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3. N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

Author

Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, van de Wetering, Koen, Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, and van de Wetering, Koen

Abstract

Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.

Published
2015

4. REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Author

Xu, G. (Guotai), Ross Chapman, J., Brandsma, I. (Inger), Yuan, J. (Jingsong), Mistrik, M. (Martin), Bouwman, R.J.P. (Peter), Bartkova, J. (Jirina), Gogola, E. (Ewa), Warmerdam, D.O. (Daniël), Barazas, M. (Marco), Jaspers, J.E. (Janneke), Watanabe, K. (Kenji), Pieterse, M. (Mark), Kersbergen, A. (Ariena), Sol, W. (Wendy), Celie, P.H. (Patrick H.), Schouten, P.C. (Philip), Van Den Broek, B. (Bram), Salman, A. (Ahmed), Nieuwland, M. (Marja), De Rink, I. (Iris), Ronde, J. (Jorma) de, Jalink, K. (Kees), Boulton, S.J. (Simon J.), Chen, J. (Junjie), Gent, D.C. (Dik) van, Bartek, J. (Jiri), Jonkers, J. (Jos), Borst, P. (Piet), Rottenberg, S. (Sven), Xu, G. (Guotai), Ross Chapman, J., Brandsma, I. (Inger), Yuan, J. (Jingsong), Mistrik, M. (Martin), Bouwman, R.J.P. (Peter), Bartkova, J. (Jirina), Gogola, E. (Ewa), Warmerdam, D.O. (Daniël), Barazas, M. (Marco), Jaspers, J.E. (Janneke), Watanabe, K. (Kenji), Pieterse, M. (Mark), Kersbergen, A. (Ariena), Sol, W. (Wendy), Celie, P.H. (Patrick H.), Schouten, P.C. (Philip), Van Den Broek, B. (Bram), Salman, A. (Ahmed), Nieuwland, M. (Marja), De Rink, I. (Iris), Ronde, J. (Jorma) de, Jalink, K. (Kees), Boulton, S.J. (Simon J.), Chen, J. (Junjie), Gent, D.C. (Dik) van, Bartek, J. (Jiri), Jonkers, J. (Jos), Borst, P. (Piet), and Rottenberg, S. (Sven)

Abstract

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Published
2015
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5. N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

Author

Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, van de Wetering, Koen, Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, and van de Wetering, Koen

Published
2015

6. N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

Author

Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, van de Wetering, Koen, Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, and van de Wetering, Koen

Published
2015

7. N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

Author

Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, van de Wetering, Koen, Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Jansen, Robert S, Addie, Ruben, Merkx, Remco, Fish, Alexander, Mahakena, Sunny, Bleijerveld, Onno B, Altelaar, Maarten, IJlst, Lodewijk, Wanders, Ronald J, Borst, P, and van de Wetering, Koen

Published
2015

8. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays.

Author

Biology of Reproductive Cells, Membrane Enzymology, Sub Membrane Enzymology begr. 01-06-12, Dep Biochemie en Celbiologie, Krumpochova, P, Sapthu, S., Brouwers, J.F.H.M., de Haas, M., de Vos, R., Borst, P., van de Wetering, K., Biology of Reproductive Cells, Membrane Enzymology, Sub Membrane Enzymology begr. 01-06-12, Dep Biochemie en Celbiologie, Krumpochova, P, Sapthu, S., Brouwers, J.F.H.M., de Haas, M., de Vos, R., Borst, P., and van de Wetering, K.

Published
2013

10. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays.

Author

Biology of Reproductive Cells, Membrane Enzymology, Sub Membrane Enzymology begr. 01-06-12, Dep Biochemie en Celbiologie, Krumpochova, P, Sapthu, S., Brouwers, J.F.H.M., de Haas, M., de Vos, R., Borst, P., van de Wetering, K., Biology of Reproductive Cells, Membrane Enzymology, Sub Membrane Enzymology begr. 01-06-12, Dep Biochemie en Celbiologie, Krumpochova, P, Sapthu, S., Brouwers, J.F.H.M., de Haas, M., de Vos, R., Borst, P., and van de Wetering, K.

Published
2013

11. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays.

Author

Biology of Reproductive Cells, Membrane Enzymology, Sub Membrane Enzymology begr. 01-06-12, Dep Biochemie en Celbiologie, Krumpochova, P, Sapthu, S., Brouwers, J.F.H.M., de Haas, M., de Vos, R., Borst, P., van de Wetering, K., Biology of Reproductive Cells, Membrane Enzymology, Sub Membrane Enzymology begr. 01-06-12, Dep Biochemie en Celbiologie, Krumpochova, P, Sapthu, S., Brouwers, J.F.H.M., de Haas, M., de Vos, R., Borst, P., and van de Wetering, K.

Published
2013

13. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors

Author

Rottenberg, S. (Sven), Vollebergh, M.A. (Marieke), Hoon, B. (Bas) de, Ronde, J. (Jorma) de, Schouten, P.C. (Philip), Kersbergen, A. (Ariena), Zander, A.R., Pajic, M. (Marina), Jaspers, J.E. (Janneke), Jonkers, M. (Martijn), Lodén, M. (Martin), Sol, W. (Wendy), Burg, E. (Eline) van der, Wesseling, J. (Jelle), Gillet, J.-P. (Jean-Pierre), Gottesman, R.F. (Rebecca), Gribnau, J.H. (Joost), Wessels, L. (Lodewyk), Linn, S.C. (Sabine), Jonkers, J. (Jos), Borst, P. (Piet), Rottenberg, S. (Sven), Vollebergh, M.A. (Marieke), Hoon, B. (Bas) de, Ronde, J. (Jorma) de, Schouten, P.C. (Philip), Kersbergen, A. (Ariena), Zander, A.R., Pajic, M. (Marina), Jaspers, J.E. (Janneke), Jonkers, M. (Martijn), Lodén, M. (Martin), Sol, W. (Wendy), Burg, E. (Eline) van der, Wesseling, J. (Jelle), Gillet, J.-P. (Jean-Pierre), Gottesman, R.F. (Rebecca), Gribnau, J.H. (Joost), Wessels, L. (Lodewyk), Linn, S.C. (Sabine), Jonkers, J. (Jos), and Borst, P. (Piet)

Abstract

The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy.

Published
2012
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14. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays

Author

Krumpochova, P., Sapthu, S., Brouwers, J.F., de Haas, M., de Vos, R.C.H., Borst, P., van de Wetering, K., Krumpochova, P., Sapthu, S., Brouwers, J.F., de Haas, M., de Vos, R.C.H., Borst, P., and van de Wetering, K.

Abstract

The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple “transportomics” approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2-/- mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.—Krumpochova, P., Sapthu, S., Brouwers, J. F., de Haas, M., de Vos, R., Borst, P., van de Wetering, K. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays

Published
2012

21. Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells.

Author

Wielinga, P.R., Reid, G., Challa, E.E., Heijden, I.M. van der, Deemter, L. van, Haas, M. de, Mol, C., Kuil, A.J., Groeneveld, E., Schuetz, J.D., Brouwer, C., Abreu, R.A. de, Wijnholds, J., Beijnen, J.H., Borst, P., Wielinga, P.R., Reid, G., Challa, E.E., Heijden, I.M. van der, Deemter, L. van, Haas, M. de, Mol, C., Kuil, A.J., Groeneveld, E., Schuetz, J.D., Brouwer, C., Abreu, R.A. de, Wijnholds, J., Beijnen, J.H., and Borst, P.

Abstract

Item does not contain fulltext, Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.

Published
2002

22. Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells.

Author

Wielinga, P.R., Reid, G., Challa, E.E., Heijden, I.M. van der, Deemter, L. van, Haas, M. de, Mol, C., Kuil, A.J., Groeneveld, E., Schuetz, J.D., Brouwer, C., Abreu, R.A. de, Wijnholds, J., Beijnen, J.H., Borst, P., Wielinga, P.R., Reid, G., Challa, E.E., Heijden, I.M. van der, Deemter, L. van, Haas, M. de, Mol, C., Kuil, A.J., Groeneveld, E., Schuetz, J.D., Brouwer, C., Abreu, R.A. de, Wijnholds, J., Beijnen, J.H., and Borst, P.

Abstract

Item does not contain fulltext, Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.

Published
2002

23. Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells.

Author

Wielinga, P.R., Reid, G., Challa, E.E., Heijden, I.M. van der, Deemter, L. van, Haas, M. de, Mol, C., Kuil, A.J., Groeneveld, E., Schuetz, J.D., Brouwer, C., Abreu, R.A. de, Wijnholds, J., Beijnen, J.H., Borst, P., Wielinga, P.R., Reid, G., Challa, E.E., Heijden, I.M. van der, Deemter, L. van, Haas, M. de, Mol, C., Kuil, A.J., Groeneveld, E., Schuetz, J.D., Brouwer, C., Abreu, R.A. de, Wijnholds, J., Beijnen, J.H., and Borst, P.

Abstract

Item does not contain fulltext, Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.

Published
2002

25. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drags as judged by interference with nucleotide trapping

Author

Membrane Enzymology, Universiteit Utrecht, Dep Scheikunde, Smith, A.J., van Helvoort, A., van Meer, G., Szabó, K., Welker, E., Szakács, G., Váradi, A., Sarkadi, B., Borst, P., Membrane Enzymology, Universiteit Utrecht, Dep Scheikunde, Smith, A.J., van Helvoort, A., van Meer, G., Szabó, K., Welker, E., Szakács, G., Váradi, A., Sarkadi, B., and Borst, P.

Published
2000

26. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drags as judged by interference with nucleotide trapping

Author

Membrane Enzymology, Universiteit Utrecht, Dep Scheikunde, Smith, A.J., van Helvoort, A., van Meer, G., Szabó, K., Welker, E., Szakács, G., Váradi, A., Sarkadi, B., Borst, P., Membrane Enzymology, Universiteit Utrecht, Dep Scheikunde, Smith, A.J., van Helvoort, A., van Meer, G., Szabó, K., Welker, E., Szakács, G., Váradi, A., Sarkadi, B., and Borst, P.

Published
2000

27. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drags as judged by interference with nucleotide trapping

Author

Membrane Enzymology, Universiteit Utrecht, Dep Scheikunde, Smith, A.J., van Helvoort, A., van Meer, G., Szabó, K., Welker, E., Szakács, G., Váradi, A., Sarkadi, B., Borst, P., Membrane Enzymology, Universiteit Utrecht, Dep Scheikunde, Smith, A.J., van Helvoort, A., van Meer, G., Szabó, K., Welker, E., Szakács, G., Váradi, A., Sarkadi, B., and Borst, P.

Published
2000

29. MDR1 P-glycoprotein is a lipid translocase of broad specifity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine

Author

van Helvoort, A., Smith, A.J., Sprong, H., Fritzsche, I., Schinkel, A.H., Borst, P., van Meer, G., van Helvoort, A., Smith, A.J., Sprong, H., Fritzsche, I., Schinkel, A.H., Borst, P., and van Meer, G.

Abstract

The human MDR1 P-glycoprotein (Pgp) extrudes a variety of drugs across the plasma membrane. The homologous MDR3 Pgp is required for phosphatidylcholine secretion into bile. After stable transfection of epithelial LLC-PK1 cells, MDR1 and MDR3 Pgp were localized in the apical membrane. At 15 degrees C, newly synthesized short-chain analogs of various membrane lipids were recovered in the apical albumin-containing medium of MDR1 cells but not control cells. MDR inhibitors and energy depletion reduced apical release. MDR3 cells exclusively released a short-chain phosphatidylcholine. Since no vesicular secretion occurs at 15 degrees C, the short-chain lipids must have been translocated by the Pgps across the plasma membrane before extraction into the medium by the lipid-acceptor albumin.

Published
1996

37. ADP-ribosyl transferase activity in Trypanosoma brucei.

Author

UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Farzaneh, F, Shall, S, Michels, Paulus, Borst, P., UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Farzaneh, F, Shall, S, Michels, Paulus, and Borst, P.

Abstract

Nuclear adenosine diphosphoribosyl transferase (ADPRT) catalyses the covalent modification of chromatin proteins by (ADP-ribose)n. This activity, which is entirely dependent on DNA containing strand breaks, is required for efficient DNA excision repair possibly because it regulates DNA ligation. ADPRT activity is also required for cytodifferentiation in a number of different cell types. We report here the presence of ADPRT activity in the blood-stream form of Trypanosoma brucei and its activation by DNA strand breaks formed by exposure to, either exogenously supplied deoxyribonuclease I, or treatment with the methylating agent, dimethylsulphate. 3-Aminobenzamide, but not its chemical analogue 3-aminobenzoic acid, is a competitive inhibitor of ADPRT activity in T. brucei. Intact trypanosomes are readily permeable to this competitive inhibitor of ADPRT activity.

Published
1985

40. Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

Author

UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Osinga, K A, Swinkels, B W, Gibson, W C, Borst, P., Veeneman, G H, Van Boom, J H, Michels, Paulus, Opperdoes, Frederik, UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Osinga, K A, Swinkels, B W, Gibson, W C, Borst, P., Veeneman, G H, Van Boom, J H, Michels, Paulus, and Opperdoes, Frederik

Abstract

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.

Published
1985

41. Common elements on the surface of glycolytic enzymes from Trypanosoma brucei may serve as topogenic signals for import into glycosomes.

Author

UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Wierenga, R K, Swinkels, B., Michels, Paulus, Osinga, K., Misset, O., Van Beeumen, J, Gibson, W C, Postma, J P, Borst, P., Opperdoes, Frederik, UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Wierenga, R K, Swinkels, B., Michels, Paulus, Osinga, K., Misset, O., Van Beeumen, J, Gibson, W C, Postma, J P, Borst, P., and Opperdoes, Frederik

Abstract

In Trypanosoma brucei, a major pathogenic protozoan parasite of Central Africa, a number of glycolytic enzymes present in the cytosol of other organisms are uniquely segregated in a microbody-like organelle, the glycosome, which they are believed to reach post-translationally after being synthesized by free ribosomes in the cytosol. In a search for possible topogenic signals responsible for import into glycosomes we have compared the amino acid sequences of four glycosomal enzymes: triosephosphate isomerase (TIM), glyceraldehyde-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and aldolase (ALDO), with each other and with their cytosolic counterparts. Each of these enzymes contains a marked excess of positive charges, distributed in two or more clusters along the polypeptide chain. Modelling of the three-dimensional structures of TIM, PGK and GAPDH using the known structural coordinates of homologous enzymes from other organisms indicates that all three may have in common two 'hot spots' about 40 A apart, which themselves include a pair of basic amino acid residues separated by a distance of about 7 A. The sequence of glycosomal ALDO, for which no three-dimensional information is available, is compatible with the presence of the same configuration on the surface of this enzyme. We propose that this feature plays an essential role in the import of enzymes into glycosomes.

Published
1987

42. Particle-bound enzymes in the bloodstream form of Trypanosoma brucei.

Author

UCL, Opperdoes, Frederik, Borst, P., Spits, H, UCL, Opperdoes, Frederik, Borst, P., and Spits, H

Abstract

We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol-3-phosphate oxidase, ATPase, isocitrate dehydrogenase, acid phosphatase and part of the hyperoxide dismutase and malate dehydrogenase activities were found to be particle-bound after fractionation of homogenates by differential centrifugation. Part of the ATPase activity was sensitive to oligomycin, an inhibitor of oxidative phosphorylation. This oligomycin-sensitive activity can serve as a specific marker for the mitochondria. More than 80% of the NAD+-linked glycerol-3-phosphate dehydrogenase in T. brucei was found to be particulate and latent. The enzyme could be activated by Triton X-100, by the combined action of sonication and salt, but not by salt alone, and partially by freezing and thawing. We conclude that the NAD+-linked glycerol-3-phosphate dehydrogenase is located inside an organelle.

Published
1977

Catalog

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