Nuclear Envelope Transmembrane Proteins (NETs) have functions in nuclear size scaling, cell migration, cell cycle control, genome regulation, and tissue differentiation1. Despite these many important functions, structural determinants of most NET functions are not yet chacterised. This PhD project aims to first perform a bioinformatic analysis on the set of several hundred NETs identified by Schirmer looking for shared domains and domains for which structural information is available or can be modelled. Based on this information and partner proteins identified previously in the Schirmer lab particular interacting regions will be selected for structural and functional investigations. Examples of potential specific complexes to investigate are three NETs for which we have already some success in protein purification trials that could be pursued for structure-function analysis: NET50, KLHL31, and NET29.
NET50 is important for nuclear size regulation in prostate and is downregulated in late-stage prostate cancer2 when this size regulation is disrupted. It also has a dehydrogenase/reductase activity for steroids. We are interested to co-crystalise sterols with the NET to better elucidate its function in nuclear size regulation and why estradiol seems to inhibit its role in nuclear size regulation.
KLHL31 has a predicted transmembrane domain that may instead form internal hydrophobic alpha helices. It is an interesting protein because it is at the nuclear envelope and cytoskeletal filaments 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.
NET29 directs gene repositioning events in adipogenesis and knockout mice have a lipodystrophic phenotype. As it binds lamin A, but not lamin A lipodystrophy mutants it may mediate lamin-linked lipodystrophy; so this interaction will be investigated.
The work will involve biochemical and structural (X-ray crystallography) characterization of NET50, KHL31, and NET29-lamin complexes 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 molecular 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.
If you wish to apply for this project, please go to this link.