Genetically Encoded Sensors for Detection of Proteases Utilizing Auto-Inhibited Coiled Coils and Split-Protein Reassembly

Persistent Link:
http://hdl.handle.net/10150/205209
Title:
Genetically Encoded Sensors for Detection of Proteases Utilizing Auto-Inhibited Coiled Coils and Split-Protein Reassembly
Author:
Shekhawat, Sujan Singh
Issue Date:
2011
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Embargo: Release after 08/24/2012
Abstract:
The detection of cellular events is central to understanding biomoleculer processes as well as aid in therapeutic intervention strategies. One of the most fascinating biomoleculer events during the life cycle of a cell is proteolytic cleavage of proteins by enzymes known as proteases. Proteases are ubiquitous and participate in essential functions such as fertilization, embryo development, cell cycle regulation, immune response, tissue remodeling and programmed cell death. As proteases are involved in fundamental cellular processes any dysregulation of protease activity is usually associated with a diseased state. Thus methods for detection of protease activity are desirable as it may facilitate the identification of many pathological conditions which are associated with the aberrant expression and activity of proteases.Towards the goal of a general and modular strategy we have utilized split protein reassembly and coiled coils to develop genetically encoded sensors for detection of proteases. We established our first generation protease design utilizing split firefly luciferase and anti-parallel coiled coils and detected Tobacco Etch Virus (TEV) as a model protease. Two further iterations of the coiled-coil design led to the development of second and third generation of protease sensors which showed substantial improvement in the sensor response and was applied towards detection of therapeutically relevant proteases such as caspase-3, prostate specific antigen (PSA), ß-secretase and calpain-1.We applied our methodolgy to develop protease biosensors for the detection of a family of cysteine protease known as caspases. Caspases are involved in programmed cell death and their misregulation is implicated in cancer as well as neurodegenerative disorders. The panel of caspase biosensors was utilized to investigate caspase cleavage specificity as well as caspase activation in mammalian cytosolic extracts and live mammalian cells. Perhaps more importantly, we discovered cross talk between members of the caspase family which perform different biological functions.Finally, we detail our progress towards mimicking a naturally occurring multicomponent complex formed during programmed cell death, known as the apoptosome which leads to the activation of caspases. We have successfully utilized principles of self assembly and multivalency to assemble multi component complexes which exhibit proteolytic activity similar to the natural apoptosome.
Type:
text; Electronic Dissertation
Keywords:
Protease; Split Protein Reassembly; Chemistry; Casapse; Coiled Coils
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Ghosh, Indraneel

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleGenetically Encoded Sensors for Detection of Proteases Utilizing Auto-Inhibited Coiled Coils and Split-Protein Reassemblyen_US
dc.creatorShekhawat, Sujan Singhen_US
dc.contributor.authorShekhawat, Sujan Singhen_US
dc.date.issued2011-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.releaseEmbargo: Release after 08/24/2012en_US
dc.description.abstractThe detection of cellular events is central to understanding biomoleculer processes as well as aid in therapeutic intervention strategies. One of the most fascinating biomoleculer events during the life cycle of a cell is proteolytic cleavage of proteins by enzymes known as proteases. Proteases are ubiquitous and participate in essential functions such as fertilization, embryo development, cell cycle regulation, immune response, tissue remodeling and programmed cell death. As proteases are involved in fundamental cellular processes any dysregulation of protease activity is usually associated with a diseased state. Thus methods for detection of protease activity are desirable as it may facilitate the identification of many pathological conditions which are associated with the aberrant expression and activity of proteases.Towards the goal of a general and modular strategy we have utilized split protein reassembly and coiled coils to develop genetically encoded sensors for detection of proteases. We established our first generation protease design utilizing split firefly luciferase and anti-parallel coiled coils and detected Tobacco Etch Virus (TEV) as a model protease. Two further iterations of the coiled-coil design led to the development of second and third generation of protease sensors which showed substantial improvement in the sensor response and was applied towards detection of therapeutically relevant proteases such as caspase-3, prostate specific antigen (PSA), ß-secretase and calpain-1.We applied our methodolgy to develop protease biosensors for the detection of a family of cysteine protease known as caspases. Caspases are involved in programmed cell death and their misregulation is implicated in cancer as well as neurodegenerative disorders. The panel of caspase biosensors was utilized to investigate caspase cleavage specificity as well as caspase activation in mammalian cytosolic extracts and live mammalian cells. Perhaps more importantly, we discovered cross talk between members of the caspase family which perform different biological functions.Finally, we detail our progress towards mimicking a naturally occurring multicomponent complex formed during programmed cell death, known as the apoptosome which leads to the activation of caspases. We have successfully utilized principles of self assembly and multivalency to assemble multi component complexes which exhibit proteolytic activity similar to the natural apoptosome.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectProteaseen_US
dc.subjectSplit Protein Reassemblyen_US
dc.subjectChemistryen_US
dc.subjectCasapseen_US
dc.subjectCoiled Coilsen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorGhosh, Indraneelen_US
dc.contributor.committeememberBandarian, Vaheen_US
dc.contributor.committeememberHruby, Victor J.en_US
dc.contributor.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberGhosh, Indraneelen_US
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