Research interests and projects

Research in the lab is focused on these topics:

1. Oxidative protein folding in the endoplasmic reticulum (ER)
About a third of all mammalian proteins are synthesized in the ER, including important cellular proteins such as antibodies, extracellular matrix proteins, hormones and cell surface receptors and channels. Most of these proteins contain disulfide bonds, and the pairing of cysteines into native disulfides is most often a prerequisite for correct protein folding.

We study the function of thiol-disulfide oxidoreductases of the protein disulfide isomerase (PDI) family, enzymes that catalyze disulfide-bond formation in newly synthesized ER proteins. As part of this overall topic we are investigating how specialized cone snail PDIs assist folding of disulfide-rich cone snail toxins (see below). We also investigate ER redox homeostasis, that is the interplay of ER redox enzymes and glutathione that modulates the overall ER redox conditions. In particular, we focus on the ER flavoprotein oxidases of the Ero1 family, and their role in controlling the redox status of PDI-family members and the ER environment. 
2. The degradation of misfolded ER proteins
Proteins that don’t fold correctly have a tendency to aggregate, and the accumulation of such misfolded proteins in the ER can pose a serious risk to eukaryotic cells. Misfolded ER proteins are retrotranslocated to the cytosol where they are degraded by the proteasome following poly-ubiquitination. This process is termed ER-associated degradation (ERAD). While important for the clearance of misfolded proteins from the ER, the ERAD system is also exploited by certain viruses to avoid immune detection and by toxins to gain access to the cytosol where they exert their deleterious functions. 

We focus on redox-dependent steps involved in substrate recognition in the ER and dislocation to the cytosol. Thus, we characterize the mechanism whereby specific proteins assist these steps of the ERAD process. At the same time we are investigating the same proteins biochemically using recombinantly expressed protein. In addition, we are taking a broader approach involving RNAi screening in mammalian cells to identify which proteins are involved in the degradation of specific ERAD substrates.

3. Oxidative folding of disulfide-rich cone snail toxins
Predatory marine cone snails are amazing creatures that kill their prey – fish, worms or other snails – by injecting them with a deadly mixture of hundreds of different disulfide-rich neuropeptides referred to as conotoxins. Conotoxins selectively target specific subtypes of receptors or ion channels throughout the nervous system, a characteristic that has lead to wide use of conotoxins in ion channel research and as therapeutic agents. The fact that conotoxins generally contain many disulfide-bonds make them hard to produce – when synthesized as linear peptides, refolding often results in misfolding because cysteines do not form native disulfide-bonds.

We have found that cone snails have evolved a hypervariable family of conotoxin-specific PDIs (csPDIs) that assist the formation of disulfide-bonds in conotoxins. We will now establish recombinant expression systems that will allow the production and molecular characterization of a variety of conotoxins and other disulfide-rich peptides of biomedical or research interest.