Howard Florey Institute Australia's Brain Research Institute

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Neurochemistry Laboratory

Research Interests

Enzymes are proteins that play an important role in regulating physiological function – they are instrumental in the generation or breakdown of key molecules that transmit communication between cells known as peptide hormones. One such enzyme is insulin-regulated aminopeptidase (IRAP), which has a number of potential tissue specific roles that are poorly understood.

These include

• participation in cognitive and memory processing in the brain
• maintenance of glucose homeostasis in insulin responsive tissues
• regulation of the peptide hormones, oxytocin and vasopressin

Our group has previously demonstrated that peptide inhibitors of IRAP elicit robust effects on accelerating learning and facilitating memory in normal animals and alleviating memory deficits in animal models of amnesia.

Current Projects

Role of IRAP in the Central Nervous System

Collaborations with Drs Ewan McNay from Yale University, Ilse Smolders from Vrije University Brussel and Patrick Kehoe from University of Bristol

We are investigating the roles of IRAP in the brain by

• determining the regional, cellular and subcellular distribution of IRAP in the brain
• investigating the effects of a series of newly developed, non-peptide inhibitors of IRAP on different forms of memory
• characterising the behavioural phenotype of the global and tissue-specific IRAP knockout mice

We postulate that the IRAP inhibitors act via one of these mechanisms to enhance learning and memory

• the compounds bind to the catalytic site of IRAP and inhibit its enzymatic activity thereby prolonging the half-life of some neuropeptide substrates that have memory-enhancing properties, or
• the compounds, upon binding to IRAP, alters the trafficking of GLUT4-containing vesicles thereby regulating the levels of the glucose transporter expressed at the cell surface resulting in an increase in glucose uptake into neurons

Development of Small Molecule Inhibitors of IRAP

Collaborations with Prof Michael Parker from St Vincent’s Institute of Medical Research

In view of the robust and dramatic effects of IRAP inhibitors on accelerating learning and facilitating memory, we propose that IRAP is a good target for the development of a new class of cognitive-enhancing agent. In collaboration with Prof Michael Parker, we have generated a computer model of the catalytic site of IRAP based on its high sequence homology with the enzyme leukotriene A4 hydrolase which has a known crystal structure. We have used this homology model to screen a chemical database for compounds with the potential to bind IRAP. After a series of sequential screening steps, we have identified a family of small molecule IRAP inhibitors. The next stage of our research is to refine the structures of these compounds to improve efficacy and pharmacokinetic properties and test for their ability to enhance memory in animal.

Structure-Activity Studies on the Catalytic Site of IRAP

Collaborations with Prof Michael Parker from St Vincent’s Institute of Medical Research

Given the therapeutic potential of IRAP inhibitors in treating memory disorders, we are investigating the key amino acid residues in the catalytic domain of IRAP that are important for substrate recognition and cleavage and also for interaction with inhibitors of the enzyme using site directed mutagenesis and also the homology model of IRAP.

Interacting Proteins of IRAP

In insulin responsive tissues, IRAP is almost exclusively co-localised with GLUT4, being retained in intracellular compartments in the basal state or translocating with GLUT4 to the plasma membrane under insulin stimulation. Although the function of IRAP in these tissues has not been elucidated, the protein is thought to be involved in the tethering of GLUT4 vesicles to intracellular compartments. This is because the 109 amino acid intracellular tail of IRAP contains sorting and trafficking motifs that are unique to this enzyme. This domain of IRAP was used as a bait to isolate proteins that may be involved in the anchoring or retention of these vesicles to intracellular compartments. We have identified 2 proteins that interact with this domain of IRAP and are currently investigating their roles in GLUT4 vesicle tethering and trafficking.

Role of IRAP in the Kidney

We have recently demonstrated that IRAP in the kidney has a different distribution to GLUT4. This is an interesting observation since IRAP is known as a marker of GLUT4 vesicles. We propose that IRAP is found in a different population of vesicles in the inner medulla of the kidney that regulates the trafficking of the water channel AQP2 (aquaporin-2). This study would involve determination of the localisation of AQP-2 and IRAP by (1) dual labelling immunohistochemistry and immunoprecipitation studies and investigating the role of IRAP in water reabsorption in the kidney. The outcome would have significant bearing on understanding the process of salt and water excretion in the kidney and therefore on the causal effects of nephrotic diabetes insipidus.

Laboratory Techniques

• Molecular Biology: construction of expression vectors, RT-PCR, sequencing and bioinformatic analysis
• Cell Biology: maintenance of cell lines, transfections and western blot analysis
• Histology: immunohistochemistry, in situ hybridization histochemistry and in vitro autoradiography
• Whole animal physiology: small animal surgery and behavioural tests

Funding

• NHMRC
• Institute for the Study of Aging
• Neurosciences Victoria
• Besen Family Trust
• Howard Florey Biomedical Foundation
• CASS Foundation

Research

• Scientific Laboratories
  • Brain Development
  • Brain Injury and Repair
  • Genomic Disorders Research Centre
  • Ion Channels and Human Disease
  • Multiple Sclerosis
  • Neuroimaging
  • Neuropeptides
    • Neurochemistry
    • Peptide
    • Peptide Neurobiology
    • Relaxin
    • Relaxin-Fibrosis
    • Neuropeptide Receptor
    • Synaptic Neurobiology
  • Systems Neurobiology
• Research Partners
  • Integrative Neuroscience Facility
  • Melbourne Neuropsychiatry Centre
• Research Prizes
• Postdoctoral Researchers