50 results on '"Cinzia Esposito"'
Search Results
2. Editorial: Novel Strategies in Drug Development Against Multifactorial Diseases
- Author
-
Cinzia Esposito, Catrine Johansson, and Simone Di Micco
- Subjects
multifactorial disease ,drug discovery ,drug resistance ,biochemistry ,drug repurposing ,Chemistry ,QD1-999 - Published
- 2022
- Full Text
- View/download PDF
3. Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function
- Author
-
Thomas Briston, Jenna M. Stephen, Luke W. Thomas, Cinzia Esposito, Yuen-Li Chung, Saiful E. Syafruddin, Mark Turmaine, Lucas A. Maddalena, Basma Greef, Gyorgy Szabadkai, Patrick H. Maxwell, Sakari Vanharanta, and Margaret Ashcroft
- Subjects
Hippel-Lindau protein (pVHL) ,hypoxia inducible factor ,mitochondria ,bioenergetics ,metabolism ,CHCHD4 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Published
- 2021
- Full Text
- View/download PDF
4. CHCHD4 regulates tumour proliferation and EMT-related phenotypes, through respiratory chain-mediated metabolism
- Author
-
Luke W. Thomas, Cinzia Esposito, Jenna M. Stephen, Ana S. H. Costa, Christian Frezza, Thomas S. Blacker, Gyorgy Szabadkai, and Margaret Ashcroft
- Subjects
Coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) ,hypoxia ,HIF-1α ,mitochondria ,respiratory chain ,disulfide relay system ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Abstract Background Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. Results Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. Conclusions CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.
- Published
- 2019
- Full Text
- View/download PDF
5. CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain
- Author
-
Luke W. Thomas, Jenna M. Stephen, Cinzia Esposito, Simon Hoer, Robin Antrobus, Afshan Ahmed, Hasan Al-Habib, and Margaret Ashcroft
- Subjects
Coiled-coil-helix-coiled-coil-helix domain-containing 4 (CHCHD4) ,Hypoxia ,HIF-1α ,Mitochondria ,Respiratory chain ,Disulfide relay system ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Abstract Background Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus, mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is critical for regulating intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers correlates with increased tumour progression and poor patient survival. Results Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I–V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). Conclusions Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology.
- Published
- 2019
- Full Text
- View/download PDF
6. VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function
- Author
-
Thomas Briston, Jenna M. Stephen, Luke W. Thomas, Cinzia Esposito, Yuen-Li Chung, Saiful E. Syafruddin, Mark Turmaine, Lucas A. Maddalena, Basma Greef, Gyorgy Szabadkai, Patrick H. Maxwell, Sakari Vanharanta, and Margaret Ashcroft
- Subjects
von Hippel-Lindau protein (pVHL) ,hypoxia inducible factor ,mitochondria ,bioenergetics ,metabolism ,CHCHD4 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.
- Published
- 2018
- Full Text
- View/download PDF
7. Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia
- Author
-
Stacey Price, Cinzia Esposito, Ultan McDermott, Rachel E. Morgan, Jamie Young, Lucas A. Maddalena, Steven P. Williams, Margaret Ashcroft, Luke W. Thomas, Thomas, Luke W [0000-0002-2246-0361], Ashcroft, Margaret [0000-0002-0066-3707], Apollo - University of Cambridge Repository, and Thomas, Luke W. [0000-0002-2246-0361]
- Subjects
Cancer microenvironment ,0301 basic medicine ,Mitochondrial DNA ,82/29 ,QH301-705.5 ,45/41 ,Medicine (miscellaneous) ,Biology ,Mitochondrion ,631/67/2327 ,Genome informatics ,Genome ,Oxidative Phosphorylation ,General Biochemistry, Genetics and Molecular Biology ,45/29 ,38/43 ,82/80 ,03 medical and health sciences ,0302 clinical medicine ,Neoplasms ,Tumor Cells, Cultured ,Humans ,CRISPR ,Viability assay ,Biology (General) ,Hypoxia ,Gene ,13/89 ,Cell Proliferation ,96/106 ,Cas9 ,Cell growth ,article ,45/77 ,Cancer metabolism ,Mitochondria ,Cell biology ,Genes, Mitochondrial ,030104 developmental biology ,030220 oncology & carcinogenesis ,Genome, Mitochondrial ,631/114/2785 ,38/39 ,CRISPR-Cas Systems ,General Agricultural and Biological Sciences ,82/1 ,Glycolysis ,631/67/327 - Abstract
Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): FP7/2007-2013, Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.
- Published
- 2021
- Full Text
- View/download PDF
8. Establishing standardized immune phenotyping of metastatic melanoma by digital pathology
- Author
-
Bettina Sobottka, Marta Nowak, Anja Laura Frei, Martina Haberecker, Samuel Merki, Mitchell P. Levesque, Reinhard Dummer, Holger Moch, Viktor Hendrik Koelzer, Rudolf Aebersold, Melike Ak, Faisal S. Al-Quaddoomi, Jonas Albinus, Ilaria Alborelli, Sonali Andani, Per-Olof Attinger, Marina Bacac, Daniel Baumhoer, Beatrice Beck-Schimmer, Niko Beerenwinkel, Christian Beisel, Lara Bernasconi, Anne Bertolini, Bernd Bodenmiller, Ximena Bonilla, Ruben Casanova, Stéphane Chevrier, Natalia Chicherova, Maya D'Costa, Esther Danenberg, Natalie Davidson, Monica-Andreea Drăganmoch, Stefanie Engler, Martin Erkens, Katja Eschbach, Cinzia Esposito, André Fedier, Pedro Ferreira, Joanna Ficek, Bruno Frey, Sandra Goetze, Linda Grob, Gabriele Gut, Detlef Günther, Pirmin Haeuptle, Viola Heinzelmann-Schwarz, Sylvia Herter, Rene Holtackers, Tamara Huesser, Anja Irmisch, Francis Jacob, Andrea Jacobs, Tim M. Jaeger, Katharina Jahn, Alva R. James, Philip M. Jermann, André Kahles, Abdullah Kahraman, Werner Kuebler, Jack Kuipers, Christian P. Kunze, Christian Kurzeder, Kjong-Van Lehmann, Sebastian Lugert, Gerd Maass, Markus G. Manz, Philipp Markolin, Julien Mena, Ulrike Menzel, Julian M. Metzler, Nicola Miglino, Emanuela S. Milani, Simone Muenst, Riccardo Murri, Charlotte K.Y. Ng, Stefan Nicolet, Patrick G.A. Pedrioli, Lucas Pelkmans, Salvatore Piscuoglio, Michael Prummer, Mathilde Ritter, Christian Rommel, María L. Rosano-González, Gunnar Rätsch, Natascha Santacroce, Jacobo Sarabia del Castillo, Ramona Schlenker, Petra C. Schwalie, Severin Schwan, Tobias Schär, Gabriela Senti, Franziska Singer, Sujana Sivapatham, Berend Snijder, Vipin T. Sreedharan, Stefan Stark, Daniel J. Stekhoven, Alexandre P.A. Theocharides, Tinu M. Thomas, Markus Tolnay, Vinko Tosevski, Nora C. Toussaint, Mustafa A. Tuncel, Marina Tusup, Audrey Van Drogen, Marcus Vetter, Tatjana Vlajnic, Sandra Weber, Walter P. Weber, Rebekka Wegmann, Michael Weller, Fabian Wendt, Norbert Wey, Andreas Wicki, Mattheus HE Wildschut, Bernd Wollscheid, Shuqing Yu, Johanna Ziegler, Marc Zimmermann, Martin Zoche, Gregor Zuend, University of Zurich, Sobottka-Brillout, Bettina, and Koelzer, Viktor Hendrik
- Subjects
Pathology ,medicine.medical_specialty ,Stromal cell ,610 Medicine & health ,Disease ,Predictive markers ,Article ,Pathology and Forensic Medicine ,1307 Cell Biology ,Immune system ,10049 Institute of Pathology and Molecular Pathology ,1312 Molecular Biology ,Medicine ,Compartment (pharmacokinetics) ,Molecular Biology ,Melanoma ,business.industry ,10177 Dermatology Clinic ,Digital pathology ,10060 Epidemiology, Biostatistics and Prevention Institute (EBPI) ,Cell Biology ,Biomarker (cell) ,2734 Pathology and Forensic Medicine ,10032 Clinic for Oncology and Hematology ,Cohort ,Imaging the immune system ,business ,CD8 - Abstract
CD8+ tumor-infiltrating T cells can be regarded as one of the most relevant predictive biomarkers in immune-oncology. Highly infiltrated tumors, referred to as inflamed (clinically “hot”), show the most favorable response to immune checkpoint inhibitors in contrast to tumors with a scarce immune infiltrate called immune desert or excluded (clinically “cold”). Nevertheless, quantitative and reproducible methods examining their prevalence within tumors are lacking. We therefore established a computational diagnostic algorithm to quantitatively measure spatial densities of tumor-infiltrating CD8+ T cells by digital pathology within the three known tumor compartments as recommended by the International Immuno-Oncology Biomarker Working Group in 116 prospective metastatic melanomas of the Swiss Tumor Profiler cohort. Workflow robustness was confirmed in 33 samples of an independent retrospective validation cohort. The introduction of the intratumoral tumor center compartment proved to be most relevant for establishing an immune diagnosis in metastatic disease, independent of metastatic site. Cut-off values for reproducible classification were defined and successfully assigned densities into the respective immune diagnostic category in the validation cohort with high sensitivity, specificity, and precision. We provide a robust diagnostic algorithm based on intratumoral and stromal CD8+ T-cell densities in the tumor center compartment that translates spatial densities of tumor-infiltrating CD8+ T cells into the clinically relevant immune diagnostic categories “inflamed”, “excluded”, and “desert”. The consideration of the intratumoral tumor center compartment allows immune phenotyping in the clinically highly relevant setting of metastatic lesions, even if the invasive margin compartment is not captured in biopsy material., The authors present a robust diagnostic algorithm based on digital pathology and image analysis that quantifies intratumoral and stromal CD8+ T-cell densities in the tumor center and invasive margin compartment in metastatic melanoma. Spatial CD8+ T-cell densities are translated into the clinically relevant immune diagnostic categories “inflamed”, “excluded”, and “desert”. Their approach also allows efficient immune phenotyping of metastatic lesions, on biopsy material or even in the absence of material from the invasive margin.
- Published
- 2021
- Full Text
- View/download PDF
9. The Tumor Profiler Study: integrated, multi-omic, functional tumor profiling for clinical decision support
- Author
-
René Holtackers, Simone Muenst, Martin Zoche, Rudolf Aebersold, Stéphane Chevrier, Tobias Schär, Bettina Sobottka, Marc Zimmermann, Abdullah Kahraman, Lucas Pelkmans, Faisal Alquaddoomi, Philip Jermann, Natascha Santacroce, Andreas Wicki, Norbert Wey, Nora C. Toussaint, Monica-Andreea Drăgan, Mattheus H.E. Wildschut, Ruben Casanova, Shuqing Yu, Markus Tolnay, Marcus Vetter, Mustafa Anil Tuncel, Ximena Bonilla, Stefan Nicolet, Gabriele Gut, Stefan G. Stark, Philipp Markolin, Bruno S. Frey, Ramona Schlenker, Rebekka Wegmann, Walter P. Weber, Lara Bernasconi, Emanuela S. Milani, Viktor H. Koelzer, Christian Rommel, Christian P. Kunze, Sylvia Herter, Cinzia Esposito, Gabriela Senti, Michael Prummer, Katja Eschbach, Bernd Wollscheid, Riccardo Murri, Salvatore Piscuoglio, Mathilde Ritter, Mitchell P. Levesque, Christian Beisel, Tim M. Jaeger, Viola Heinzelmann-Schwarz, Gunnar Rätsch, Severin Schwan, Marina Bacac, Reinhard Dummer, Joanna Ficek, Sandra Goetze, Tatjana Vlajnic, Martin Erkens, Ilaria Alborelli, Ulrike Menzel, Vinko Tosevski, Markus G. Manz, Werner Kuebler, Detlef Günther, Julian M. Metzler, Daniel J. Stekhoven, Christian Kurzeder, Anja Frei, Tamara Huesser, Marta Nowak, Melike Ak, Francis Jacob, Gregor Zuend, Berend Snijder, Martina Haberecker, Pirmin Haeuptle, Anja Irmisch, Maya D'Costa, Linda Grob, Natalia Chicherova, Bernd Bodenmiller, Johanna Ziegler, Per-Olof Attinger, Jack Kuipers, Katharina Jahn, Nicola Miglino, Natalie R. Davidson, Jacobo Sarabia del Castillo, André Fedier, Fabian Wendt, Esther Danenberg, Pedro Ferreira, María Lourdes Rosano-Gonzalez, Sebastian Lugert, Andrea Jacobs, Charlotte K.Y. Ng, Alva Rani James, Tinu M. Thomas, André Kahles, Gerd Maass, Julien Mena, Jonas B. Albinus, Daniel Baumhoer, Sonali Andani, Petra C. Schwalie, Anne Bertolini, Marina Tusup, Franziska Singer, Alexandre Theocharides, Sandra Weber, Beatrice Beck-Schimmer, Sujana Sivapatham, Kjong-Van Lehmann, Stefanie Engler, Holger Moch, Niko Beerenwinkel, Vipin T. Sreedharan, Michael Weller, Audrey Van Drogen, Patrick G. A. Pedrioli, University of Zurich, Rätsch, Gunnar, and Levesque, Mitchell Paul
- Subjects
0301 basic medicine ,Decision support system ,Cancer Research ,Computer science ,Observational Trial ,Clinical Decision-Making ,610 Medicine & health ,Computational biology ,Clinical decision support system ,1307 Cell Biology ,03 medical and health sciences ,0302 clinical medicine ,Neoplasms ,10049 Institute of Pathology and Molecular Pathology ,Humans ,Profiling (information science) ,1306 Cancer Research ,Prospective Studies ,Precision Medicine ,business.industry ,Computational Biology ,10177 Dermatology Clinic ,10060 Epidemiology, Biostatistics and Prevention Institute (EBPI) ,Cell Biology ,Decision Support Systems, Clinical ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,10032 Clinic for Oncology and Hematology ,2730 Oncology ,Personalized medicine ,business ,11493 Department of Quantitative Biomedicine - Abstract
The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.
- Published
- 2021
- Full Text
- View/download PDF
10. The Tumor Profiler Study: Integrated, multi-omic, functional tumor profiling for clinical decision support
- Author
-
Lucas Pelkmans, Daniel J. Stekhoven, Rudolf Aebersold, Ximena Bonilla, Bernd Bodenmiller, Bettina Sobottka, Rebekka Wegmann, Ulrike Menzel, Stéphane Chevrier, Jack Kuipers, Kjong-Van Lehmann, Franziska Singer, Andrea Jacobs, Mitchell P. Levesque, Sandra Goetze, Anja Irmisch, Marina Bacac, Holger Moch, Emanuela S. Milani, Christian Beisel, Julien Mena, Niko Beerenwinkel, Anja Frei, Viktor H. Koelzer, Michael Prummer, Gabriele Gut, Bernd Wollscheid, Sujana Sivapatham, Markus Tolnay, Stefanie Engler, Francis Jacob, Jacobo Sarabia del Castillo, Nora C. Toussaint, Reinhard Dummer, Berend Snijder, Joanna Ficek, Ruben Casanova, Gunnar Rätsch, Mustafa Anil Tuncel, and Cinzia Esposito
- Subjects
Treatment response ,Targeted ngs ,Profiling (information science) ,Digital pathology ,Genomics ,Computational biology ,Omics ,Clinical decision support system ,Predictive biomarker - Abstract
Recent technological advances allow profiling of tumor samples to an unparalleled level with respect to molecular and spatial composition as well as treatment response. We describe a prospective, observational clinical study performed within the Tumor Profiler (TuPro) Consortium that aims to show the extent to which such comprehensive information leads to advanced mechanistic insights of a patient’s tumor, enables prognostic and predictive biomarker discovery, and has the potential to support clinical decision making. For this study of melanoma, ovarian carcinoma, and acute myeloid leukemia tumors, in addition to the emerging standard diagnostic approaches of targeted NGS panel sequencing and digital pathology, we perform extensive characterization using the following exploratory technologies: single-cell genomics and transcriptomics, proteotyping, CyTOF, imaging CyTOF, pharmacoscopy, and 4i drug response profiling (4i DRP). In this work, we outline the aims of the TuPro study and present preliminary results on the feasibility of using these technologies in clinical practice showcasing the power of an integrative multi-modal and functional approach for understanding a tumor’s underlying biology and for clinical decision support.
- Published
- 2020
11. CHCHD4 regulates a proliferation-EMT switch in tumour cells, through respiratory complex I mediated metabolism
- Author
-
Gyorgy Szabadkai, Christian Frezza, Ana S. H. Costa, Cinzia Esposito, Margaret Ashcroft, Thomas S. Blacker, Jenna M. Stephen, and Luke W. Thomas
- Subjects
0303 health sciences ,biology ,Cell growth ,Chemistry ,Respiratory chain complex ,Respiratory chain ,Vimentin ,mTORC1 ,Gene signature ,Hedgehog signaling pathway ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,030220 oncology & carcinogenesis ,biology.protein ,Intracellular ,030304 developmental biology - Abstract
BACKGROUNDMitochondrial metabolism involves oxidative phosphorylation (OXPHOS) via the respiratory chain and is required for the maintenance of tumour cell proliferation and regulation of epithelial–mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study we interrogate the role of CHCHD4-regulated respiratory chain activity and metabolism in tumour cell proliferation and EMT-related phenotypes.RESULTSWe show that CHCHD4 is essential for the proliferation of tumour cells irrespective of their aetiology. In human tumours, elevated CHCHD4 expression is correlated with a mitochondrial OXPHOS gene signature and with a proliferative gene signature associated with the mTORC1 signalling pathway. Elevated CHCHD4 increases tumour cell proliferation, in a manner that is dependent on complex I (CI) activity, glutamine consumption and mTORC1 activation. CHCHD4 expression is inversely correlated with EMT gene expression both in vitro and in vivo. Finally, we show CHCHD4 regulates the intracellular distribution of the EMT marker vimentin, in a CI-mediated manner.CONCLUSIONSCHCHD4 regulates tumour cell proliferation and metastatic (EMT-related) phenotypes through its control of CI-mediated mitochondrial metabolism.
- Published
- 2019
- Full Text
- View/download PDF
12. CHCHD4 regulates tumour proliferation and EMT-related phenotypes, through respiratory chain-mediated metabolism
- Author
-
Gyorgy Szabadkai, Margaret Ashcroft, Cinzia Esposito, Christian Frezza, Jenna M. Stephen, Luke W. Thomas, Ana S. H. Costa, Thomas S. Blacker, Ashcroft, Margaret [0000-0002-0066-3707], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Cell ,respiratory chain ,Respiratory chain ,HIF-1α ,mTORC1 ,Mitochondrion ,Biology ,Coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) ,lcsh:RC254-282 ,03 medical and health sciences ,0302 clinical medicine ,complex I ,disulfide relay system ,hypoxia ,mitochondria ,tumour growth ,tumour metabolism ,Gene expression ,medicine ,Cell growth ,Research ,Respiratory chain complex ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,3. Good health ,Cell biology ,Psychiatry and Mental health ,030104 developmental biology ,medicine.anatomical_structure ,Cell culture ,030220 oncology & carcinogenesis - Abstract
Background Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. Results Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. Conclusions CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells. Electronic supplementary material The online version of this article (10.1186/s40170-019-0200-4) contains supplementary material, which is available to authorized users.
- Published
- 2019
13. CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain
- Author
-
Cinzia Esposito, Afshan Ahmed, Margaret Ashcroft, Jenna M. Stephen, Luke W. Thomas, Hasan Al-Habib, Simon Hoer, Robin Antrobus, Ashcroft, Margaret [0000-0002-0066-3707], and Apollo - University of Cambridge Repository
- Subjects
Respiratory chain ,HIF-1α ,Oxidative phosphorylation ,Mitochondrion ,lcsh:RC254-282 ,03 medical and health sciences ,0302 clinical medicine ,Tumour metabolism ,Stable isotope labeling by amino acids in cell culture ,Complex I ,medicine ,Glycolysis ,Hypoxia ,030304 developmental biology ,0303 health sciences ,Chemistry ,Research ,Tumour growth ,Hypoxia (medical) ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Cell biology ,Coiled-coil-helix-coiled-coil-helix domain-containing 4 (CHCHD4) ,Mitochondria ,Mitochondrial respiratory chain ,030220 oncology & carcinogenesis ,medicine.symptom ,Intracellular ,Disulfide relay system - Abstract
BACKGROUNDTumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) is critical for maintaining intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers, correlates with increased tumour progression and poor patient survival.RESULTSHere, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I-V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS).CONCLUSIONSOur study highlights an important role for CHCHD4 in regulating tumour cell metabolism, and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology.
- Published
- 2019
14. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
- Author
-
Anna Seelig, Manuel Hellstern, Cinzia Esposito, and Matthias Zwick
- Subjects
0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,biology ,ATP-binding cassette transporter ,Cell Biology ,respiratory system ,Anion channel activity ,Biochemistry ,Cystic fibrosis transmembrane conductance regulator ,respiratory tract diseases ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,ATP hydrolysis ,Membrane Biology ,Chloride channel ,biology.protein ,Biophysics ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Adenosine triphosphate - Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.
- Published
- 2016
15. How phosphorylation and ATPase activity regulate anion flux through the cystic fibrosis transmembrane conductance regulator (CFTR)
- Author
-
Matthias Zwick, Cinzia Esposito, Manuel Hellstern, and Anna Seelig
- Subjects
Adenosine Triphosphatases ,Anions ,Hydrolysis ,Cystic Fibrosis Transmembrane Conductance Regulator ,Cell Biology ,Biochemistry ,Catalysis ,Oxidative Phosphorylation ,Cell Line ,Adenosine Triphosphate ,Cricetinae ,Mutation ,Animals ,Additions and Corrections ,Molecular Biology ,Glycolysis - Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.
- Published
- 2016
16. Dynamic sorting of lipids and proteins in membrane tubes with a moving phase boundary
- Author
-
Michael C. Heinrich, Aiwei Tian, Cinzia Esposito, and Tobias Baumgart
- Subjects
1,2-Dipalmitoylphosphatidylcholine ,Microscopy, Atomic Force ,Models, Biological ,Polar membrane ,Quantitative Biology::Cell Behavior ,Quantitative Biology::Subcellular Processes ,Computer Simulation ,Physics::Biological Physics ,Models, Statistical ,Multidisciplinary ,biology ,Membrane transport protein ,Vesicle ,Proteins ,Biological Transport ,Membrane transport ,Lipids ,Cell biology ,Transport protein ,Protein Transport ,Membrane ,Physical Sciences ,Phosphatidylcholines ,biology.protein ,Biophysics ,Thermodynamics ,Membrane biophysics ,Signal Transduction ,Elasticity of cell membranes - Abstract
Cellular organelle membranes maintain their integrity, global shape, and composition despite vigorous exchange among compartments of lipids and proteins during trafficking and signaling. Organelle homeostasis involves dynamic molecular sorting mechanisms that are far from being understood. In contrast, equilibrium thermodynamics of membrane mixing and sorting, particularly the phase behavior of binary and ternary model membrane mixtures and its coupling to membrane mechanics, is relatively well characterized. Elucidating the continuous turnover of live cell membranes, however, calls for experimental and theoretical membrane models enabling manipulation and investigation of directional mass transport. Here we introduce the phenomenon of curvature-induced domain nucleation and growth in membrane mixtures with fluid phase coexistence. Membrane domains were consistently observed to nucleate precisely at the junction between a strongly curved cylindrical (tube) membrane and a pipette-aspirated giant unilamellar vesicle. This experimental geometry mimics intracellular sorting compartments, because they often show tubular-vesicular membrane regions. Nucleated domains at tube necks were observed to present diffusion barriers to the transport of lipids and proteins. We find that curvature-nucleated domains grow with characteristic parabolic time dependence that is strongly curvature-dependent. We derive an analytical model that reflects the observed growth dynamics. Numerically calculated membrane shapes furthermore allow us to elucidate mechanical details underlying curvature-dependent directed lipid transport. Our observations suggest a novel dynamic membrane sorting principle that may contribute to intracellular protein and lipid sorting and trafficking.
- Published
- 2010
17. Lipid Bilayer Domain Fluctuations as a Probe of Membrane Viscosity
- Author
-
Frank L. H. Brown, Cinzia Esposito, Tobias Baumgart, and Brian A. Camley
- Subjects
Time Factors ,Lipid Bilayers ,Analytical chemistry ,Biophysics ,01 natural sciences ,Models, Biological ,Cell membrane ,Viscosity ,0103 physical sciences ,medicine ,Membrane fluidity ,Lipid bilayer phase behavior ,010306 general physics ,Lipid bilayer ,010304 chemical physics ,Chemistry ,Biophysical Letter ,Vesicle ,Spectrum Analysis ,Relaxation (NMR) ,Cell Membrane ,Membrane ,medicine.anatomical_structure ,Chemical physics ,Hydrodynamics - Abstract
We argue that membrane viscosity, ηm, plays a prominent role in the thermal fluctuation dynamics of micron-scale lipid domains. A theoretical expression is presented for the timescales of domain shape relaxation, which reduces to the well-known ηm = 0 result of Stone and McConnell in the limit of large domain sizes. Experimental measurements of domain dynamics on the surface of ternary phospholipid and cholesterol vesicles confirm the theoretical results and suggest domain flicker spectroscopy as a convenient means to simultaneously measure both the line tension, σ, and the membrane viscosity, ηm, governing the behavior of individual lipid domains.
- Published
- 2010
- Full Text
- View/download PDF
18. Are Acantholysis and Transglutaminase Inhibition Related Phenomena?
- Author
-
A. Lo Schiavo, Anna Cozzolino, Vincenzo Ruocco, Raffaele Porta, Maria Luisa Lombardi, Cinzia Esposito, C., Esposito, V., Ruocco, A., Cozzolino, A., Lo Schiavo, M. L., Lombardi, Porta, Raffaele, Esposito, C, Ruocco, Vincenzo, Cozzolino, A, LO SCHIAVO, Ada, Lombardi, Ml, and Porta, R.
- Subjects
Keratinocytes ,Captopril ,Biochemical Phenomena ,Tissue transglutaminase ,Cystamine ,Angiotensin-Converting Enzyme Inhibitors ,Human skin ,Dermatology ,Binding, Competitive ,Biochemistry ,chemistry.chemical_compound ,Enalapril ,Cell Adhesion ,medicine ,Humans ,Breast ,Disulfides ,Sulfhydryl Compounds ,Enzyme Inhibitors ,Cells, Cultured ,Skin ,Transglutaminases ,integumentary system ,biology ,Acantholysis ,Tiopronin ,medicine.disease ,In vitro ,Pemphigus ,medicine.anatomical_structure ,chemistry ,Enzyme inhibitor ,biology.protein ,Keratinocyte - Abstract
Background: The loss of intercellular cohesion among keratinocytes (acantholysis) may be considered the histologic marker of pemphigus. Many drugs, especially thiol drugs, proved to be able to provoke in vitro acantholysis by biochemical mechanisms interfering with the disulfide and thiol group balance. As to nonthiol drugs, the pathomechanism of acantholysis is still unexplained. Objective: To explain the molecular mechanism of enalapril-induced acantholysis a potential link between transglutaminase (TGase) activity and the effects of this drug was investigated. Methods: TGase activity in extracts from human breast skin cultured in the presence of thiopronine, captopril and enalapril were evaluated in vitro. The acantholytic potential of cystamine, a known TGase inhibitor, was also investigated. Results: Enalapril, the most powerful acantholytic drug in vitro, was found to inhibit both the purified enzyme and the TGase activity in the extracts from cultured human breast skin explants. Kinetic studies showed that enalapril inhibition was competitive with respect to the amino acceptor substrate and uncompetitive with respect to the amino donor substrate. Moreover, an acantholytic effect of cystamine on explants of normal human skin was shown. Conclusions: These results suggest that acantholysis and the inhibition of TGase activity could be two related phenomena.
- Published
- 1996
19. Membrane charge dependent states of the beta-amyloid fragment Abeta (16-35) with differently charged micelle aggregates
- Author
-
Anna Ramunno, Anna Maria D'Ursi, Vittorio Limongelli, Ettore Novellino, Manuela Grimaldi, Cinzia Esposito, Mario Scrima, Gerardino D'Errico, Giuseppe Vitiello, M., Grimaldi, M., Scrima, C., Esposito, Vitiello, Giuseppe, A., Ramunno, Limongelli, Vittorio, D'Errico, Gerardino, Novellino, Ettore, and A. M., D'Ursi
- Subjects
Circular dichroism ,Amyloid peptide ,spectroscopy ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Molecular Sequence Data ,Static Electricity ,Biophysics ,Peptide ,Micelle ,Biochemistry ,Protein structure ,NMR spectroscopy ,Static electricity ,micelle ,Organic chemistry ,Computer Simulation ,Amino Acid Sequence ,Spin-labeled peptide ,Spin label ,Protein Structure, Quaternary ,Micelles ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Circular Dichroism ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,amyloid ,Cell Biology ,Random coil ,Peptide Fragments ,Membrane ,chemistry ,Micelle solution ,Indicators and Reagents ,Spin Labels ,EPR spectroscopy - Abstract
Abeta (16-35) is the hydrophobic central core of beta-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, beta-amyloid peptides undergo a conformational transition from random coil or alpha-helical monomers, to highly toxic beta-sheet oligomers and aggregate fibrils. The behavior of beta-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Abeta (16-35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles "doped" with small amounts of SDS. To analyze the tendency of Abeta (16-35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Abeta (16-35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Abeta (16-35) undergoes a conformational transition from a soluble helical-kink-helical structure, to a U-turn shaped conformation that resembles protofibril models.
- Published
- 2009
20. Bending Stiffness Depends on Curvature of Ternary Lipid Mixture Tubular Membranes
- Author
-
Benjamin R. Capraro, Aiwei Tian, Tobias Baumgart, and Cinzia Esposito
- Subjects
Lipid Bilayers ,Analytical chemistry ,Biophysics ,Thermodynamics ,Complex Mixtures ,Curvature ,01 natural sciences ,Quantitative Biology::Cell Behavior ,Quantitative Biology::Subcellular Processes ,03 medical and health sciences ,0103 physical sciences ,010306 general physics ,Lipid bilayer ,030304 developmental biology ,0303 health sciences ,Physics::Biological Physics ,Chemistry ,Cell Membrane ,Membrane ,Lipids ,Biomechanical Phenomena ,Tube bending ,Membrane curvature ,Bending stiffness ,Linear Models ,Emulsions ,Ternary operation ,Membrane biophysics - Abstract
Lipid and protein sorting and trafficking in intracellular pathways maintain cellular function and contribute to organelle homeostasis. Biophysical aspects of membrane shape coupled to sorting have recently received increasing attention. Here we determine membrane tube bending stiffness through measurements of tube radii, and demonstrate that the stiffness of ternary lipid mixtures depends on membrane curvature for a large range of lipid compositions. This observation indicates amplification by curvature of cooperative lipid demixing. We show that curvature-induced demixing increases upon approaching the critical region of a ternary lipid mixture, with qualitative differences along two roughly orthogonal compositional trajectories. Adapting a thermodynamic theory earlier developed by M. Kozlov, we derive an expression that shows the renormalized bending stiffness of an amphiphile mixture membrane tube in contact with a flat reservoir to be a quadratic function of curvature. In this analytical model, the degree of sorting is determined by the ratio of two thermodynamic derivatives. These derivatives are individually interpreted as a driving force and a resistance to curvature sorting. We experimentally show this ratio to vary with composition, and compare the model to sorting by spontaneous curvature. Our results are likely to be relevant to the molecular sorting of membrane components in vivo.
- Published
- 2009
21. The role of cell penetrating peptides (CPPs) in membrane lipid phase behavior: a novel aspect elucidating peptide-mediated delivery
- Author
-
Cinzia, Esposito, AiWei, Tian, Mario, Scrima, Karolina, Kieres, Pietro, Campiglia, Anna Maria, D'Ursi, and Tobias, Baumgart
- Subjects
Membrane Lipids ,Cell Membrane Permeability ,Drug Delivery Systems ,Microscopy, Confocal ,Microscopy, Fluorescence ,Molecular Sequence Data ,Amino Acid Sequence ,Peptides - Published
- 2009
22. The role of Cell Penetrating Peptides (CPPs) in membrane lipid phase behavior: a novel aspect elucidating peptide-mediated delivery
- Author
-
Pietro Campiglia, Cinzia Esposito, Anna Maria D'Ursi, Tobias Baumgart, Mario Scrima, Aiwei Tian, and Karolina Kieres
- Subjects
chemistry.chemical_classification ,Membrane ,medicine.anatomical_structure ,Biochemistry ,Chemistry ,Phase (matter) ,Cell ,medicine ,Peptide ,Lipid raft - Published
- 2009
23. The role of Cell Penetrating Peptides (CPPs) in membrane lipid phase behavior: a novel aspect elucidating peptide-mediated deliveryPeptides for Youth
- Author
-
Cinzia, Esposito, Aiwei, Tian, Mario, Scrima, Karolina, Kieres, Campiglia, Pietro, D'Ursi, Anna Maria, and Tobias, Baumgart
- Published
- 2009
24. Structures and micelle locations of the nonlipidated and lipidated C-terminal membrane anchor of 2’,3’-cyclic nucleotide-3’-phosphodiesterase
- Author
-
Anna Maria D'Ursi, Gerardino D'Errico, Mario Scrima, Paolo Rovero, Annamaria Tedeschi, Alfonso Carotenuto, Anna Maria Malfitano, Maurizio Bifulco, Cinzia Esposito, Esposito, C., Scrima, M., Carotenuto, Alfonso, Tedeschi, A., Rovero, P., D'Errico, Gerardino, Malfitano, A. M., Bifulco, M., and A. M. D. ’. U. R. S. I.
- Subjects
Models, Molecular ,Spectrometry, Mass, Electrospray Ionization ,Magnetic Resonance Spectroscopy ,Lipid-anchored protein ,Peptide ,Biochemistry ,2',3'-Cyclic-nucleotide 3'-phosphodiesterase ,Prenylation ,micelle ,lipidated peptide ,Nucleotide ,Computer Simulation ,Chromatography, High Pressure Liquid ,Micelles ,chemistry.chemical_classification ,Chemistry ,C-terminus ,Circular Dichroism ,Cell Membrane ,Site-directed spin labeling ,NMR ,Protein Structure, Tertiary ,Membrane ,2',3'-Cyclic-Nucleotide Phosphodiesterases ,Peptides ,Protein Binding - Abstract
2,3'-Cyclic nucleotide-3'-phosphodiesterase (CNP) is a myelin-associated protein, an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate is still unknown. Recently, it was found that CNP is a possible linker protein between microtubules and the plasma membranes. Since CNP is modified post-translationally by an isoprenylation process at its C terminus, the prenylation is hypothesized to be a requisite process, which permanently anchors CNP to the plasma membrane. This study investigates the molecular mechanism of the interaction between CNP and the plasma membrane, proposing a general model to interpret the structural bases of prenylated proteins binding to the membrane. A 13 residue, C-terminal CNP fragment, C13, was demonstrated to be directly responsible for CNP membrane anchoring. C13 and its lipidated derivative (LIPO-C13) were subjected to conformational analysis in membrane mimetic environments, by means of CD and NMR spectroscopies. The orientation of C13 in relation to the membrane was investigated by NMR and EPR spin labeling studies. Our structural investigation shows that the presence of the lipidic tail is essential for the peptide to be folded and correctly positioned on the membrane surface. A general model is proposed in which the post-translational lipidation is an important biomolecular trick to enlarge the hydrophobic surface and to enable the contact of the protein with membrane.
- Published
- 2008
25. Obestatin conformational features: a strategy to unveil obestatin's biological role?
- Author
-
Pietro Campiglia, Cinzia Esposito, Anna Maria D'Ursi, Isabel Gomez-Monterrey, Ettore Novellino, Mario Scrima, M., Scrima, P., Campiglia, C., Esposito, GOMEZ MONTERREY, ISABEL MARIA, Novellino, Ettore, and A. M. D. ’. U. r. s., I.
- Subjects
Models, Molecular ,Food intake ,Protein Conformation ,Molecular Sequence Data ,Biophysics ,Neuropeptide ,Biochemistry ,Protein Structure, Secondary ,In vivo ,Animals ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Protein secondary structure ,Nuclear Magnetic Resonance, Biomolecular ,G protein-coupled receptor ,Chemistry ,Circular Dichroism ,Cell Biology ,Obestatin ,Ghrelin ,Peptide Fragments ,Protein Structure, Tertiary ,Rats ,Rat Stomach - Abstract
Obestatin and its derivative Ob(11-23) are recently discovered peptides produced in the rat stomach. They have proven to be involved in the regulation of energy balance, inhibiting feeding, causing reductions in food intake, body weight and jejunal contraction in rodents. The G-protein coupled receptor, GPR39, was originally proposed as being an obestatin target receptor, but this remains controversial. As such, the molecular mechanism for obestatin's effects in vivo is still uncertain. Here we report the CD and NMR conformational analysis of obestatin and Ob(11-23). Both peptides assume a regular secondary structure in the C-terminal region of the molecule. In this region, structural elements similar to other GPCR binding neuropeptides support the identity of obestatin as a new and functionally autonomous GPCR ligand. Conversely sequence and conformational specificity point to a new farmacoforic structure, on which innovative derivatives with a potential role in the treatment of obesity can be designed and synthetized.
- Published
- 2007
26. Antibodies generated in cats by a lipopeptide reproducing the membrane-proximal external region of the feline immunodeficiency virus transmembrane enhance virus infectivity
- Author
-
Anna Maria D'Ursi, Mario Scrima, Cinzia Esposito, Elisa Zabogli, Paolo Rovero, Giulia Freer, Mauro Bendinelli, and Simone Giannecchini
- Subjects
Microbiology (medical) ,Feline immunodeficiency virus ,Magnetic Resonance Spectroscopy ,Protein Conformation ,viruses ,Lipoproteins ,Clinical Biochemistry ,Immunology ,Context (language use) ,Immunodeficiency Virus, Feline ,Antibodies, Viral ,Virus ,Veterinary Immunology ,chemistry.chemical_compound ,Neutralization Tests ,Immunology and Allergy ,Animals ,Micelles ,Infectivity ,Membrane Glycoproteins ,biology ,Immunogenicity ,Circular Dichroism ,Cell Membrane ,Virion ,Lipopeptide ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Virology ,Transmembrane protein ,Specific Pathogen-Free Organisms ,chemistry ,biology.protein ,Cats ,Immunization ,Antibody ,Peptides - Abstract
The immunogenicity of a lipoylated peptide (lipo-P59) reproducing the membrane-proximal external region (MPER) of the transmembrane glycoprotein of feline immunodeficiency virus (FIV) was investigated with cats. In the attempt to mimic the context in which MPER is located within intact virions, lipo-P59 was administered in association with membrane-like micelles. Analyses showed that in this milieu, lipo-P59 had a remarkable propensity to be positioned at the membrane interface, displayed a large number of ordered structures folded in turn helices, and was as active as lipo-P59 alone at inhibiting FIV infectivity in vitro. The antibodies developed differed from the ones previously obtained by immunizing cats with the nonlipoylated version of the peptide (G. Freer, S. Giannecchini, A. Tissot, M. F. Bachmann, P. Rovero, P. F. Serres, and M. Bendinelli, Virology 322:360-369, 2004) in epitope specificity and in the fact that they bound FIV virions. However, they too lacked virus-neutralizing activity and actually enhanced FIV infectivity for lymphoid cell cultures. It is concluded that the use of MPER-reproducing oligopeptides is not a viable approach for vaccinating against FIV.
- Published
- 2007
27. Driving forces in the delivery of penetratin conjugated G protein fragment
- Author
-
Anna Maria D'Ursi, Maria R. Mazzoni, Francesca Porchia, Paolo Rovero, Laura Giusti, G. Caliendo, Cinzia Esposito, Stefania Albrizio, Ettore Novellino, Gerardino D'Errico, Albrizio, Stefania, L., Giusti, D'Errico, Gerardino, C., Esposito, F., Porchia, G., Caliendo, Novellino, Ettore, M. R., Mazzoni, P., Rovero, and A. M., D’Ursi
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Stereochemistry ,G protein ,Phosphorylcholine ,Static Electricity ,chimera peptide ,Peptide ,Cell-Penetrating Peptides ,Fluorescence ,Permeability ,chemistry.chemical_compound ,Surface-Active Agents ,Drug Discovery ,Fluorescence microscope ,GTP-Binding Protein alpha Subunits, Gs ,Sodium dodecyl sulfate ,Micelles ,chemistry.chemical_classification ,Vesicle ,Circular Dichroism ,Proteins ,Sodium Dodecyl Sulfate ,Membranes, Artificial ,Membrane transport ,Peptide Fragments ,Membrane ,chemistry ,Microscopy, Fluorescence ,Dipalmitoylphosphatidylcholine ,drug delivery ,liposome ,Molecular Medicine ,Carrier Proteins ,Peptides ,Hydrophobic and Hydrophilic Interactions - Abstract
A42 is a chimera peptide consisting of Galphas(374-394)C379A--the 21-mer C terminus of the Galphas protein, able of adenosine inhibitory activity--and penetratin--the 16 residue fragment, derived from the homeodomain of the Drosophila transcription factor Antennapedia. A42 is able to cross cell membranes and to inhibit A2A and A2B adenosine and beta-adrenergic receptor stimulated camps (D'Ursi et al. Mol. Pharmacol. 2006, 69, 727-36). Here we present an extensive biophysical study of A42 in different membrane mimetics, with the objective to evaluate the molecular mechanisms which promote the membrane permeation. Fluorescence, CD, and NMR data were acquired in the presence of negatively charged and zwitterionic sodium dodecyl sulfate and dodecylphosphocholine surfactants. To validate the spectroscopic results in a larger scale, fluorescence microscopy experiments were performed on negatively charged and zwitterionic dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles. Our results show that the internalization of A42 is mainly driven by electrostatic interactions, hydrophobic interactions playing only a secondary, sinergistic role. The distribution of the charges along the molecule has an important role, highlighting that internalization is a process which requires a specific matching of peptide and membrane properties.
- Published
- 2007
28. Physicochemical characterization of a peptide deriving from the glycoprotein gp36 of the feline immunodeficiency virus and its lipoylated analogue in micellar systems
- Author
-
Anna Maria D'Ursi, Simone Giannecchini, Paolo Rovero, Mauro Bendinelli, Gerardino D'Errico, Cinzia Esposito, Maria Rosaria Armenante, C., Esposito, D'Errico, Gerardino, M. R., Armenante, S., Giannecchini, M., Bendinelli, P., Rovero, and A. M., D'Ursi
- Subjects
Feline immunodeficiency virus ,Protein Conformation ,Lipoproteins ,Phosphorylcholine ,Biophysics ,Peptide ,Immunodeficiency Virus, Feline ,Conformational analysi ,Antiviral Agents ,Biochemistry ,Lipopeptide ,Diffusion ,chemistry.chemical_compound ,Viral Envelope Proteins ,Nuclear Magnetic Resonance, Biomolecular ,Micelles ,Glycoproteins ,chemistry.chemical_classification ,biology ,Circular Dichroism ,Electron Spin Resonance Spectroscopy ,Sodium Dodecyl Sulfate ,Cell Biology ,biology.organism_classification ,In vitro ,FIV ,Conformational analysis ,Spectrometry, Fluorescence ,Membrane ,chemistry ,Viral replication ,Cell culture ,Micelle interface ,Glycoprotein - Abstract
P59 is the Trp-rich 20-mer peptide ( 767 L–G 786 ), partial sequence of the membrane-proximal external region (MPER) of the FIV gp36. It has potent antiviral activity, possibly due to a mechanism that inhibits the fusion of the virus with the cell membranes. In the hypothesis that a lipophilic tail could enhance the adhesion of P59 to the membrane so improving its antiviral activity, we synthesized its lipoylated analogue lipo-P59. Fluorescence, CD and NMR investigations in membrane mimicking environments (such as SDS and DPC micelles) were aimed to assess the potential of the lipo-P59 lipophilic tail to affect the biophysical and conformational behaviour of the peptide. In vitro inhibitory assays using lymphoid cell cultures to check the antiviral activity of peptides were also performed. The data show that the biophysical properties and the conformational preferences of the peptides are not dramatically affected by the hydrophobic tail, suggesting that the lipopeptide is capable of preserving all the biophysical peculiarities. Similarly, antiviral experimental data show that the membrane-anchored lipo-P59 peptide is also effective in inhibiting virus replication. Moreover, the lipophilic tail allows P59 to preserve its antiviral activity even in conditions in which the non lipoylated peptide is devoid of activity. In accordance with the unusual high Trp presence, the peptides confirm the preference to be positioned on the membrane interface. Furthermore, the data point out a peculiarity of interaction of the peptides with SDS as compared with DPC.
- Published
- 2006
29. Exploring interaction of β-amyloid segment (25-35) with membrane models through paramagnetic probes
- Author
-
M. Francesca Ottaviani, Mario Scrima, Gerardino D'Errico, Annamaria Tedeschi, Paolo Rovero, Cinzia Esposito, Anna Maria D'Ursi, C., Esposito, A., Tedeschi, M., Scrima, D'Errico, Gerardino, M. F., Ottaviani, P., Rovero, and A. M., D’Ursi
- Subjects
Circular dichroism ,micelles ,Synthetic membrane ,Peptide ,Biochemistry ,Structural Biology ,Drug Discovery ,Drug Interactions ,Senile plaques ,Molecular Biology ,Peptide sequence ,Pharmacology ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Circular Dichroism ,β-amyloid(25–35) ,Organic Chemistry ,Electron Spin Resonance Spectroscopy ,P3 peptide ,General Medicine ,Peptide Fragments ,Peptide Conformation ,Amino acid ,electron paramagnetic resonance ,chemistry ,Liposomes ,liposome ,Biophysics ,Molecular Medicine - Abstract
The accumulation of β-amyloid peptides into senile plaques is one of the hallmarks of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the β-sheet oligomerization process of β-amyloid. Aβ(25–35), the sequence of which is GSNKGAIIGLM, is a highly toxic segment of amyloid β (Aβ)-peptides, which forms fibrillary aggregates. In the present work, two spin-labelled Aβ(25–35) analogues containing the nitroxide group of the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe at the N- or the C-terminus of the peptide sequence, respectively, were synthesized in order to investigate the peptide-membrane interaction. The orientation and associated changes of the peptide conformation in the presence of different artificial membrane models (micelles, liposomes) were evaluated by electron paramagnetic resonance and circular dichroism techniques. The results of this study allowed us to propose a model in which the C-terminal portion of the peptide is highly associated to the membrane, while the N-terminal part extends into the aqueous phase with occasional contacts with the lipid head-group region. Interestingly, the interaction of the C-terminal portion of the peptide is particularly enhanced in the presence of sodium dodecyl sulfate (SDS) molecules. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.
- Published
- 2006
30. A membrane-permeable peptide containing the last 21 residues of the G alpha(S) carboxyl terminus inhibits G(S)-coupled receptor signaling in intact cells: Correlations between peptide structure and biological activity
- Author
-
Laura Giusti, Annamaria D'ursi, Antonio Lucacchini, Claudia Gargini, Francesca Porchia, Paolo Rovero, Cinzia Esposito, Stefania Albrizio, Maria Rosa Mazzoni, G. Caliendo, Ettore Novellino, D'Ursi, Am, Giusti, L, Albrizio, Stefania, Porchia, F, Esposito, C, Caliendo, G, Gargini, C, Novellino, Ettore, Lucacchini, A, Rovero, P, and Mazzoni, Mr
- Subjects
Magnetic Resonance Spectroscopy ,Protein Conformation ,Peptide ,Adenosine-5'-(N-ethylcarboxamide) ,Cell-Penetrating Peptides ,PC12 Cells ,Adenylyl cyclase ,chemistry.chemical_compound ,structural studies ,MULTIPLE ,Cyclic AMP ,GTP-Binding Protein alpha Subunits, Gs ,POSITION ,Internalization ,Receptor ,Protein secondary structure ,media_common ,chemistry.chemical_classification ,SECONDARY STRUCTURE ,Chemistry ,peptide ,Biochemistry ,NMR-SPECTROSCOPY ,Molecular Medicine ,A(2A) ADENOSINE RECEPTOR ,Signal transduction ,Signal Transduction ,PROTEIN ALPHA-SUBUNITS ,ENDOTHELIAL-CELLS ,ADENYLYL-CYCLASE ,MICELLES ,DOMAINS ,G-protein ,media_common.quotation_subject ,Recombinant Fusion Proteins ,Endocytosis ,Permeability ,Structure-Activity Relationship ,Animals ,Humans ,Pharmacology ,Cell Membrane ,Isoproterenol ,Membrane Proteins ,Proteins ,NMR ,Peptide Fragments ,Rats ,N-terminus ,Endothelium, Vascular ,Carrier Proteins ,Peptides - Abstract
Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of G alpha(s). This G alpha(s) peptide was an effective inhibitor of 5'-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect. The maximal efficacy of NECA was substantially reduced when PC12 cells were treated with the chimeric peptide, suggesting that it competes with G alpha(s) for interaction with receptors. The peptide inhibited neither G(q)- nor G(i)-coupled receptor signaling. The use of a carboxy-fluorescein derivative of the peptide proved its ability to cross the plasma membrane of live cells. NMR analysis of the chimeric peptide structure in a membrane-mimicking environment showed that the G alpha(s) fragment assumed an amphipathic alpha-helical conformation tailored to make contact with key residues on the intracellular side of the receptor. The N-terminal penetratin portion of the molecule also showed an alpha-helical structure, but hydrophobic and hydrophilic residues formed clustered surfaces at the N terminus and center of the fragment, suggesting their involvement in the mechanism of penetratin internalization by endocytosis. Our biological data supported by NMR analysis indicate that the membrane-permeable G alpha(s) peptide is a valuable, nontoxic research tool to modulate G(s)-coupled receptor signal transduction in cell culture models.
- Published
- 2006
31. Retro-Inverso Analogue of the Antiviral Octapeptide C8 Inhibits Feline Immunodeficiency Virus in Serum
- Author
-
Cinzia Esposito, Alfonso Carotenuto, Maria Rosaria Armenante, Mauro Bendinelli, Paolo Rovero, Simone Giannecchini, Anna Maria D'Ursi, Armida Di Fenza, D'Ursi, A. M., Giannecchini, S., DI FENZA, A., Esposito, C., Armenante, M. R., Carotenuto, Alfonso, Bendinelli, M., and Rovero, P.
- Subjects
Models, Molecular ,Feline immunodeficiency virus ,Magnetic Resonance Spectroscopy ,Molecular Conformation ,Retroviridae Proteins ,Peptide ,Immunodeficiency Virus, Feline ,Virus Replication ,Antiviral Agents ,Virus ,chemistry.chemical_compound ,Drug Stability ,Drug Discovery ,Peptide synthesis ,Animals ,chemistry.chemical_classification ,Membrane Glycoproteins ,biology ,Tryptophan ,Biological activity ,biology.organism_classification ,Virology ,In vitro ,Enzyme ,chemistry ,Lentivirus ,Cats ,Molecular Medicine ,Oligopeptides - Abstract
We described the antiviral activity of an octapeptide corresponding to a Trp-rich domain of feline immunodeficiency virus (FIV) transmembrane glycoprotein. To overcome the limited enzymatic stability of short peptides, the retroinverso analogue was prepared and tested for inhibitory activity of FIV in the presence or absence of normal cat serum. Differently from the unmodified peptide, the retroinverso analogue maintains strong inhibitory activity in serum. NMR studies showed that it displays crucial conformational features believed to be important for antiviral activity.
- Published
- 2003
32. Bending Stiffness and Curvature Coupling of Ternary Lipid Mixtures
- Author
-
Tobias Baumgart, Cinzia Esposito, Benjamin R. Capraro, and Aiwei Tian
- Subjects
Quantitative Biology::Biomolecules ,Physics::Biological Physics ,Phase boundary ,Chemistry ,Biophysics ,Analytical chemistry ,Mechanics ,Curvature ,Quantitative Biology::Cell Behavior ,Quantitative Biology::Subcellular Processes ,Membrane ,Membrane curvature ,Bending stiffness ,Perpendicular ,Ternary operation ,Elasticity of cell membranes - Abstract
There exists a wide range of curvature gradients within and between cellular organelles. Differences between membrane morphologies play important roles in cell homeostasis, for example, in the sorting and trafficking of membrane components, as well as in controlling the activities of membrane associated proteins. To better understand the mechanisms by which curvature regulates cellular functions, here, we investigate membrane curvature coupling to membrane composition and mechanical properties.We find that bending stiffness depends on membrane curvature of micro-scale homogeneous ternary lipid mixtures. Curvature gradients were generated by lipid tethers with controllable radius pulled from giant vesicles, and bending stiffness was obtained from tether radius and membrane tension measurements. As curvature increases, bending energy overcomes mixing entropy such that highly flexible lipid groups are sorted into the tube from the flat membrane. The sorting is enhanced as composition approaches the neighborhood of the mixing-demixing critical point, through two trajectories: parallel and perpendicular to the phase boundary. An expression that predicts bending stiffness to be a quadratic function of curvature in ternary mixture is derived, from which curvature sorting efficiency is obtained. We then interpret the sorting efficiency to be the ratio of a driving force for and a resistance to sorting. In addition, we estimate the bending stiffness of ternary mixtures at zero curvature, finding consistency with our measurements from the micropipette aspiration method.
- Published
- 2010
33. GTPase and transglutaminase are associated in the secretion of the rat anterior prostate
- Author
-
Emilio Chiosi, G. Illiano, Annamaria Spina, Anna Cozzolino, Cinzia Esposito, Raffaele Porta, Loredana Mariniello, M Pagano, Spina, Annamaria, Esposito, C, Pagano, M, Chiosi, Emilio, Mariniello, L, Cozzolino, A, Porta, R, Illiano, G., Spina, A. M., Esposito, C., Pagano, M., Chiosi, E., Mariniello, Loredana, Cozzolino, A., and Porta, Raffaele
- Subjects
Male ,GTP' ,Tissue transglutaminase ,Biophysics ,GTPase ,Biology ,Biochemistry ,Chromatography, Affinity ,GTP Phosphohydrolases ,Prostate ,medicine ,Animals ,Secretion ,Rats, Wistar ,Molecular Biology ,chemistry.chemical_classification ,Transglutaminases ,Hydrolysis ,Substrate (chemistry) ,Cell Biology ,Molecular biology ,Rats ,Molecular Weight ,Enzyme ,medicine.anatomical_structure ,Secretory protein ,chemistry ,biology.protein ,Chromatography, Gel ,Electrophoresis, Polyacrylamide Gel ,Guanosine Triphosphate - Abstract
We have found that in the secretion of rat anterior prostate, a hydrolyzing activity on GTP is present with a high affinity for the substrate; ATP, GDP, and ADP are not substrates for enzymatic activity. At the same time we have shown that GTP is a negative modulator for the well-known type IV transglutaminase activity present in the prostatic secretion. The hydrolyzing activity on GTP appears to be due to two molecular species: a high-molecular-weight GTPase, having electrophoretical mobility higher than 100 kDa, and a low-molecular-weight GTPase, of about 30 kDa. The two enzymatic activities are associated in the prostatic secretion with the transglutaminase (type IV). We describe an experimental procedure to separate them.
- Published
- 1999
34. The in vitro effect of hydroxychloroquine on skin morphology and transglutaminase
- Author
-
A. Lo Schiavo, M.L. Lombardi, Cinzia Esposito, Vincenzo Ruocco, Ronni Wolf, Wolf, R, LO SCHIAVO, Ada, Lombardi, Ml, Esposito, C, and Ruocco, Vincenzo
- Subjects
medicine.medical_specialty ,Tissue transglutaminase ,Human skin ,Dermatology ,Antimalarials ,Reference Values ,Culture Techniques ,Psoriasis ,Internal medicine ,Keratin ,Humans ,Medicine ,Barrier function ,Skin ,Hydroxychloroquine Sulfate ,chemistry.chemical_classification ,Transglutaminases ,Dose-Response Relationship, Drug ,integumentary system ,biology ,business.industry ,Hydroxychloroquine ,Models, Theoretical ,medicine.disease ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Immunology ,biology.protein ,business ,Keratinocyte ,medicine.drug - Abstract
Background Antimalarials are some of the most notorious drugs which may induce psoriasis, with 25% of all reported cases being associated with them. Antimalarials do not induce psoriasis de novo, but trigger subclinical psoriasis. In a previous report, we suggested that antimalarials exert their effect by interfering with the epidermal transglutaminase (TGase) activity. Objective on verify this hypothesis by examining the effect of hydroxychloroquine sulfate (HCQS) on cultured human skin and on TGase activity in vitro. Materials and methods Skin samples from normal donors were cultured in the presence of HCQS for 4 days, and then processed for microscopic examination. TGase activity was assayed in the presence of HCQS and compared with blanks. Results Significant changes in epidermal morphology were seen in all explants cultured in the presence of HCQS at all concentrations employed. Areas of enhanced and irregular keratinization were observed in the upper epidermis, while a loss of cell polarity, with keratinocyte crowding and disarray, was seen in the lower epidermis. In addition, we observed intraepidermal splitting at different levels and dermo-epidermal detachments. HCQS showed a concentration-dependent inhibition of TGase activity. Conclusions We suggest that HCQS causes an initial break in the barrier function of the epidermis by inhibiting TGase activity; this is followed by a physiologic response of the epidermis aimed at barrier restoration. This rather nonspecific stimulus to epidermal proliferation is probably sufficient to trigger psoriasis in predisposed individuals or aggravate it in psoriatic patients.
- Published
- 1997
35. Substance P inactivation by transglutaminase in vitro
- Author
-
Raffaele Porta, Antonio Calignano, Loredana Mariniello, Paola Persico, Piero Pucci, Gennaro Marino, F. Mancuso, Cinzia Esposito, Persico, P, Calignano, Antonio, Mancuso, F, Marino, Gennaro, Pucci, Pietro, Esposito, C, Mariniello, Loredana, and Porta, Raffaele
- Subjects
medicine.medical_specialty ,Physiology ,Guinea Pigs ,Molecular Sequence Data ,Histamine Antagonists ,Neuropeptide ,Spermine ,Substance P ,Pharmacology ,Biochemistry ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Endocrinology ,In vivo ,Internal medicine ,medicine ,Animals ,Edema ,Amino Acid Sequence ,Receptor ,Peptide modification ,Transglutaminases ,Chemistry ,Extremities ,Muscle, Smooth ,Biological activity ,In vitro ,Liver ,Histamine ,Muscle Contraction - Abstract
Gamma(glutamyl5)spermine derivative of substance P (Spm-SP) was synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The spermine adduct of the neuropeptide was purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The biological activities of Spm-SP were tested by assaying, in comparison with substance P, its ability to induce both the contractions of smooth muscle in vitro and the edema formation in vivo. Spm-SP was shown not to elicit contractile responses in the isolated rat stomach strip and duodenum and not to antagonize the spasmogenic effect evoked by the native neuropeptide. Furthermore, Spm-SP was unable, when administered into rats by plantar injection, either to provoke an acute inflammatory response in the hind limb or to antagonize the edema formation induced by a concurrent administration of substance P. These results indicate that the introduction of a large size hydrophilic moiety at the glutamine5 level negatively affects the ability of the neuropeptide to bind to its receptor(s), thus supporting the view that the hydrophobic middle portion of substance P plays a key role in receptor recognition.
- Published
- 1992
36. Transglutaminase-catalyzed modifications of SV-IV, a major protein secreted from the rat seminal vesicle epithelium
- Author
-
S. Metafora, Gianfranco Peluso, Loredana Mariniello, Cinzia Esposito, Raffaele Porta, and V. Gentile
- Subjects
Male ,Seminal Plasma Proteins ,Prostatic Secretory Proteins ,Tissue transglutaminase ,Spermidine ,Lysine ,Guinea Pigs ,In Vitro Techniques ,Biochemistry ,Epithelium ,chemistry.chemical_compound ,Seminal vesicle ,medicine ,Animals ,Transglutaminases ,biology ,Prostate ,Proteins ,Seminal Vesicles ,Rats, Inbred Strains ,Molecular biology ,Rats ,medicine.anatomical_structure ,chemistry ,Liver ,Uteroglobin ,biology.protein ,Polyamine ,Chromatography, Liquid - Abstract
One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (gamma-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.
- Published
- 1990
37. Driving Forces in the Delivery of Penetratin Conjugated G Protein Fragment.
- Author
-
Stefania Albrizio, Laura Giusti, Gerardino D'Errico, Cinzia Esposito, Francesca Porchia, Gabriella Caliendo, Ettore Novellino, Maria R. Mazzoni, Paolo Rovero, and Anna M. D'Ursi
- Published
- 2007
- Full Text
- View/download PDF
38. Development of Antiviral Fusion Inhibitors: Short Modified Peptides Derived from the Transmembrane Glycoprotein of Feline Immunodeficiency Virus.
- Author
-
Anna Maria D'Ursi, Simone Giannecchini, Cinzia Esposito, Maria Claudia Alcaro, Olimpia Sichi, Maria Rosaria Armenante, Alfonso Carotenuto, Anna Maria Papini, Mauro Bendinelli, and Paolo Rovero
- Published
- 2006
- Full Text
- View/download PDF
39. Homology between rabbit uteroglobin and the rat seminal vesicle sperm-binding protein: Prediction of structural features of glutamine substrates for transglutaminase
- Author
-
G. Peluso, Raffaele Porta, Antonio Facchiano, S. Metafora, Cinzia Esposito, Francesco Facchiano, S., Metafora, F., Facchiano, A., Facchiano, C., Esposito, G., Peluso, and Porta, Raffaele
- Subjects
chemistry.chemical_classification ,biology ,Tissue transglutaminase ,Protein subunit ,Binding protein ,Biochemistry ,Homology (biology) ,Glutamine ,Enzyme ,Seminal vesicle ,medicine.anatomical_structure ,chemistry ,Uteroglobin ,medicine ,biology.protein - Abstract
A comparison of both amino acid composition and sequence of the rabbit uteroglobin (UG) subunit and the rat seminal vesicle sperm-binding protein (rSBP) by computer analysis indicates homology between the two polypeptide chains. These findings are supported by immunological studies showing the occurrence of similar antigenic determinants. In addition, our data indicate the glutamine-9 of the rat seminal vesicle sperm-binding protein and glutamine-40 of UG as the possible glutamine residues involved when the proteins act as transglutaminase (TGase) substrates. The latter results represent an interesting approach to determining the general structural features of the acyl donor site in the TGase-catalyzed reaction.
- Published
- 1987
40. Flicker Spectroscopy of Thermal Lipid Bilayer Domain Boundary Fluctuations
- Author
-
Aiwei Tian, Corinne N. Johnson, Cinzia Esposito, Svetlana Melamed, Tobias Baumgart, and Shang-You Tee
- Subjects
Capillary wave ,Membranes ,Hot Temperature ,1,2-Dipalmitoylphosphatidylcholine ,Chemistry ,Bilayer ,Spectrum Analysis ,Relaxation (NMR) ,Lipid Bilayers ,Biophysics ,Analytical chemistry ,Biological membrane ,Membranes, Artificial ,Quantitative Biology::Subcellular Processes ,Domain wall (magnetism) ,Membrane ,Cholesterol ,Models, Chemical ,Chemical physics ,Liposomes ,Phosphatidylcholines ,Gamma Cameras ,Hydrodynamic theory ,Lipid bilayer - Abstract
This contribution describes measurements of lipid bilayer domain line tension based on two-dimensional thermal undulations of membranes with liquid ordered/liquid disordered phase coexistence and near-critical composition at room temperature. Lateral inhomogeneity of lipid and protein composition is currently a subject of avid research aimed at determining both fundamental properties and biological relevance of membrane domains. Line tension at fluid lipid bilayer membrane domain boundaries controls the kinetics of domain growth and therefore regulates the size of compositional heterogeneities. High line tension promotes membrane domain budding and fission. Line tension could therefore be an important control parameter regulating functional aspects of biological membranes. Here the established method of fluid domain flicker spectroscopy is applied to examine thermal domain wall fluctuations of phase-separated bilayer membranes. We find a Gaussian probability distribution for the first few excited mode amplitudes, which permits an analysis by means of appropriately specialized capillary wave theory. Time autocorrelation functions are found to decay exponentially, and relaxation times are fitted by means of a hydrodynamic theory relating line tensions and excited mode relaxation kinetics. Line tensions below 1 pN are obtained, with these two approaches yielding similar results. We examine experimental artifacts that perturb the Fourier spectrum of domain traces and discuss ways to identify the number of modes that yield reliable line tension information.
- Full Text
- View/download PDF
41. New gas-liquid chromatographic method for biologically important indoleamines
- Author
-
M. Camardella, Cinzia Esposito, Raffaele Porta, G. Della Pietra, Porta, Raffaele, Esposito, C., Camardella, M., and DELLA PIETRA, G.
- Subjects
Chromatography ,Chemistry ,5 methoxytryptamine, acetylserotonin methyltransferase, serotonin, tryptamine ,Organic Chemistry ,General Medicine ,Biochemistry ,Gas liquid chromatographic ,drug determination, drug mixture, gas liquid chromatography, indoleamine n methyltransferase, methodology, n methyltryptamine, theoretical study ,Analytical Chemistry - Abstract
In connection with studies on indoleamine-N-methyltransferase a procedure is developed for determing tryptamine, N-methyltryptamine, 5-methoxy-tryptamine and 5-hydroxytryptamine simultaneously by means of flame ionization detection, without derivatization and in a total analysis time of 13 min.
- Published
- 1978
42. Inhibition of indolethylamine-N-methyltransferase by aliphatic diamines
- Author
-
Cinzia Esposito, Raffaele Porta, G. Della Pietra, M. Camardella, Porta, Raffaele, M., Camardella, C., Esposito, and G., Della Pietra
- Subjects
Male ,Tryptamine ,chemistry.chemical_classification ,Reaction mechanism ,Spermidine ,Chemistry ,Stereochemistry ,Biophysics ,Substrate (chemistry) ,Methyltransferases ,Cell Biology ,Diamines ,Biochemistry ,Tryptamines ,Molecular Weight ,Kinetics ,chemistry.chemical_compound ,Non-competitive inhibition ,Enzyme ,Indolethylamine N-methyltransferase ,Animals ,Spermine ,Rabbits ,Lung ,Molecular Biology - Abstract
Summary Aliphatic diamines exert a dead-end inhibition on the S-adenosylmethionine dependent indolethylamine-N-methyltransferase from rabbit lung Kinetic studies show that this inhibition is uncompetitive with respect to S-adenosylmethionine and competitive with respect to tryptamine. The K i values of the most effective diaminic inhibitors (1,8-diaminooctane and 1,7-diaminoheptane) indicate a similar affinity to tryptamine for the enzyme. A reaction mechanism “Ordered BiBi” with S-adenosylmethionine as first substrate bound is suggested.
- Published
- 1977
43. Transglutaminase-catalyzed crosslinking of an immunosuppressive and anti-inflammatory protein secreted from the rat seminal vesicles
- Author
-
Paola Persico, Gianfranco Peluso, Cinzia Esposito, Raffaele Porta, Salvatore Metafora, Zappia V., Galletti P., Porta R., Wold F., Porta, Raffaele, Esposito, C., Persico, P., Peluso, G., and Metafora, S.
- Subjects
endocrine system ,biology ,urogenital system ,Seminal Plasma Proteins ,Tissue transglutaminase ,Immunogenicity ,Sperm ,Cell biology ,Mammalian reproduction ,medicine.anatomical_structure ,Antigen ,Immunity ,Immunology ,biology.protein ,medicine ,Gamete ,reproductive and urinary physiology - Abstract
It is well known that the mammalian reproduction system is potentially hazardous because of male gamete immunogenicity. In fact, it has been demonstrated by a variety of immunological techniques that the epididymal sperm cells possess non-self specific, highly immunogenic autoantigens and alloantigens on their surface. Therefore, sperm antigens should be able to elicit a dangerous immunological response in the female genital tract where the sperm cells should be rejected. However, even though spermatozoa possess components which are antigenically foreign to the female mammal, the deposition of sperm cells at natural mating does not cause sterilizing anti-sperm immunity. The surface antigenic characteristics of the epididymal sperm cells change following their mixing in the ejaculate with the secretions of the male accessory sexual glands; in fact, the male gametes lose their immunological reactivity in the seminal plasma, adapting themselves to avoid rejecton by the immunocompetent elements normally occurring in the female genital tract.2
- Published
- 1988
44. FAB Mass Spectrometric Detection of ε( γ -Glutamyl)Lysine Crosslinks and ( γ -Glutamyl)Polyamine Derivatives Produced by Transglutaminase in Vitro
- Author
-
Antonio Malorni, Cinzia Esposito, Piero Pucci, Raffaele Porta, Salvatore Metafora, and Gennaro Marino
- Subjects
chemistry.chemical_compound ,Biochemistry ,biology ,chemistry ,Tissue transglutaminase ,Yield (chemistry) ,Lysine ,biology.protein ,Proteolytic enzymes ,Fast atom bombardment ,Polyamine ,In vitro ,Adduct - Abstract
The identification of both acyl donor and acceptor substrates, as well as the detection of e( γ -glutamyl)lysine crosslinks and ( γ-glutamyl) amine derivatives, are some of the most important aspects in the field of the transglutaminase (TGase)-mediated reactions1–5. These reactions have been revealed so far using both direct and indirect methodologies. The main direct method is based upon chromatographic isolation and identification of the isopeptide e( γ -glutamyl)lysine or of ( γ -glutamyl)amine derivatives after exhaustive proteolytic digestion of the reaction products. On the other hand, the detection of either polymers or radioactive amine-protein adducts by SDS-PAGE, together with the protein crosslinking inhibition by small molecular weight amines, represent the most commonly used indirect methodologies. However, both procedures suffer from severe limitations since a single analytical system does not yield unambiguous results.
- Published
- 1988
45. Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts
- Author
-
G. Illiano, A. Di Donato, Raffaele Porta, Cinzia Esposito, Giulio Draetta, A. De Santis, Porta, Raffaele, A., DE SANTIS, C., Esposito, G. F., Draetta, A., DI DONATO, and G., Illiano
- Subjects
Tissue transglutaminase ,Spermidine ,Phosphatidylethanolamine N-Methyltransferase ,Biophysics ,Phospholipid ,Adenylate kinase ,Biology ,Biochemistry ,Cyclase ,Guinea pig ,chemistry.chemical_compound ,Adenosine Triphosphate ,Animals ,Carbon Radioisotopes ,Columbidae ,Molecular Biology ,Transglutaminases ,integumentary system ,Erythrocyte Membrane ,Caseins ,Cell Biology ,Methylation ,Methyltransferases ,Kinetics ,Membrane ,chemistry ,Adenylyl Cyclase Inhibitors ,biology.protein ,Calcium ,Phosphatidyl-N-Methylethanolamine N-Methyltransferase ,Cyclase activity ,Phosphorus Radioisotopes ,Adenylyl Cyclases - Abstract
Summary We report the occurrence in pigeon erythrocytes of a soluble Ca 2+ -dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of l-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca 2+ concentration.
- Published
- 1986
46. B-lipotropin 61-76 and 61-91 fragments act as transglutaminase substrates in vitro
- Author
-
Raffaele Porta, Gianfranco Peluso, V. Gentile, Alfredo Fusco, Salvatore Metafora, Cinzia Esposito, Porta, R, Gentile, Vittorio, Esposito, C, Fusco, A, Peluso, G, Metafora, S., Porta, Raffaele, V., Gentile, C., Esposito, A., Fusco, G., Peluso, and S., Metafora
- Subjects
medicine.hormone ,endocrine system ,Tissue transglutaminase ,Stereochemistry ,Lipotropin ,Spermine ,In Vitro Techniques ,Substrate Specificity ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Residue (chemistry) ,Endocrinology ,Labelling ,medicine ,alpha-Endorphin ,chemistry.chemical_classification ,Transglutaminases ,biology ,Endocrine and Autonomic Systems ,beta-Endorphin ,General Medicine ,Acceptor ,In vitro ,Enzyme ,Neurology ,chemistry ,Biochemistry ,biology.protein ,Endorphins ,hormones, hormone substitutes, and hormone antagonists - Abstract
Both alpha- and beta-endorphin were shown to incorporate radioactive polyamines during incubation in the presence of purified transglutaminase and Ca2+, spermine acting as the best acyl acceptor substrate. The endorphin labelling is dependent on the time of exposure to the enzyme and on the substrate concentration. In the absence of acyl acceptor polyamines, isotopically labelled beta-endorphin gives rise to high molecular weight radioactive polymer(s). Moreover, when different proteins acting as transglutaminase substrates are added, beta-endorphin produces heterologous structures. These data indicate that the glutamine-71 of the beta-lipotropin chain, contained in both alpha- and beta-endorphin, functions as acyl donor substrate for the enzyme and that at least one beta-endorphin lysyl residue can serve as acyl acceptor.
- Published
- 1988
47. Sperm maturation in human semen: role of transglutaminase-mediated reactions
- Author
-
Salvatore Metafora, Michelangelo Iannone, Alfredo Fusco, Cinzia Esposito, Raffaele Porta, A. De Santis, Porta, Raffaele, C., Esposito, A., Desanti, A., Fusco, M., Iannone, and S., Metafora
- Subjects
Adult ,Male ,Antigenicity ,Tissue transglutaminase ,Seminal Plasma Proteins ,Semen ,Biology ,Reference Values ,medicine ,Humans ,Ejaculation ,chemistry.chemical_classification ,Transglutaminases ,Spermatozoon ,urogenital system ,Cell Biology ,General Medicine ,Sperm ,Spermatozoa ,Cell biology ,Sperm Maturation ,Kinetics ,Enzyme ,medicine.anatomical_structure ,Reproductive Medicine ,Biochemistry ,chemistry ,biology.protein ,Gamete - Abstract
A Ca2�-dependent, transglutaminase-like activity has been detected both free in the human seminal plasma and bound on the spermatozoon surface. A marked variability of the two enzymatic activities in the semen of different normal subjects was observed; but limited changes occurred in various ejaculates of the same in dividual. Moreover, we report evidence of the ability of several seminal plasma proteins to act as acyl donor substrates for endogenous transglutaminase, whereas human ejaculated spermatozoa have been shown to possess polyamine-binding sites specifically involved in transglutaminase-catalyzed reactions. It is postulated that semen transglutaminase may play a role in suppressing sperm antigenicity and in the male gamete’s acquiring biological features of a fully differentiated and fertile cell.
- Published
- 1986
48. Purification and Structural Characterization of In Vitro Synthesized (γ-Glutamyl) Spermidine Conjugates of a Major Protein Secreted from the Rat Seminal Vesicles
- Author
-
Gennaro Marino, Raffaele Porta, Cinzia Esposito, Piero Pucci, Salvatore Metafora, and Antonio Malorni
- Subjects
chemistry.chemical_classification ,medicine.diagnostic_test ,biology ,Proteolysis ,Proteolytic enzymes ,Spermine ,Molecular biology ,Amino acid ,Spermidine ,chemistry.chemical_compound ,Phospholipase A2 ,chemistry ,Biochemistry ,medicine ,biology.protein ,Secretion ,Polyamine - Abstract
Very little is known about the physiological meaning of spermidine (Spd) and spermine (Spm) occurrence in mammal seminal plasma where polyamines are secreted in large amounts by the prostate (1–3). One postulated role for semen polyamines is to serve as acyl acceptor substrates for transglutaminase (TGase; EC 2.3.2.13). In fact, it has been demonstrated that rat seminal clot proteins have covalently bound polyamines and upon proteolytic digestion released (γ-glutamyl)polyamine derivatives (4). MDreover, N-mono- and N, N-bis-(γ-glutamyl)polyamine derivatives were found after proteolysis of rat vesicular secretion proteins that have been incubated with coagulating gland extracts (5). Some of us have recently shown the ability of one of the major proteins secreted from the rat seminal vesicle epithelium to covalently bind radioactive Spd in the presence of either purified guinea pig liver TGase or crude secretory fluid produced by the rat coagulating gland (6). Such a protein, named SV-IV, is the fourth major band observed on the SDS-PAGE pattern of the proteins present in the rat seminal vesicle secretion. The sequence of its 90 amino acids (Mr= 9, 758) and the main features of the gene coding for it are known (6–9). We have demonstrated that SV-IV possesses immunosuppressive and anti-inflanmatory activities (6, 10, 11). Microgram amounts of SV-IV are able to inhibit both the mitogen-induced lymphocyte blastogenesis and the mixed lymphocyte reaction; the observed anti-inflaitmatory activity, comparable to that of dexamethasone, seems to be due to the block of arachidonic acid cascade at level of the enzyme phospholipase A2.
- Published
- 1988
49. Beta-endorphin modification by transglutaminase in vitro: identification by FAB/MS of glutamine-11 and lysine-29 as acyl donor and acceptor sites
- Author
-
Salvatore Metafora, Gennaro Marino, Raffaele Porta, Antonio Malorni, Piero Pucci, Cinzia Esposito, Pucci, Pietro, Malorni, A, Marino, Gennaro, Metafora, S, Esposito, C, and Porta, Raffaele
- Subjects
Tissue transglutaminase ,Stereochemistry ,Acylation ,Glutamine ,Lysine ,Molecular Sequence Data ,Biophysics ,Spermine ,Peptide ,Biochemistry ,Mass Spectrometry ,Residue (chemistry) ,chemistry.chemical_compound ,Humans ,Amino Acid Sequence ,Molecular Biology ,chemistry.chemical_classification ,Transglutaminases ,biology ,beta-Endorphin ,Cell Biology ,Acceptor ,Enzyme ,chemistry ,biology.protein - Abstract
FAB-Mapping strategy was successfully exploited to characterize the reaction products of the transglutaminase-mediated modifications of human beta-endorphin in vitro. The GLN-11 residue of the neuropeptide was shown to be an effective acyl donor site for the enzyme, being able to bind spermine in the presence of Ca2+. Moreover, only one out of five lysyl residues (LYS-29) was demonstrated to act as acyl acceptor crosslinking with GLN-11.
- Published
- 1988
50. Biological activities of CNBr fragments of a major protein secreted from the rat seminal vesicle epithelium
- Author
-
Piero Pucci, Maria Eugenia Schininà, Gianfranco Peluso, Cinzia Esposito, Gennaro Marino, F. Mancuso, Salvatore Metafora, Raffaele Porta, Porta, Raffaele, Esposito, C, Schinina, Me, Mancuso, F, Marino, Gennaro, Pucci, Pietro, Peluso, G, and Metafora, S.
- Subjects
Male ,Tissue transglutaminase ,Molecular Sequence Data ,SV-IV ,Spectrometry, Mass, Fast Atom Bombardment ,Biochemistry ,Peptide Mapping ,Epithelium ,transglutaminase ,Residue (chemistry) ,chemistry.chemical_compound ,In vivo ,medicine ,Animals ,Amino Acid Sequence ,Cyanogen Bromide ,Rats, Wistar ,Methionine ,Transglutaminases ,biology ,Chemistry ,Seminal Plasma Proteins ,Prostatic Secretory Proteins ,Proteins ,Seminal Vesicles ,Fast atom bombardment ,Mixed lymphocyte reaction ,Molecular biology ,In vitro ,Peptide Fragments ,Rats ,medicine.anatomical_structure ,platelet aggregation ,biology.protein - Abstract
Two fragments of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met-70) occurring in its polypeptide chain. After their purification by reversed-phase chromatography, SV-IV/A (71-70 fragment) and SV-IV/B (71–90 fragment) were assayed as transglutaminase substrates, and their anti-inflammatory, anti-thrombotic and immunosuppressive properties were evaluated in comparison with native SV-IV. Both fragments retained the SV-IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln-9 and Gln-86 as acyl donor sites, and to Lys-59, -79 and -80 as acyl acceptor sites. In contrast, only SV-IV/A was shown to possess, like SV-IV, the property of inhibiting both the intensity of the carrageenin-induced rat foot edema and the platelet aggregation induced in vivo by different agents. Finally, the two protein fragments were found to be completely unable to inhibit both the mitogen-induced proliferation of human T cells and the mixed lymphocyte reaction.
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.