Achieving Specificity in Redox Signaling and Redox Regulation of Protein Function

Redox signaling is crucial for cells to maintain homeostasis. The goal of this research is to understand the molecular mechanisms that underlie redox signal propagation and how changes in the cellular redox state affect signaling pathways. This work can be divided into three themes. First, we study how the oxidation of proteins is achieved both efficiently and specifically. Secondly, we study examples of how the oxidation of specific proteins can affect their function in important signaling pathways. Thirdly, we examine how redox regulation can affect protein aggregation.
How H2O2 leads to selective and efficient oxidation of specific thiols on specific proteins is an important open question in redox biology. We explore peroxiredoxin-based redox relays on a proteome-wide scale (chapter 2) to investigate whether this serves as a mechanism for specific and efficient transmission of oxidative signals. We demonstrate that all 2-cys peroxiredoxin isoforms can form numerous cysteine-dependent heterodimers with target proteins, and that each isoform displays a preference for a subset of these binding partners. This suggests that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.
We then focus on the cysteine-dependent redox regulation of several signaling proteins. We show that the PP2A-regulating protein TIPRL is highly sensitive to oxidation and forms three different disulfide-dependent homodimers involving C14 and C87 in response to endogenous levels of H2O2. Preliminary data suggest a role for TIPRL oxidation in regulating phosphatase activity of PP2A-C.
Chapter 4 describes how the tumor suppressor p16INK4A can form β-amyloid-like fibrils under physiological conditions, triggered by oxidation and subsequent S-S-dependent homodimerization of its single cysteine. p16INK4A amyloid formation abolishes its function as a CDK4/6 inhibitor. This example highlights the role of the cellular redox state as an important regulator of fibril formation. We use the term ‘oxaggregation’ to specify the critical dependence on reversible cysteine oxidation as a trigger for protein aggregation.
Excessive ROS can lead to random protein damage, including protein unfolding and aggregation, processes that have been associated with aging. Conversely, H2O2 is an essential second messenger important for healthy cell physiology. Chapter 5 discusses how these seemingly opposite effects of H2O2 as a signaling molecule and as driver of age-related protein aggregation can be united in one hypothesis. We give an overview of the intricate relationship between redox signaling and protein aggregation.
Chapter 6 describes an example of how the cellular redox state is intimately integrated with cell cycle progression. We show that cell cycle regulators CDK4 and cyclinD form a temporary covalently linked complex under oxidizing conditions, mediated by the formation of a disulfide bond involving C135 in CDK4. This stabilizes otherwise electrostatic, non-covalent interactions between these proteins and leads to increased kinase activity of the CDK4-cyclinD complex. We discuss how the redox sensitive cysteine at the CDK4/cyclinD interface could provide a potential target for novel covalent cytostatic drugs.
Finally, we discuss the implications of and concepts emerging from the work in this thesis (chapter 7).