Kretz Lab

The objective of this project is to explore the biochemical and structural mechanisms that contribute to the regulation of ADAMTS13. ADAMTS13 exists as a latent protease that undergoes substrate-induced activation through a mechanism that is not well understood. Because ADAMTS13 is an important regulator of blood clotting, understanding the mechanisms that control its activity will allow us to design novel therapies to treat thrombosis.

Figure: VWF recognition by ADAMTS13. (A) ADAMTS13 circulates in a closed conformation that masks important exosites required for binding to VWF. (B) VWF is a large multimeric protein that circulates in a compact “birds nest” configuration. (C) Under conditions of high shear stress (such as at the site of vessel injury), VWF unfolds into a long string like form, exposing critical binding sites for ADAMTS13. As the VWF multimer accumulates platelets, its central A2 domain can denature to expose the cleavage site for ADAMTS13.

Approximately two per cent of the human genome encodes for a protease. Many of these proteases are poorly understood, having no known role in biology. Therefore, new tools are required to explore the role of proteases in biological processes. We are developing and applying a high throughput technique to define protease substrates specificity, termed deep protease profiling. This technique is based on the coupling of traditional substrate phage display with high throughput next generation DNA sequencing. This approach allows us to quantitatively determine the activity of a protease towards >3.2 million peptide sequences in a single reaction and then use computational methods to understand the rules that govern its substrate specificity. The long-term goals of this project are to develop a tool kit of substrates with absolute specificity toward its target protease and to identify protease activity in complex biological mixtures. Such technologies will shed new light on the role of proteases in biology and may lead to the develop of novel diagnostic tools to detect protease activity in biological settings.

Mutations in VWF that impair its clotting function result in the bleeding disorder von Willebrand Disease. While genome sequencing efforts identify genetic variants in VWF, the major obstacle is in the interpretation of variants. This is due to a lack of evidence about the functional impact of variants. Traditional phenotyping efforts suffer from low throughput and cannot keep up with the demand. The result is a growing list of variants of unknown significance (VUS). In fact, approximately 41 per cent of the 72,000 missense variants annotated in the ClinVar database are classified as VUS. In this project, we are applying Deep mutational scanning (DMS) in order to catalogue the impact of every possible single amino acid substitution at each position of VWF on its function. To do this, we have developed a cell secretion assay in order to quantify the impact of missense mutations on VWF biosynthesis and secretion from HEK293 cells.

Figure: Mutagenesis libraries are designed in silico and synthesized as an oligonucleotide pool. Pooled oligo libraries are assembled into full length VWF expression vector, fused to eGFP. Libraries are stably transfected into HEK293 cells, and sorted into bins according to their intracellular eGFP signal. Bins are submitted for throughput DNA sequencing to quantify variant abundance within each bin. Data are expressed as heat maps representing cell abundance scores for every variants at each position.