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3. Human fetal brain self-organizes into long-term expanding organoids

Author

Hendriks, Delilah, Pagliaro, Anna, Andreatta, Francesco, Ma, Ziliang, van Giessen, Joey, Massalini, Simone, López-Iglesias, Carmen, van Son, Gijs J.F., DeMartino, Jeff, Damen, J. Mirjam A., Zoutendijk, Iris, Staliarova, Nadzeya, Bredenoord, Annelien L., Holstege, Frank C.P., Peters, Peter J., Margaritis, Thanasis, Chuva de Sousa Lopes, Susana, Wu, Wei, Clevers, Hans, and Artegiani, Benedetta

Published
2024
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5. Human fetal brain self-organizes into long-term expanding organoids

Author

CMM, CMM Groep Burgering, Cancer, Groep Holstege, Hendriks, Delilah, Pagliaro, Anna, Andreatta, Francesco, Ma, Ziliang, van Giessen, Joey, Massalini, Simone, López-Iglesias, Carmen, van Son, Gijs J.F., DeMartino, Jeff, Damen, J. Mirjam A., Zoutendijk, Iris, Staliarova, Nadzeya, Bredenoord, Annelien L., Holstege, Frank C.P., Peters, Peter J., Margaritis, Thanasis, Chuva de Sousa Lopes, Susana, Wu, Wei, Clevers, Hans, Artegiani, Benedetta, CMM, CMM Groep Burgering, Cancer, Groep Holstege, Hendriks, Delilah, Pagliaro, Anna, Andreatta, Francesco, Ma, Ziliang, van Giessen, Joey, Massalini, Simone, López-Iglesias, Carmen, van Son, Gijs J.F., DeMartino, Jeff, Damen, J. Mirjam A., Zoutendijk, Iris, Staliarova, Nadzeya, Bredenoord, Annelien L., Holstege, Frank C.P., Peters, Peter J., Margaritis, Thanasis, Chuva de Sousa Lopes, Susana, Wu, Wei, Clevers, Hans, and Artegiani, Benedetta

Published
2024

6. Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG)

Author

Sub Biomol.Mass Spectrometry & Proteom., Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Zou, Yang, Pronker, Matti F, Damen, J Mirjam A, Heck, Albert J R, Reiding, Karli R, Sub Biomol.Mass Spectrometry & Proteom., Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Zou, Yang, Pronker, Matti F, Damen, J Mirjam A, Heck, Albert J R, and Reiding, Karli R

Published
2024

7. The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors

Author

Sub Molecular Microbiology, Afd Biomol.Mass Spect. and Proteomics, Molecular Microbiology, Chen, Xiaoyi, Moran Torres, Juan P., Jan Vonk, Peter, Damen, J. Mirjam A., Reiding, Karli R., Dijksterhuis, Jan, Lugones, Luis G., Wösten, Han A.B., Sub Molecular Microbiology, Afd Biomol.Mass Spect. and Proteomics, Molecular Microbiology, Chen, Xiaoyi, Moran Torres, Juan P., Jan Vonk, Peter, Damen, J. Mirjam A., Reiding, Karli R., Dijksterhuis, Jan, Lugones, Luis G., and Wösten, Han A.B.

Published
2024

8. Human fetal brain self-organizes into long-term expanding organoids

Author

Afd Biomol.Mass Spect. and Proteomics, Faculteit Diergeneeskunde, Biomolecular Mass Spectrometry and Proteomics, Hendriks, Delilah, Pagliaro, Anna, Andreatta, Francesco, Ma, Ziliang, van Giessen, Joey, Massalini, Simone, López-Iglesias, Carmen, van Son, Gijs J.F., DeMartino, Jeff, Damen, J. Mirjam A., Zoutendijk, Iris, Staliarova, Nadzeya, Bredenoord, Annelien L., Holstege, Frank C.P., Peters, Peter J., Margaritis, Thanasis, Chuva de Sousa Lopes, Susana, Wu, Wei, Clevers, Hans, Artegiani, Benedetta, Afd Biomol.Mass Spect. and Proteomics, Faculteit Diergeneeskunde, Biomolecular Mass Spectrometry and Proteomics, Hendriks, Delilah, Pagliaro, Anna, Andreatta, Francesco, Ma, Ziliang, van Giessen, Joey, Massalini, Simone, López-Iglesias, Carmen, van Son, Gijs J.F., DeMartino, Jeff, Damen, J. Mirjam A., Zoutendijk, Iris, Staliarova, Nadzeya, Bredenoord, Annelien L., Holstege, Frank C.P., Peters, Peter J., Margaritis, Thanasis, Chuva de Sousa Lopes, Susana, Wu, Wei, Clevers, Hans, and Artegiani, Benedetta

Published
2024

10. Glycoproteomics-Compatible MS/MS-Based Quantification of Glycopeptide Isomers

Author

Maliepaard, Joshua C L, Damen, J Mirjam A, Boons, Geert-Jan P H, Reiding, Karli R, Maliepaard, Joshua C L, Damen, J Mirjam A, Boons, Geert-Jan P H, and Reiding, Karli R

Abstract

Glycosylation is an essential protein modification occurring on the majority of extracellular human proteins, with mass spectrometry (MS) being an indispensable tool for its analysis, that not only determines glycan compositions, but also the position of the glycan at specific sites via glycoproteomics. However, glycans are complex branching structures with monosaccharides interconnected in a variety of biologically relevant linkages, isomeric properties that are invisible when the readout is mass alone. Here, we developed an LC-MS/MS-based workflow for determining glycopeptide isomer ratios. Making use of isomerically defined glyco(peptide) standards, we observed marked differences in fragmentation behavior between isomer pairs when subjected to collision energy gradients, specifically in terms of the galactosylation/sialylation branching and linkage. These behaviors were developed into component variables that allowed for relative quantification of isomerism within mixtures. Importantly, at least for small peptides, the isomer quantification appeared to be largely independent from the peptide portion of the conjugate, allowing a broad application of the method.

Published
2023

11. Glycoproteomics-compatible MS/MS-based quantification of glycopeptide isomers

Author

Afd Biomol.Mass Spect. and Proteomics, Afd Chemical Biology and Drug Discovery, Chemical Biology and Drug Discovery, Biomolecular Mass Spectrometry and Proteomics, Maliepaard, Joshua C.L., Damen, J. Mirjam A., Boons, Geert-Jan P.H., Reiding, Karli R., Afd Biomol.Mass Spect. and Proteomics, Afd Chemical Biology and Drug Discovery, Chemical Biology and Drug Discovery, Biomolecular Mass Spectrometry and Proteomics, Maliepaard, Joshua C.L., Damen, J. Mirjam A., Boons, Geert-Jan P.H., and Reiding, Karli R.

Published
2023

17. GAGE mediates radio resistance in cervical cancers via the regulation of chromatin accessibility

Author

Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Nin, Dawn Sijin, Wujanto, Caryn, Tan, Tuan Zea, Lim, Diana, Damen, J Mirjam A, Wu, Kuan-Yi, Dai, Ziyu Melvin, Lee, Zheng-Wei, Idres, Shabana Binte, Leong, Yiat Horng, Jha, Sudhakar, Ng, Joseph Soon-Yau, Low, Jeffrey J H, Chang, Shih-Chung, Tan, David Shao Peng, Wu, Wei, Choo, Bok Ai, Deng, Lih-Wen, Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Nin, Dawn Sijin, Wujanto, Caryn, Tan, Tuan Zea, Lim, Diana, Damen, J Mirjam A, Wu, Kuan-Yi, Dai, Ziyu Melvin, Lee, Zheng-Wei, Idres, Shabana Binte, Leong, Yiat Horng, Jha, Sudhakar, Ng, Joseph Soon-Yau, Low, Jeffrey J H, Chang, Shih-Chung, Tan, David Shao Peng, Wu, Wei, Choo, Bok Ai, and Deng, Lih-Wen

Published
2021

18. Cysteine Deprivation Targets Ovarian Clear Cell Carcinoma Via Oxidative Stress and Iron-Sulfur Cluster Biogenesis Deficit

Author

Novera, Wisna, Lee, Zheng-Wei, Nin, Dawn Sijin, Dai, Melvin Zi-Yu, Binte Idres, Shabana, Wu, Hui, Damen, J Mirjam A, Tan, Tuan Zea, Sim, Arthur Yi Loong, Long, Yun Chau, Wu, Wei, Huang, Ruby Yun-Ju, Deng, Lih-Wen, Novera, Wisna, Lee, Zheng-Wei, Nin, Dawn Sijin, Dai, Melvin Zi-Yu, Binte Idres, Shabana, Wu, Hui, Damen, J Mirjam A, Tan, Tuan Zea, Sim, Arthur Yi Loong, Long, Yun Chau, Wu, Wei, Huang, Ruby Yun-Ju, and Deng, Lih-Wen

Abstract

Aims: Current treatment options for ovarian clear cell carcinoma (OCCC) are limited to combination of platinum-based and other cytotoxic agents to which patients respond poorly due to intrinsic chemoresistance. There is therefore an urgent need to develop alternative therapeutic strategies for OCCC. Results: Cysteine deprivation suppresses OCCC growth in vitro and in vivo with no apparent toxicity. Modes of cell death induced by cysteine deprivation in OCCC are determined by their innate metabolic profiles. Cysteine-deprived glycolytic OCCC is abolished primarily by oxidative stress-dependent necrosis and ferroptosis, which can otherwise be prevented by pretreatment with antioxidative agents. Meanwhile, OCCC that relies on mitochondria respiration for its bioenergetics is suppressed through apoptosis, which can otherwise be averted by pretreatment with cysteine precursor alone, but not with antioxidative agents. Cysteine deprivation induces apoptosis in respiring OCCC by limiting iron-sulfur (Fe-S) cluster synthesis in the mitochondria, without which electron transport chain may be disrupted. Respiring OCCC responds to Fe-S cluster deficit by increasing iron influx into the mitochondria, which leads to iron overload, mitochondria damage, and eventual cell death. Innovation/Conclusion: This study demonstrates the importance of cysteine availability in OCCC that is for its antioxidative property and its less appreciated role in mitochondria respiration. Regardless of OCCC metabolic profiles, cysteine deprivation abolishes both glycolytic and respiring OCCC growth in vitro and in vivo. Conclusion: This study highlights the therapeutic potential of cysteine deprivation for OCCC.

Published
2020

19. Cysteine Deprivation Targets Ovarian Clear Cell Carcinoma Via Oxidative Stress and Iron-Sulfur Cluster Biogenesis Deficit

Author

Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Novera, Wisna, Lee, Zheng-Wei, Nin, Dawn Sijin, Dai, Melvin Zi-Yu, Binte Idres, Shabana, Wu, Hui, Damen, J Mirjam A, Tan, Tuan Zea, Sim, Arthur Yi Loong, Long, Yun Chau, Wu, Wei, Huang, Ruby Yun-Ju, Deng, Lih-Wen, Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Novera, Wisna, Lee, Zheng-Wei, Nin, Dawn Sijin, Dai, Melvin Zi-Yu, Binte Idres, Shabana, Wu, Hui, Damen, J Mirjam A, Tan, Tuan Zea, Sim, Arthur Yi Loong, Long, Yun Chau, Wu, Wei, Huang, Ruby Yun-Ju, and Deng, Lih-Wen

Published
2020

21. Cysteine Deprivation Targets Ovarian Clear Cell Carcinoma Via Oxidative Stress and Iron−Sulfur Cluster Biogenesis Deficit

Author

Novera, Wisna, primary, Lee, Zheng-Wei, additional, Nin, Dawn Sijin, additional, Dai, Melvin Zi-Yu, additional, Binte Idres, Shabana, additional, Wu, Hui, additional, Damen, J. Mirjam A., additional, Tan, Tuan Zea, additional, Sim, Arthur Yi Loong, additional, Long, Yun Chau, additional, Wu, Wei, additional, Huang, Ruby Yun-Ju, additional, and Deng, Lih-Wen, additional

Published
2020
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22. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

Author

Soni, Siddarth, Raaijmakers, Antonia J A, Raaijmakers, Linsey M, Damen, J Mirjam A, van Stuijvenberg, Leonie, Vos, Marc A, Heck, Albert J R, van Veen, Toon A B, Scholten, Arjen, Soni, Siddarth, Raaijmakers, Antonia J A, Raaijmakers, Linsey M, Damen, J Mirjam A, van Stuijvenberg, Leonie, Vos, Marc A, Heck, Albert J R, van Veen, Toon A B, and Scholten, Arjen

Abstract

AIMS: Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue.METHODS AND RESULTS: General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF) and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2), Nexilin (NEXN), Popeye-domain-containg-protein 2 (POPDC2) and thioredoxin-related-transmembrane-protein 2 (TMX2)) and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes.CONCLUSION: The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart.

Published
2016

23. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

Author

Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Soni, Siddarth, Raaijmakers, Antonia J A, Raaijmakers, Linsey M, Damen, J Mirjam A, van Stuijvenberg, Leonie, Vos, Marc A, Heck, Albert J R, van Veen, Toon A B, Scholten, Arjen, Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spect. and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Soni, Siddarth, Raaijmakers, Antonia J A, Raaijmakers, Linsey M, Damen, J Mirjam A, van Stuijvenberg, Leonie, Vos, Marc A, Heck, Albert J R, van Veen, Toon A B, and Scholten, Arjen

Published
2016

24. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

Author

Medische Fysiologie, Circulatory Health, Soni, Siddarth, Raaijmakers, Antonia J A, Raaijmakers, Linsey M, Damen, J Mirjam A, van Stuijvenberg, Leonie, Vos, Marc A, Heck, Albert J R, van Veen, AAB, Scholten, Arjen, Medische Fysiologie, Circulatory Health, Soni, Siddarth, Raaijmakers, Antonia J A, Raaijmakers, Linsey M, Damen, J Mirjam A, van Stuijvenberg, Leonie, Vos, Marc A, Heck, Albert J R, van Veen, AAB, and Scholten, Arjen

Published
2016

25. Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG).

Author

Yang Zou, Pronker, Matti F., Damen, J. Mirjam A., Heck, Albert J. R., and Reiding, Karli R.

Subjects
*PLASMIN, *CHO cell, *FUCOSYLATION, *NEOVASCULARIZATION inhibitors, *BLOOD proteins
Abstract

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotypedependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG. [ABSTRACT FROM AUTHOR]

Published
2024
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27. Structural characterization and immunogenicity in wildtype and immune tolerant mice of degraded recombinant human interferon alpha2b

Author

Hermeling, Suzanne, Caetano, Liliana Aranha, Damen, J. Mirjam A., Slijper, Monique, Schellekens, Huub, Crommelin, Daan J. A., and Jiskoot, Wim

Subjects
Degraded recombinant human interferon alpha2b, Immunogenicity, Biopharmaceutics
Abstract

Purpose - To study the influence of protein structure on the immunogenicity in wildtype and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). Methods - RhIFNα2b was degraded by metal catalyzed oxidation (M), crosslinking with glutaraldehyde (G), oxidation with hydrogen peroxide (H) and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reversed-phase HPLC, SDS-PAGE, Western blotting and mass spectrometry. The immunogenicity of the products was evaluated in wildtype mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by ELISA or surface plasmon resonance. Results - M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Native (N) rhIFNα2b was immunogenic in the wildtype mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The antirhIFNα2b antibody levels in the wildtype mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ~ N-rhIFNα2b >> B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. Conclusions - RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Published
2005

31. Degradation of azaglycinamido residues in model tripeptides derived from goserelin

Author

Hoitink, Marnix A., Beijnen, Jos H., Bult, Auke, Damen, J. Mirjam A., Houwen, Oeds A. G. J. van der, Kruijtzer, John A. W., Tibben, Matthijs M., Wiese, Gerard, and Underberg, Willy J. M.

Abstract

Three model tripeptides, N-acetyl-Tyr-Pro-azaGly-NH2 (NYPaG), Tyr-Pro-azaGly-NH2 (YPaG), and Tyr-Pro-Gly-NH2(YPG), were subjected to a systematic degradation study to get information about the degradation of the azaglycinamido residue. The degradation products were characterized with LC-MS. Main degradation products of NYPaG possess partially or totally eliminated azaglycinamido residues, while YPaG and YPG are exhibit cyclo(Tyr-Pro) formation, a diketopiperazine. The influence of the pH on the degradation rate constant kobs was investigated for NYPaG and YPaG in the pH range 0.4–11. An U-shaped profile with an inflexion around pH 9 was found for NYPaG while the degradation rate of YPaG was independent of the pH. NYPaG apparently was subject to proton-, solvent-, and hydroxyl-catalyzed degradation reactions whereas YPaG only underwent solvent-catalyzed reactions. Some influence of acetate and phosphate ions on kobs was found for YPaG. Arrhenius plots of NYPaG and YPaG were found to be linear. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 108–114, 2000

Published
2000
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32. Cysteine Deprivation Targets Ovarian Clear Cell Carcinoma Via Oxidative Stress and Iron-Sulfur Cluster Biogenesis Deficit.

Author

Novera W, Lee ZW, Nin DS, Dai MZ, Binte Idres S, Wu H, Damen JMA, Tan TZ, Sim AYL, Long YC, Wu W, Huang RY, and Deng LW

Subjects
Apoptosis, Cell Survival, Female, Ferroptosis, Glutathione metabolism, Humans, Mitochondria metabolism, Models, Biological, Necrosis metabolism, Adenocarcinoma, Clear Cell metabolism, Cysteine metabolism, Iron metabolism, Ovarian Neoplasms metabolism, Oxidative Stress, Sulfur metabolism
Abstract

Aims: Current treatment options for ovarian clear cell carcinoma (OCCC) are limited to combination of platinum-based and other cytotoxic agents to which patients respond poorly due to intrinsic chemoresistance. There is therefore an urgent need to develop alternative therapeutic strategies for OCCC. Results: Cysteine deprivation suppresses OCCC growth in vitro and in vivo with no apparent toxicity. Modes of cell death induced by cysteine deprivation in OCCC are determined by their innate metabolic profiles. Cysteine-deprived glycolytic OCCC is abolished primarily by oxidative stress-dependent necrosis and ferroptosis, which can otherwise be prevented by pretreatment with antioxidative agents. Meanwhile, OCCC that relies on mitochondria respiration for its bioenergetics is suppressed through apoptosis, which can otherwise be averted by pretreatment with cysteine precursor alone, but not with antioxidative agents. Cysteine deprivation induces apoptosis in respiring OCCC by limiting iron-sulfur (Fe-S) cluster synthesis in the mitochondria, without which electron transport chain may be disrupted. Respiring OCCC responds to Fe-S cluster deficit by increasing iron influx into the mitochondria, which leads to iron overload, mitochondria damage, and eventual cell death. Innovation/Conclusion: This study demonstrates the importance of cysteine availability in OCCC that is for its antioxidative property and its less appreciated role in mitochondria respiration. Regardless of OCCC metabolic profiles, cysteine deprivation abolishes both glycolytic and respiring OCCC growth in vitro and in vivo . Conclusion: This study highlights the therapeutic potential of cysteine deprivation for OCCC.

Published
2020
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33. NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network.

Author

Bovenschen N, de Koning PJ, Quadir R, Broekhuizen R, Damen JM, Froelich CJ, Slijper M, and Kummer JA

Subjects
Caspases physiology, Cell Death immunology, Cytoskeletal Proteins metabolism, Cytoskeleton enzymology, Cytoskeleton immunology, Cytoskeleton metabolism, Cytoskeleton pathology, HeLa Cells, Humans, Jurkat Cells, Microtubules enzymology, Microtubules metabolism, Perforin physiology, Signal Transduction immunology, Substrate Specificity immunology, Cytotoxicity, Immunologic, Granzymes physiology, Killer Cells, Natural enzymology, Killer Cells, Natural immunology, Microtubules immunology, Microtubules pathology, Tubulin metabolism
Abstract

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.

Published
2008
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