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Studying in vitro and in vivo protease processing in models of cancer cell death and viral infection by N-terminal COFRADIC

Francis Impens (UGent)
(2010)
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Abstract
Being an irreversible protein modification, proteolytic processing has an important role in many biological processes. Together with proteases and their inhibitors, protease substrates are the key components of proteolytic networks. Knowledge on the substrate repertoire of individual proteases is therefore crucial for a thorough understanding of protease biology. Over the last decade several screening methods for proteome-wide identification of protease substrates were developed. These methods are based on targeted, N-terminal proteomics and include the N-terminal COFRADIC technology developed in the lab in which my PhD project was performed. During my PhD project N-terminal COFRADIC was used to identify unknown protease cleavage sites in two model systems. In a first study, I tried to gain novel insight into the proteases involved in cell death induced by the chemotherapeutic agent paclitaxel or taxol. Amongst others, my results shed new light on the caspase-dependency of this cell death model. In a second series of screens, protease processing during HIV-1 infection was studied. Unknown protease cleavage sites were found both in infected cells and purified HIV-1 virions. Furthermore, novel information on non-processing events was obtained from these experiments. Finally, several in vitro substrate catalogues were generated for proteases linked to both these previous studies and a novel experimental design to compile these catalogues was developed. In the latter procedure laborious manual annotation of cleavage sites was replaced by software-based quantification and annotation. Taken together, this thesis project illustrates the power of N-terminal proteomics in protease research, but also indicates its limitations in analyzing in vivo protease cleavage sites. Nevertheless, many novel interesting results were obtained, providing clues for further research in the future.
Keywords
degradomics, taxol, proteomics, mass spectrometry, proteases, HIV-1

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MLA
Impens, Francis. Studying in Vitro and in Vivo Protease Processing in Models of Cancer Cell Death and Viral Infection by N-Terminal COFRADIC. Ghent University. Faculty of Medicine and Health Sciences, 2010.
APA
Impens, F. (2010). Studying in vitro and in vivo protease processing in models of cancer cell death and viral infection by N-terminal COFRADIC. Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium.
Chicago author-date
Impens, Francis. 2010. “Studying in Vitro and in Vivo Protease Processing in Models of Cancer Cell Death and Viral Infection by N-Terminal COFRADIC.” Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
Chicago author-date (all authors)
Impens, Francis. 2010. “Studying in Vitro and in Vivo Protease Processing in Models of Cancer Cell Death and Viral Infection by N-Terminal COFRADIC.” Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
Vancouver
1.
Impens F. Studying in vitro and in vivo protease processing in models of cancer cell death and viral infection by N-terminal COFRADIC. [Ghent, Belgium]: Ghent University. Faculty of Medicine and Health Sciences; 2010.
IEEE
[1]
F. Impens, “Studying in vitro and in vivo protease processing in models of cancer cell death and viral infection by N-terminal COFRADIC,” Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium, 2010.
@phdthesis{967546,
  abstract     = {{Being an irreversible protein modification, proteolytic processing has an important role in many biological processes. Together with proteases and their inhibitors, protease substrates are the key components of proteolytic networks. Knowledge on the substrate repertoire of individual proteases is therefore crucial for a thorough understanding of protease biology. Over the last decade several screening methods for proteome-wide identification of protease substrates were developed. These methods are based on targeted, N-terminal proteomics and include the N-terminal COFRADIC technology developed in the lab in which my PhD project was performed.
During my PhD project N-terminal COFRADIC was used to identify unknown protease cleavage sites in two model systems. In a first study, I tried to gain novel insight into the proteases involved in cell death induced by the chemotherapeutic agent paclitaxel or taxol. Amongst others, my results shed new light on the caspase-dependency of this cell death model. In a second series of screens, protease processing during HIV-1 infection was studied. Unknown protease cleavage sites were found both in infected cells and purified HIV-1 virions. Furthermore, novel information on non-processing events was obtained from these experiments. Finally, several in vitro substrate catalogues were generated for proteases linked to both these previous studies and a novel experimental design to compile these catalogues was developed. In the latter procedure laborious manual annotation of cleavage sites was replaced by software-based quantification and annotation.
Taken together, this thesis project illustrates the power of N-terminal proteomics in protease research, but also indicates its limitations in analyzing in vivo protease cleavage sites. Nevertheless, many novel interesting results were obtained, providing clues for further research in the future.}},
  author       = {{Impens, Francis}},
  keywords     = {{degradomics,taxol,proteomics,mass spectrometry,proteases,HIV-1}},
  language     = {{eng}},
  pages        = {{VII, 200}},
  publisher    = {{Ghent University. Faculty of Medicine and Health Sciences}},
  school       = {{Ghent University}},
  title        = {{Studying in vitro and in vivo protease processing in models of cancer cell death and viral infection by N-terminal COFRADIC}},
  year         = {{2010}},
}