Primary Supervisor: Professor Alessandro Rufini, Leicester Cancer Research Centre (LCRC)
Secondary supervisor: Dr Joanna Fox, Wellcome Trust fellow, Leicester Institute for Structural and Chemical Biology (LISCB)
PhD project title: Structural-guided understanding of the functions of proline metabolic enzymes in health and disease.
University of Registration: University of Leicester
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.
BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology & Pharmaceuticals
Techniques that will be undertaken during the project:
- Purification of proteins for in vitro work
- Enzymatic assay
- Cell culture
- MTT-based Cell survival assay
- Cell proliferation assay
- FACS analysis
Contact: Professor Alessandro Rufini, University of Leicester