Old Yellow Enzyme Research Overview

Protection of the Actin Cytoskeleton From Oxidative Stress

Oxidative metabolism produces damaging free-radicals that attack important macromolecules such as chromatin and protein complexes. The resulting oxidative damage is a main cause of aging and cell death (apoptosis) but is also the main cause of the painful and deadly event called crisis in sickle cell patients. It turns out that our central player, the actin cytoskeleton, is a critically important target of oxidative stress. As a result of damaging free-radicals, actin can accumulate in cells in an oxidized form with inappropriate disulfide bonds that result in stabilization of the actin cytoskeleton. It is precisely this stabilization that locks sickled red blood cells into a sickled shape leading them to block capillaries, cause organ damage and mortality in people with sickle cell anemia. Our lab has discovered that cells have an enzyme, called "Old Yellow Enzyme", whose function is to reduce and repair these bad disulfide bonds in actin. We have also found that mutating the actin residues that form these disufide bonds actually makes cells super-resistant to oxidative stress, providing a possible avenue of research into how to reverse the aging process. Below is shown the structure of Old Yellow Enzyme (Panel B) and the structure of actin (Panel C). The cysteines that undergo oxidative damage in actin are shown in yellow in Panel B and the regions of contact between Oye2p and actin are indicated in red.

Oyefig1

The structure of Oye2p is shown in B and the structure of actin is shown in C. The red region of Oye2p is sufficient to interact with actin while the red regions on actin are amino acids required for the interaction with Oye2p. The redox regulated cysteines of actin are labeled and shown in yellow.

Oyefig2 Visualization of the actin cytoskeleton with rhodamine-phalloidin shows the clear difference between the wild type strain (Panel A at right) and a oye deletion strain (Panel B at right). The excessive actin elaboration seen in the oye deletion strain correlates with an increased sensitivity to oxidative stress and can be completely suppressed by mutating either of the redox regulated cysteines to alanine. In fact, the Cys-Ala substations make cells super-resistant to oxidative stress confirming that actin is a critically important target of oxidative stress.

More recently, Michelle Farah, working in our lab, has found that oye deletion cells undergo accelerated cell aging and death associated with many of the classic markers of apoptosis including ROs elevation, nuclear, and DNA fragmentation. The figure below is from her recent publication and shows a chronological aging assay. Note that the oye deletion cell culture shows a much more rapid loss of cell viability than the wild type straing and that this can be completely suppressed by the Cys-Ala mutations in the redox reactive cysteines.

f4oyedth

Chronological aging assay. Cells were grown to stationary phase, counted at the indicated times and plated to determine the proportions of viable cells.

Our current working model for the physiological role of the actin redox cycle is cartooned below. We believe that the actin disulfides act as sensors of the ROS load in cells. If the oxidative stress exceeds the ability of Oye2p to keep the actin reduced, then oxidized actin accumulates and forms stable actin filaments. By an unknown mechanism, actin stablization then leads to a further elevation of ROS contributing to a positive feedback loop that trigger programmed cell death.

RosLoad

Model for the Role of actin oxidation in a positive feedback loop that leads to a ROS-triggered programmed cell death

References:
Haarer BK and Amberg DC. (2004) Old Yellow Enzyme Protects the Actin Cytoskeleton from Oxidative Stress. Molec. Biol. Cell; 15: 4522-4531.

Farah ME and Amberg DC. (2007) Conserved actin cysteine residues are oxidative stress sensors that can regulate cell death in yeast. Molec. Biol. Cell. Mol Biol Cell, Apr;18(4):1359-65. [Epub 2007 Feb 7]