Barts and The London School of Medicine and Dentistry at Queen Mary University of London and A*STAR PhD Studentship: Functional and structural studies of rod-like structures regulated by the dose and de-ubiquitinating activity of USP25, a gene with roles in Down Syndrome associated diseases
Down Syndrome (DS), caused by trisomy of human chromosome 21 (T21), is the most common genetic cause of intellectual disability, neurodevelopmental pathology, and Alzheimer’s dementia (AD). Altered poly-ubiquitinated protein profiles were reported in DS and DS-AD brains. We found that knockdown and over-expression of chr21-encoded Ubiquitin-specific-protease-25 (USP25) significantly inversely correlated with overall levels of poly-ubiquitinated cellular proteins. Over-expression of human USP25 in all cell lines attempted, generated cytoplasmic rod-like structures , typically 0.5 µm in diameter and 2-5 µm long, whose frequency was positively correlated with the transfection level of USP25. We found similar peri-nuclear cytoplasmic rods in human and mouse liver tissues, with an increased presence in DS and mouse DS model (Tc1). Deletion and mutation of USP25 revealed that the linker region bridging UBA and the tandem UIM domains, as well as intact de-ubiquitinating catalytic activity of USP25, were essential for the formation of rods. Combination of different inactive mutants partly restored the USP25’s rod-making and de-ubiquitinating capability, suggesting homo-dimers as basic structural and functional units of USP25. Fluorescence Recovery After Bleaching (FRAP) was extremely rapid, demonstrating a vivid exchange of USP25 molecules between rods and cytoplasm, and super-resolution microscopy revealed a helical or cochleate-like fine structure of the USP25-rods. An antibody raised against USP25 detected intra-nuclear rods (INR) in brain cells. Normal human foetal brain stem and hippocampi contained 15% of INR containing cells, (up to 40% in MBP+ cells), whose frequency was identical on contralateral sides. Significantly higher percentage of INR-containing primary human foetal brain cells were found in DS compared to normal cultures. This was reproduced by quantitation of INR-containing cells in hippocampus. Complete knockout of USP25 in a human NSC line significantly decreased the size and intensity of INRs. Nizetic Lab has recently also obtained a USP25 complete knockout mouse which is showing behavioural phenotypes.
Using Nizetic lab cellular models, and Manser lab expertise in Proximity labelling technique (termed BioID), this project aims to (i) identify the USP25-interacting proteins that participate in the rod-like-structure formation, (ii) elucidate the principles for the USP25 aggregation into a regular and regulated structure, and (iii) exploit the same structure as a hub for sequestration and concentration of proteins or peptides of interest that can be rapidly exchanged with the appropriate sub-cellular compartment.
All applicants should hold a masters level degree at first /distinction level in biomedical or natural sciences field (or a related discipline). Applicants should have a good knowledge of English and ability to express themselves clearly in both speech and writing. The successful candidate must be strongly motivated for doctoral studies, must have demonstrated the ability to work independently and to perform critical analysis.
Candidates are asked to possess fundamental knowledge and skills in two or more of the following areas:
• Excellent background in cellular and/or molecular biology. Good knowledge of microscopy-imaging techniques, such as confocal microscopy, super-resolution and/or electron microscopy would be desirable.
• Prior experience/education in cellular biology and/or protein biochemistry.
• Hands on experience using structural biology software/modelling would be an advantage.
A*STAR Research Attachment Programme (ARAP): This PhD programme is a collaboration between A*STAR and Queen Mary University of London (QMUL) to provide research opportunities for PhD students at A*STAR Research Institutes. Under this programme, PhD students from overseas universities will spend two years at A*STAR Research Institutes under the joint supervision of staff of the A*STAR Research Institutes and QMUL.
The primary supervisor is Professor Dean Nizetic: https://www.qmul.ac.uk/blizard/staff/centre-for-genomics-and-child-health/staff/dean-nizetic.html
The co-supervisor in Singapore is Dr Edward Manser: https://www.a-star.edu.sg/imcb/Science/Investigators/Senior-Pls/Senior-Pls-Profile/ResearchListID/4
Interviews are expected to take place in April
Starting date: September 2019 (dates can be flexible)