Heterologous expression and functional characterization of human TRPA1 in Saccharomyces cerevisiae

Sammanfattning: Understanding Sense Perception We all need to perceive sensations such as pain; cold or pungent chemicals in order survive. In elucidating sense perception, we investigate TRPA1 which is a major player in sensation. Transient Receptor Potential (TRP) is a large family of ion channels that are found in membrane of biological cells. They were first discovered in fruit fly in 1969 and this discovery has helped in understanding our sense perception. Many more members of the group have been discovered in humans since then, one of which is TRPA1. It is the only member of the TRPA subfamily that is found in mammalians cells and it has been implicated in disease such as diabetes, pain, cough, inflammation among others. This makes TRPA1 a very important protein to study. Although, a lot has been done in investigating the mode of action, mechanism, regulation and activation of this protein, yet there is still more to be known. Especially, the step by step events that occur when chemicals bind to this protein to activate it needs to be elucidated. In order to investigate the function and characteristics of this protein, we aimed at cloning the gene and expressing it in yeast cells. Then activate the protein in vivo in yeast with mustard oil and cinnamaldehyde. This is expected to inhibit growth of the cells due to influx of cations. We also tried to trace the influx of cations into the cells by cobalt staining. At end of the project we were able to clone the gene onto an expression vector p425GPD and transferred it into yeast. We succeeded in activating the protein in the cells by mustard oil and cinnamaldehyde as additives in growth medium, as judged by growth lethality to the expressing cells. This means that we can now use the assay technique to find mutants that are insensitive to activation at determined concentration. Mapping of such mutations will thus help to determine the site of action of mustard oil and cinnamaldehyde. This may be useful in the long run to design drugs that can regulate activation of the protein, and hence may have medical application to treat associated diseases. In conclusion, the yeast growth inhibition assay is useful for investigation of the function and characteristics of TRPA1. Our discovery set the stage for finding the binding site at which the mustard oil and cinnamaldehyde interact with this protein to activate it. Identification of the binding sites will add another piece of information to the molecular mechanism behind/responsible for sense perception. Supervisor: Urban Johanson 45 Credits, Master´s Degree Project in Molecular Genetics, 2011 Department of Biochemistry and Structural Biology, Centre for Molecular Protein Science, Lund University Poplar science summary: Understanding Sense Perception Transient Receptor Potential (TRP) is a large family of ion channels that are found in membrane of biological cells. They were first discovered in fruit fly in 1969 and this discovery has helped in understanding our sense perception. Many more members of the group have been discovered in humans since then, one of which is TRPA1. It is the only member of the TRPA subfamily that is found in mammalians cells and it has been implicated in disease such as diabetes, pain, cough, inflammation among others. This makes TRPA1 a very important protein to study. Although, a lot has been done in investigating the mode of action, mechanism, regulation and activation of this protein, yet there is still more to be known. Especially, the step by step events that occur when chemicals bind to this protein to activate it needs to be elucidated. In order to investigate the function and characteristics of this protein, we aimed at cloning the gene and expressing it in yeast cells. Then activate the protein in vivo in yeast with mustard oil and cinnamaldehyde. This is expected to inhibit growth of the cells due to influx of cations. We also tried to trace the influx of cations into the cells by cobalt staining. At end of the project we were able to clone the gene onto an expression vector p425GPD and transferred it into yeast. We succeeded in activating the protein in the cells by mustard oil and cinnamaldehyde as additives in growth medium, as judged by growth lethality to the expressing cells. This means that we can now use the assay technique to find mutants that are insensitive to activation at determined concentration. Mapping of such mutations will thus help to determine the site of action of mustard oil and cinnamaldehyde. This may be useful in the long run to design drugs that can regulate activation of the protein. Hence may have medical application to treat associated diseases. In conclusion, the yeast growth inhibition assay is useful for investigation of the function and characteristics of TRPA1. Our discovery set the stage for finding the binding site at which the mustard oil and cinnamaldehyde interact with this protein to activate it. Urban Johanson 45 Credits Master´s Degree Project in Molecular Biology: Molecular Genetics and Biotechnology Centre for Molecular Protein Science, Lund University