Scott Eron

Abstract:

The process of programmed cell death, or apoptosis, is a fundamental biological process necessary for all multicellular eukaryotes and is critical for organismal development, differentiation, eliminating damaged cells, and maintaining homeostasis of cell populations. Dysfunctions in this delicate cascade have been linked to a plethora of diseases including cancer, autoimmune disorders, and classic neurodegenerative diseases such as Alzheimer’s and Huntington’s. The driving force behind apoptosis are a family of proteolytic enzymes known as caspases. Once activated, these cysteine-reactive proteases cleave a multitude of substrates, dismembering the cell and sentencing it to a structured extinction. Due to their cell death potential, they are prominent drug targets. Each caspase is regulated uniquely, so understanding individual regulation is imperative to harnessing their abilities. Unfortunately, an incomplete understanding of the mechanisms of regulation has hampered drug development.

Our lab studies the control of caspases and elucidates the mechanisms of their regulation. One manner in which nature manages caspase activity is through phosphorylation. There is a dynamic interplay between caspases and kinases (enzymes that promote phosphorylation), each attempting to inactivate the other. The winner of this battle ultimately controls whether a cell lives or dies. We have explored the effects of phosphorylation of the executioner caspase-7 through a variety of biophysical and biochemical techniques. Kinetic analysis of phosphomimetic variants pinpointed the critical phosphorylation site, S239, which decimated caspase activity. The crystal structure of this phosphomimetic at 2.2 Å illustrated that phosphorylation directly blocks interactions of the substrate with the caspase-7 substrate binding groove. We have developed a new approach for investigating the battle between caspase-7 and PAK2. Ultimately, this approach has revealed that the cleavage state and the conformation of caspase-7 influences the sites of phosphorylation.

In addition to phosphorylation, metal-mediated regulation is natively used to manage caspase activity. Discovery of metal binding sites and elucidating mechanisms of inhibition suggests new means for turning the switch for apoptosis on or off. Moreover, metal binding may illuminate distinct allosteric sites that allow for control over each caspase individually. Our recent work dives into the interactions of caspases and various biologically relevant metals. We have observed that zinc is the only biologically relevant metal able to inhibit caspases, and it does so with surprisingly tight potency. We have characterized this inhibitory interaction with executioner caspases-3 and -7 as well as the upstream initiator caspase-8. Kinetic analysis, microscale thermophoresis, thermal shift assays, and crystallography have together suggested that various caspases bind zinc at distinct locations, and use unique mechanisms to inhibit each caspase, which we have observed crystallographically.

Our investigation of caspase regulation through phosphorylation and zinc binding has led to a greater understanding of caspases and how their function can be controlled. Together this data propels us towards therapeutically relevant means to manage programmed cell death. By obtaining a complete map of caspase regulation and allostery we may be able to control the life of individual cells – whether that be delivering a caspase to cancer cells for programmed cell death, or creating inhibitors that mimic phosphorylation and suppress apoptosis.