Structure determination of bHLH/PAS proteins
Mammalian bHLH/PAS proteins have traditionally been very difficult to express and purify, thus structural information is scant.
However, recent breakthroughs were achieved in solving partial structures for the bHLH/PAS heterodimers of Clock/BMAL, a protein complex which controls circadian rhythm, HIFa/ARNT and NPAS1 and 3 proteins.
This honours project will use analogous strategies to express, purify, crystallise and solve structures of the above mammalian bHLH/PAS proteins.
Dr John Bruning has previously determined structures for ligand binding domains of steroid hormone and nuclear receptors, a transcription factor family with interesting parallels to the set of bHLH/PAS proteins we study.
Pure, recombinant proteins will also be used in drug screening programs.
Study the biochemical responses to environmental and physiological stress
The projects in our lab use many techniques of modern cell and molecular biology. These include:
- point mutant knock-in and knock-out gene targeting in mice;
- generation of stable cell lines which exhibit inducible expression of cDNAs for ectopic expression of proteins;
- inducible expression of shRNA for gene knockdown;
- RNA analysis by real time PCR and RNA-seq;
- isolation of protein complexes by Ab bound resins and mass spec identification of proteins residing within the complexes;
- ChIP assay to determine DNA sites bound by bHLH/PAS proteins (including ChIP-sequencing for global analysis of DNA binding sites);
- a range of expression and purification systems to obtain quantities of protein suitable for drug screening, dissection and culture of primary neurons from rodent embryos;
- FACS sorting of cell populations, analysis of point mutant proteins in cell based; and
- in vitro biochemical assays.
Basic-Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) transcription factors function broadly throughout life, playing key roles during embryonic development, and then subsequently maintaining homeostasis in the adult.
Some family members are critical for sensing and responding to various forms of environmental or physiological stress, such as toxin invasion, oxygen deficiency or hyperactivity of neurons.
Defects in signalling through bHLH/PAS proteins can initiate or exacerbate a range of pathologies, including cancer, stroke, neurological disorders and early onset obesity. Members of the bHLH/PAS family which we study include:
- Single Minded 1 (SIM1), which functions in hypothalamic neuron development and satiety signalling. Mutations in Sim1 can result in hyperphagia (excessive eating syndrome) and early onset obesity.
- Single Minded 2 (SIM2) is essential for postnatal survival and exhibits a neuronal expression partially overlapping that of SIM1, but we know little of its function in the brain. Its aberrant expression increases tumour development in prostate cancers, where it may cross couple with HIF activities.
- NPAS4 is brain specific, induced by neuron activity, and aids inhibitory synapse formation. NPAS4 is neuroprotective and prevents excitotoxicity during neuron signalling.
- Aryl Hydrocarbon (Dioxin) Receptor is critical for sensing infiltration of environmental pollutants and, more recently, has been established to be an important factor in differentiation and function of specific cell types of the immune system.
- Hypoxia Inducible Factors (HIF-1a & HIF-2a) are essential for embryonic angiogenesis, glycolytic metabolism and adaption to low oxygen stress.
Our projects use gene knock out and point mutant knock-in mouse models, in conjunction with genetic manipulation of cultured cells, to elucidate normal and pathogenic functions of bHLH/PAS transcription factors.
We seek to understand the signalling mechanisms which control their activities and define their direct target genes. Ultimately we hope to gain insights which will enable us to design novel strategies to treat their associated pathologies.