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Structural-guided understanding of the functions of proline metabolic enzymes in health and disease


   Leicester Cancer Research Centre

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  Prof A Rufini  No more applications being accepted  Competition Funded PhD Project (Students Worldwide)

Leicester United Kingdom Cancer Biology Cell Biology Microbiology Molecular Biology Structural Biology

About the Project

This proposal aims at investigating the structural and enzymatic properties of the proline metabolism enzyme pyrroline-5-carboxylate reductase-1 (PYCR1). NAD(P)H-dependent PYCR enzymes catalyse the last step in proline biosynthesis by reducing the precursor pyrroline-5-carboxylate to the final product proline. There are three PYCRs: mitochondrial PYCR1 and 2 are highly homologous (84%), whereas cytosolic PYCR3 is the least conserved (45%). The three-dimensional structure of PYCR1 has been resolved through X-ray crystallography and shows that the functional enzymatic unit is composed by a homodimer. In addition, experimental evidence indicates that five PYCR1 dimers combine together to form a decameric toroid structure of 350kDa. Whether the other members of the family share similar structural arrangements remains unknown. Furthermore, the precise subcellular localization of PYCR1 and 2 within the mitochondria and their potential interactions are poorly defined.

Genetic mutations in the PYCR1 gene (located on chromosome 17q25.3), including numerous missense alterations in highly conserved residues, cause the autosomal recessive cutis laxa (ARCL) syndrome. Patients affected by ARCL experience severe developmental defects including wrinkled skin, progeroid features, craniofacial dysmorphism, early onset cataract, impaired cognitive ability and epilepsy. Notwithstanding their clinical relevance, the impact of missense mutations on PYCR1 structure and activity remains largely unexplored.

In an effort to clarify the structural properties of the proline biosynthesis enzymes in health and disease, this studentship will achieve the following aims:

AIM1:

Determine how the structure of the PYCR family of protein influences its function, by (i) clarifying the cellular localisation, stoichiometry and composition of the native decamer structure both in healthy and cancer cells, using a combination of mass spectrometry, confocal microscopy and gold-labelling electron microscopy; (ii) resolve the three-dimensional and quaternary structure by Cryo-EM to gain information about the larger multi-sub unit structure.

AIM2:

Generate ARCL-linked PYCR1 mutant proteins and assess the consequences of amino acid substitutions on protein structure and function. We will initially predict the impact of the affected residues using in silico structural modelling. Impactful substitutions likely to affect decamer assembly will be further investigated using Cryo-EM and an in vitro enzymatic assay, which has been developed in collaboration with Leicester Drug Discovery and Diagnostics (LD3) team. The amino acid substitutions to be examine will include, but will not be limited to, Arg119Cys (c.355C>G) and Arg251His (c.752G>A), which have been detected in distinct families affected by ARCL and are localized in the region that mediates formation of the decameric structure.

This project will also benefit from numerous collaborations. Within the UoL we will liaise with Dr Robert Britton (Leicester Cancer Research Centre), a chemist with expertise in structural biology, and Professor Carr Mark (LISCB) a leading figure in the field of structural biology. Moreover, we have a collaboration with Professor Eleonora Candi at the University of Tor Vergata in Rome with the purpose of investigating the role of proline metabolism in the development and physiology of human epidermis.

Entry requirements:

• Those who have a 1st or a 2.1 undergraduate degree in a relevant field are eligible.

• Evidence of quantitative training is required. For example, AS or A level Maths, IB Standard or Higher Maths, or university level maths/statistics course.

• Those who have a 2.2 and an additional Masters degree in a relevant field may be eligible.

• Those who have a 2.2 and at least three years post-graduate experience in a relevant field may be eligible.

• Those with degrees abroad (perhaps as well as postgraduate experience) may be eligible if their qualifications are deemed equivalent to any of the above

• University English language requirementsapply. https://le.ac.uk/study/research-degrees/entry-reqs/eng-lang-reqs/ielts-65

For further information please contact [Email Address Removed]

Application advice:

To apply please refer the application instructions at https://le.ac.uk/study/research-degrees/funded-opportunities/bbsrc-mibtp

You will need to apply for the PhD place and also submit your online application notification to MIBTP. Links for both are on the above web page.

Project / Funding Enquiries: For further information please contact [Email Address Removed]

Application enquiries to [Email Address Removed]


Funding Notes

All MIBTP students will be provided with a 4 years studentship.
Tuition Fees at UK fee rates
- a tax free stipend of at least £15,295 p.a (to rise in line with UKRI recommendation)
- a travel allowance in year 1
- a travel / conference budget
- a generous consumables budget
- use of a laptop for the duration of the programme
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