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Breaking the rule: blocking protein degradation by N-terminal tagging as a strategy to cure disease


   School of Biology

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  Dr CM Czekster  No more applications being accepted  Competition Funded PhD Project (Students Worldwide)

About the Project

Background and project details: Proteins in a cell can last less than a minute to several days. Not only does the cell need to control protein levels but it must also eliminate proteins that are misfolded or abnormal in any way. To do so, the cellular machinery employs a variety of different strategies in eukaryotic and prokaryotic organisms. A common strategy in both groups of organisms is the modification of the N-terminus of a protein to be degraded following the “N-terminal rule”. This rule states that the half-life of a protein is related to the identity of its N-terminal residue or residues.
To modify the N-terminus of a protein and destabilize it targeting it for degradation, proteins called aminoacyl tRNA-protein transferases add destabilizing amino acids to their protein targets.1 In prokaryotes, two enzymes are known, leucyl/phenylalanyl-tRNA protein transferase (LF-TR) and arginyl-tRNA protein transferase (R-TR). In eukaryotes (including humans), there is only an R-transferase protein. These enzymes use aminoacyl-tRNAs as substrates, and catalyse peptide bond formation to add destabilizing amino acids to the N-termini of proteins and peptide substrates.
Importantly, both LF-TR and R-TR have been implicated in disease. LF-TR is essential for growth of Pseudomonas aeruginosa, while R-TR plays a role in several human conditions such as cancer and cardiovascular disease.2 Because LF-TR is absent in mammals it could be an ideal target for anti P. aeruginosa drugs. However, there is scarce information available about mammalian R-TR enzymes, and potential inhibitors could be affecting both bacterial LF-TR and mammalian R-TR.

Therefore, the goal of this project is to characterize two aminoacyl tRNA-protein transferases, the LF-TR from P. aeruginosa, and the R-TR from mice (Mus musculus).

Expected outcomes for the student
1) Produce LF-TR from P. aeruginosa and R-TR from Mus musculus.
2) Determine the in vitro activity of both enzymes, and their substrate specificity using peptide substrates.
3) Determine crystal structures of both proteins free and bound to substrate/product mimics.
4) Perform experiments using cell extracts to determine possible protein substrates for both proteins.
5) Test substrate analogues as inhibitors for LF-TR and R-TR.

Techniques and training: The Czekster laboratory routinely utilizes x-ray generators, liquid dispensing robots, automated crystal imaging instrumentation, plate readers capable of UV, fluorescence and luminescence readings, high pressure liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC). We also use a variety of instrumentation to characterize binding and fast kinetics, as we possess quenched-flow and stopped-flow apparatus, circular dichroism, isothermal titration calorimetry, and surface plasmon resonance instruments. This expertise and instrumentation covers all bases for protein production, characterization, as well as biochemical and biophysical assays that the project requires. We work closely with the mass spectrometry facility in St Andrews, and will perform our proteomic experiments in house. The student will acquire training in most, if not all, of these techniques.

Opportunities for training: Outstanding training in molecular biology, enzymology, microbiology, x-ray crystallography, isothermal titration calorimetry and mass spectrometry will be provided.3 Students will participate in journal clubs, lab meetings and internal seminars, as well as in external regional and/or international meetings, presenting posters and delivering talks, acquiring fundamental transferable skills.

-Technical skills: in enzymology, assay development, chemical and structural biology, biophysical techniques, mass spectrometry.
-Analytical skills: design and interpret experiments, as well as write manuscripts, prepare and deliver talks.
-Communication skills: student will present their work to diverse audiences, and will be expected to attend and present at one international conference during their PhD.
-Engagement with the public: The Czekster lab currently runs a public engagement program with primary schools, and the student will have the opportunity to take part.
-Career development: university offers access to the program GRADskills, a suite of workshops, networking events and activities designed for research postgraduate students and delivered by the Centre for Academic, Professional and Organisational Development (CAPOD). Student will be encouraged to take part.

Funding Notes

The successful applicant will have at minimum a 2.1 degree and/or a MSc in Biochemistry, or Chemistry.
Applicants are encouraged to contact the PI Clarissa Czekster prior to application by e-mail ([Email Address Removed]) with any questions or to discuss further details of the project.

Funding: Fees and stipend is provided for 3.5 years.

Applications received by the initial 1 December deadline are now under consideration. Applications are invited now for anyone applying for Chinese Scholarship Council (CSC) funding only.

References

1. Watanabe, K. et al (2007) Protein-based peptide-bond formation by aminoacyl-tRNA protein transferase. Nature 449, 867-871.
2. Liu, Y. et al (2016) Physiological functions and clinical implications of the N-end rule pathway. Frontiers in Medicine, 10:258-270.
3. Czekster, C.M. et al (2017) Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates. Nature Communications, 8.

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