Chemical biology to investigate protein oxidative modifications

The research in the Ahn Laboratory is centered around protein modifications that occur in response to endogenous reactive species, including reactive oxygen species (ROS) and reactive nitrogen species (RNS). Our interest in protein modifications comes from the significance of ROS in human health and disease. The traditional view of ROS is that they are by-products of metabolism whose accumulation is detrimental to cellular function. It is widely accepted that ROS are linked to various diseases in heart and skeletal muscle, as well as cancer development and aging process. However, many years of research revealed two seemingly contradictory effects of ROS in cells. High levels of ROS are detrimental and contribute to diseases, whereas low levels of ROS are necessary in the processes of cellular proliferation, migration, inflammation, and apoptosis. Many new data support the emerging field of redox signaling, highlighting ROS as important signaling molecules.

It is becoming clear that ROS induce various types of protein cysteine oxidations, including sulfenylation, disulfide formation, and glutathionylation (Picture below). These protein cysteine modifications are considered molecular mechanisms by which ROS contribute to redox signaling and/or oxidative stress. We are interested in understanding the significance of protein oxidative modifications in human health and disease. Our research program is based on two open biological questions. First, what are the important proteins that undergo oxidative modifications in response to oxidative stimulus? Second, what are the functional consequences of protein oxidative modifications?

To address these questions, we use various interdisciplinary approaches, including synthetic chemistry, protein engineering and biochemistry, enzyme kinetics, and cell biology (Picture below). Our research work can be divided into the following directions.

  • Development of biochemical tools that enable the sensitive and specific detection of protein oxidative modifications.
  • Identification of proteins that undergo oxidative modification under metabolic stress conditions.
  • Investigation of functional effects upon protein oxidative modifications in biological models,including cancer and muscle dysfunction.
  • Development of chemical inhibitors for regulatory enzymes that modulate ROS or protein oxidative modifications and that may be used in therapeutic application.

Protein glutathionylation

Our recent research focused on investigation of protein glutathionylation. Glutathionylation is disulfide bond formation of a protein cysteine residue with intracellular glutathione that occurs in response to oxidative stimuli. There are many examples that highlight the significance of protein glutathionylation in human health and disease. However, the complexity arises from the fact that many cysteine residues in multiple proteins are glutathionylated simultaneously, depending on the levels of ROS, glutathione, and redox enzymes in the cell. Many of the cysteine oxoforms are reversible, labile, and interconvertible. Thus, it is still challenging to identify and examine specific oxoforms, and demonstrate their biological significance.

1) Chemical approach to investigating protein glutathionylation

We have recently developed a biochemical approach, called clickable glutathione, that labels glutathione with a clickable group to identify and characterize protein glutathionylation. The clickable group provides a chemical tag that allows for sensitive, selective, and versatile detection of glutathionylation. We are applying our approach for protein identification in various ROS-relevant disease models.

2) Identification and functional studies of glutathionylated proteins under metabolic stress

ROS are significant factors that contribute to heart and muscle diseases, neuronal damage, and cancer development and progression. It is widely accepted that an intracellular level of ROS is significantly influenced by cellular metabolic state, or nutrient and oxygen availability. We are currently investigating two biological systems, cardiomyocytes and cancer cells, for identification and functional analysis of glutathionylated proteins under metabolic stress, including hypoxia, nutrition-deprivation, ischemia, and ischemic reperfusion. With the mechanistic studies, we further aim to develop potential therapeutic methods against diseases associated with oxidative stress.

Small-molecule or peptide-derived inhibitors for redox-sensitive enzymes

We are interested in developing small-molecule or peptide-derived inhibitors for redox-sensitive enzymes that contains the reactive cysteine residues and are implicated in cancer models. We are developing the potential inhibitors by employing various approaches, including the synthetic organic chemistry, molecular modeling, the rational-design, the peptide-library screening, and the phage-display screening.