbara Leiting, Jonathan A. Ellman, Richard T. Cummings, Kellyann D. Pryor, Joseph K. Wu, Frank Marsilio, Reshma A. Patel, Charles S. Craik, Nancy A. ThornberryAbstract:There is currently intense interest in the emerging group of Proline-specific Dipeptidases, and their roles in the regulation of biological processes. Dipeptidyl peptidase IV (DPP-IV) is involved in glucose metabolism by contributing to the regulation of glucagon family peptides and has emerged as a potential target for the treatment of metabolic diseases. Two other Prolinespecific Dipeptidases, DPP-VII (also known as quiescent cell Proline Dipeptidase) and DPP-II, have unknown functions and have recently been suggested to be identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) [Araki, Li, Yamamoto, Haneda, Nishi, Kikkawa and Ohkubo (2001) J. Biochem. 129, 279‐288; Fukasawa, Fukasawa, Higaki, Shiina, Ohno, Ito, Otogoto and Ota (2001) Biochem. J. 353, 283‐290]. To facilitate the identification of selective sub
Ingrid De Meester - One of the best experts on this subject based on the ideXlab platform.
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Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell Proline Dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7).
The Biochemical journal, 2005Co-Authors: Marie-berthe Maes, Kristel Senten, Pieter Van Der Veken, Koen Augustyns, Kambiz Gilany, Barbara Leiting, Anne-marie Lambeir, Simon Scharpé, Ingrid De MeesterAbstract:The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K(i) approximately 0.9 microM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell Proline Dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/K(m) clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing Proline at the P1 position and lysine at P2. The importance of the P1' group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/K(m)). Lys-Pro-pNA (k(cat)/K(m) 4.1x10(6) s(-1) x M(-1)) and Ala-Pro-pNA (kcat/K(m) 2.6x10(6) s(-1) x M(-1)) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/K(m) 0.4x10(6) s(-1) x M(-1)).
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Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell Proline Dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7)
Biochemical Journal, 2005Co-Authors: Marie-berthe Maes, Kristel Senten, Pieter Van Der Veken, Koen Augustyns, Kambiz Gilany, Barbara Leiting, Anne-marie Lambeir, Simon Scharpé, Ingrid De MeesterAbstract:The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (Ki~0.9 μM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell Proline Dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/Km clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing Proline at the P1 position and lysine at P2. The importance of the P1′ group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/Km). Lys-Pro-pNA (kcat/Km 4.1×106 s−1·M−1) and Ala-Pro-pNA (kcat/Km 2.6×106 s−1· M−1) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/Km 0.4×106 s−1·M−1).
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human lymphocyte x prolyl aminopeptidase aminopeptidase p like protein a new member of the Proline peptidase family
Advances in Experimental Medicine and Biology, 1997Co-Authors: Greet Vanhoof, Katinka Schatteman, Filip Goossens, Dirk Hendriks, Maria Aparecida Juliano, Ingrid De Meester, Luiz Juliano, Simon ScharpéAbstract:Peptidases are grouped according to their mechanism of catalysis in serine-type, cysteine-type, aspartic-type and metallo-type peptidases. Amongst these groups, the metallopeptidases represent the most diverse group, comprising 25 different families (1,2). Most of the Zn-binding metallopeptidases, named zincins, have the HEXXH motif for Zn-binding. Some other Zn-binding motifs have been defined, as there are the HXXEH, the HXXE, and the HXH motif (3). X-prolyl aminopeptidase (aminopeptidase P, EC 3.4.11.9), is an aminopeptidase that has been reported to bind zinc, and is activated by manganese ions, but does not contain any of these Zn-binding motifs. Aminopeptidase P from Escherichia coli was classified in the peptidase family M24, a family of metallopeptidases in which the ligands for metal ion binding are predominantly carboxylic acids (2). Apart from E. coli aminopeptidase P, this family comprises E. coli methionyl aminopeptidase (EC 3.4.11.18) and E. coli and human Proline Dipeptidase (EC 3.4.13.9). We have been studying the soluble form of aminopeptidase P in human lymphocytes and platelets (4,5), and were interested in determining the nucleotide sequence that would provide useful information for the development of potent and specific inhibitors. Aminopeptidase P is a Proline-specific metallo-aminopeptidase that catalyses specifically the removal of any unsubstituted N-terminal amino acid that is adjacent to a penultimate Proline residue. Due to its specificity towards Proline, it has been suggested that aminopeptidase P is important
Murali Chiravuri - One of the best experts on this subject based on the ideXlab platform.
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vesicular localization and characterization of a novel post Proline cleaving aminoDipeptidase quiescent cell Proline Dipeptidase
Journal of Immunology, 2000Co-Authors: Murali Chiravuri, Henry Lee, Suzanne Mathieu, Fernando A Agarraberes, Brigitte T HuberAbstract:A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell Proline Dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized with a propeptide and undergoes N -glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of post-translational regulation.
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homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell Proline Dipeptidase
Journal of Biological Chemistry, 2000Co-Authors: Murali Chiravuri, Henry Lee, Suzanne Mathieu, Brigitte T HuberAbstract:Quiescent cell Proline Dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.
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sequence purification and cloning of an intracellular serine protease quiescent cell Proline Dipeptidase
Journal of Biological Chemistry, 1999Co-Authors: Robert Underwood, Murali Chiravuri, Henry Lee, Tracy Schmitz, Alisa K Kabcenell, Kurt Yardley, Brigitte T HuberAbstract:We recently observed that specific inhibitors of post-Proline cleaving aminoDipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell Proline Dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-Proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-Proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.
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a novel apoptotic pathway in quiescent lymphocytes identified by inhibition of a post Proline cleaving aminoDipeptidase a candidate target protease quiescent cell Proline Dipeptidase
Journal of Immunology, 1999Co-Authors: Murali Chiravuri, Robert Underwood, Tracy Schmitz, Kurt Yardley, Yogeshwar Dayal, Brigitte T HuberAbstract:The vast majority of lymphocytes in vivo persist in a quiescent state. These resting lymphocytes are maintained through a cellular program that suppresses apoptosis. We show here that quiescent PBMC, but not activated PBMC or transformed lymphocytes, die in the presence of highly specific post-Proline aminoDipeptidase inhibitors. This form of death has the hallmarks of apoptosis, such as phosphatidylserine externalization and loss of mitochondrial transmembrane potential. However, it differs from apoptosis induced by gamma irradiation in the same cells or by Fas ligation in transformed lymphocytes in terms of caspase involvement. In addition, the aminoDipeptidase inhibitor-induced cell death, but not gamma-irradiation-mediated apoptosis, can be prevented by inhibition of the proteasome complex. The target of these inhibitors is not CD26/DPPIV, but probably a novel serine protease, quiescent cell Proline Dipeptidase, that we have recently isolated and cloned. These studies will yield a better understanding of the requirements and the mechanisms that mediate quiescent lymphocyte homeostasis in vivo.
Simon Scharpé - One of the best experts on this subject based on the ideXlab platform.
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Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell Proline Dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7).
The Biochemical journal, 2005Co-Authors: Marie-berthe Maes, Kristel Senten, Pieter Van Der Veken, Koen Augustyns, Kambiz Gilany, Barbara Leiting, Anne-marie Lambeir, Simon Scharpé, Ingrid De MeesterAbstract:The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K(i) approximately 0.9 microM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell Proline Dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/K(m) clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing Proline at the P1 position and lysine at P2. The importance of the P1' group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/K(m)). Lys-Pro-pNA (k(cat)/K(m) 4.1x10(6) s(-1) x M(-1)) and Ala-Pro-pNA (kcat/K(m) 2.6x10(6) s(-1) x M(-1)) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/K(m) 0.4x10(6) s(-1) x M(-1)).
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Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell Proline Dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7)
Biochemical Journal, 2005Co-Authors: Marie-berthe Maes, Kristel Senten, Pieter Van Der Veken, Koen Augustyns, Kambiz Gilany, Barbara Leiting, Anne-marie Lambeir, Simon Scharpé, Ingrid De MeesterAbstract:The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (Ki~0.9 μM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell Proline Dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/Km clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing Proline at the P1 position and lysine at P2. The importance of the P1′ group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/Km). Lys-Pro-pNA (kcat/Km 4.1×106 s−1·M−1) and Ala-Pro-pNA (kcat/Km 2.6×106 s−1· M−1) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/Km 0.4×106 s−1·M−1).
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human lymphocyte x prolyl aminopeptidase aminopeptidase p like protein a new member of the Proline peptidase family
Advances in Experimental Medicine and Biology, 1997Co-Authors: Greet Vanhoof, Katinka Schatteman, Filip Goossens, Dirk Hendriks, Maria Aparecida Juliano, Ingrid De Meester, Luiz Juliano, Simon ScharpéAbstract:Peptidases are grouped according to their mechanism of catalysis in serine-type, cysteine-type, aspartic-type and metallo-type peptidases. Amongst these groups, the metallopeptidases represent the most diverse group, comprising 25 different families (1,2). Most of the Zn-binding metallopeptidases, named zincins, have the HEXXH motif for Zn-binding. Some other Zn-binding motifs have been defined, as there are the HXXEH, the HXXE, and the HXH motif (3). X-prolyl aminopeptidase (aminopeptidase P, EC 3.4.11.9), is an aminopeptidase that has been reported to bind zinc, and is activated by manganese ions, but does not contain any of these Zn-binding motifs. Aminopeptidase P from Escherichia coli was classified in the peptidase family M24, a family of metallopeptidases in which the ligands for metal ion binding are predominantly carboxylic acids (2). Apart from E. coli aminopeptidase P, this family comprises E. coli methionyl aminopeptidase (EC 3.4.11.18) and E. coli and human Proline Dipeptidase (EC 3.4.13.9). We have been studying the soluble form of aminopeptidase P in human lymphocytes and platelets (4,5), and were interested in determining the nucleotide sequence that would provide useful information for the development of potent and specific inhibitors. Aminopeptidase P is a Proline-specific metallo-aminopeptidase that catalyses specifically the removal of any unsubstituted N-terminal amino acid that is adjacent to a penultimate Proline residue. Due to its specificity towards Proline, it has been suggested that aminopeptidase P is important
