Joel Mackay and Jacqui Matthews – with collaborators
The proper regulation of gene expression is a fundamental requirement for the development and ongoing homoeostasis of every organism. Although five decades of work have led to great advances, many of the mechanistic principles underlying gene regulation remain mysterious.
However, the development in recent years of many powerful methodologies across the disciplines of structural, molecular and cell biology – ranging from cryoEM to CRISPR/Cas9 gene editing and next-generation sequencing – offers us the opportunity to make significant advances in understanding gene regulation.
In collaboration with labs in Australia and around the world, we are combining biophysics, biochemistry and molecular and cell biology to identify the molecular underpinnings of gene regulation – with a particular focus on transcriptional regulation. We hope that the results of these endeavours will begin to provide a molecular framework that describes how the proteins involved in regulating a process such as blood cell development cooperate to bring about the expression of the appropriate genes.
Joel Mackay with Toby Passioura, Richard Payne and Louise Walport (Crick Institute)
Antibodies are extremely powerful molecular scaffolds because of their ability to specifically recognize a vast array of targets while retaining the same three-dimensional shape. This property (among others) has led to the development of a rapidly increasing number of antibody-based drugs. However, because of their large size and their requirement for disulphide bonds, they are not well suited for intracellular targets.
We are using RaPID mRNA display technology to develop proteins and cyclic peptides based around other scaffolds that can zero in on a variety of protein targets of interest to us – mostly proteins involved in regulating transcription in normal human biology or in disease states. RaPID allows the creation of exceedingly large libraries (10^12 to 10^14 members) of polypeptides and also permits the incorporation of unnatural amino acids. Together, these features yield massive chemical diversity – just what we think we need to create ligands that can modulate the function of challenging protein targets (like transcription factors!). Such ligands could be powerful tools for dissecting protein function and – ultimately – could be valuable starting points for a new generation of protein-based therapeutics.
LIM domains are zinc-binding modules that are found in many proteins, including a number of transcription factors. Proteins such as LMO2 and LMO4 (LIM-only 2 and 4) consist almost entirely of two LIM domains.
These proteins are involved in regulating the development of various cell types. Inappropriate expression of LMO2 in T-cells is linked with the development of T-cell leukemias, while LMO4 has been implicated in breast cancer.
Given that LIM domains are thought to be protein-protein interaction motifs, we believe that LMO proteins act as bridges, bringing together a number of other proteins (including LDB1, TAL1, and the breast cancer related protein BRCA1) to form regulatory complexes.
We are using a range of biophysical, molecular biological and biochemical approaches to characterize these complexes. We hope to use this information to design specific molecules to inhibit the aberrant activity of these proteins in human disease.