Nuclear Envelope Transmembrane Proteins (NETs) have functions in nuclear size scaling, nucleo-cytoskeletal connections, cell cycle control, and genome organisation/ regulation during differentiation1. Despite these many important functions, structural determinants of most of NET functions are yet to be characterised. This PhD project aims to first perform a bioinformatic analysis on the wide set of several hundred NETs looking for any domains for which structural information is available and modelling the potential of those occurring in multiple NETs for shared functional interactions. Based on this information and partner proteins identified previously in the Schirmer lab particular co-structures may be selected for investigation. There are two NETs for which we have already some success in protein purification trials that will likely be pursued to carry out structure-function analysis: NET50 and KLHL31. NET50, has been reported on as a biomarker for prostate cancer as it is strongly and reproducibly downregulated in late-stage androgen resistant prostate cancer2.
NET50 has been tested in vitro and shown to act as a dehydrogenase/ reductase for steroids and retinols. Data from a graduating student shows that NET50 functions in nuclear size regulation and that its loss correlates with the change in nuclear size associated with increased metastasis and cell migration in late-stage prostate cancer. We are interested to co-crystalise sterols with the NET to better elucidate its functions.
KLHL31 is probably not an actual NET as it appears that what was initially predicted to be a transmembrane domain forms internal hydrophobic alpha helices. Nonetheless, KLHL31 is an interesting protein as it is at the nuclear envelope in interphase cells, but relocates onto kinetochores during mitosis and the scission point in cytokinesis. Thus far there are no reports on this interesting protein and the Arulanandam lab has generated many important structures of other kinetochore proteins3.
The work will involve biochemical and structural (X-ray crystallography) characterization of NET50 and KHL31 in the Arulanandam lab and structure-guided mutants will be used in cell-based assays in the Schirmer lab to obtain functional insights.
The proposed project will give the student a great opportunity to interweave bioinformatic and wetlab approaches and to learn a multitude of different wetlab approaches, including biochemistry, structural biology and cell biology in a collaborative setting that includes weekly joint lab meetings.
1 Heras et al., and Schirmer, E. C. (2013) Tissue specificity in the nuclear envelope supports its functional complexity. Nucleus 4(6). 460-477. PMID: 24213376
2. Seibert et al., and Oldermatt, A. (2015) A role for the dehydrogenase DHRS7 (SDR34C1) in prostate cancer. Cancer Med 4. 1717-29. PMID: 26311046
3. Jeyaprakash et al., and Conti, E. Structure of a Survivin-Borealin-INCENP Core Complex Reveals How Chromosomal Passengers Travel Together. Cell (2007) 131, 271-285.
4. Abad et al., and Jeyaprakash. A. A. Structural Basis for Microtubule Recognition by the Human Kinetochore Ska Complex. Nat Commun (2014) Jan13; 5:2964. Doi:10.1038/ncomms3964.