Periodic Reporting for period 5 - REDOXCYCLE (The molecular interface between cell cycle and redox regulation)
Período documentado: 2021-07-01 hasta 2021-12-31
“To divide or not to divide”, this is potentially one of the most important questions cells in our body face every day. Controlled cell proliferation is not only essential to build multicellular organisms as complex as humans from a single fertilized oocyte but later in the adult organisms to maintain tissues such as the skin, the gut, or the hematopoietic system, which have to be continuously renewed. On the other hand, cells must also be able to stop proliferating and go into a dormant cellular state, named quiescence, from which they can re-enter a proliferative mode in response to the appropriate stimuli. For maintaining a healthy organism, the balance between proliferation and quiescence is of utmost importance and failures in doing so can result in severe consequences such as cancer where often cells lost the ability to become quiescence.
A multitude of cancer cells are not only characterized by continuous proliferation and the inability to enter quiescence but also by increased levels of reactive oxygen species (ROS). On the molecular level, intracellular ROS are by-products of the aerobic metabolism, hence their occurrence is strongly correlated with the metabolic state of the cell. ROS include e.g. the superoxide anion, hydrogen peroxide, and hydroxyl radicals, and are a crucial part of the intracellular redox system that is crucial for cellular and thus also organismal homeostasis.
In recent years, emerging evidence indicates that ROS can positively influence the proliferation potential of cells, e.g. by affected the decision between proliferation and quiescence. However thus far, we only have a limited understanding of how cell cycle and redox regulation are coordinated. An excellent example why we need to fill this gap is illustrated by our failure to target the redox system in cancer treatment using antioxidants. While cancer cells fully take advantage of elevated levels of ROS to constitutively activate proliferative pathways, they counter adverse effects of ROS by increasing the level of antioxidants. Thus, the use of antioxidants in treatment can have undesirable consequences. Another good example is the emerging evidence that ROS also function as signalling molecules in normal physiological conditions targeting cysteine residues on cell cycle proteins. This indicates that much more subtle changes in redox signalling can affect proliferation and create an intracellular environment that promotes tumorigenesis.
To reveal how the decision between proliferation and quiescence and proliferation in general influenced by the redox system, in particular by ROS, REDOXCYCLE aims to answer three key questions at the edge of cell cycle and redox research:
i) How do changes of the intracellular redox potential initiate, reinforce, and maintain cell cycle decisions?
ii) Which components of the cell cycle machinery are targeted by the redox system on the molecular level?
iii)What distinguishes the physiological cell cycle-redox response from pathological responses that promote cancer?
Our work on REDOXCYCLE revealed that the levels of ROS increase during the progression from G1 to S and G2/M phases as a result of increased mitochondrial activity. A main target of mitochondrial ROS is cyclin-dependent kinase 2 (CDK2), whose oxidation is crucial for full CDK2 activity and genome replication in S phase. Thus, cancer cells that often display elevated levels of ROS could exploit CDK2 activity regulation to promote cell proliferation. Beyond CDK2 our work revealed a multitude of further cell cycle regulators involved in tumorigenesis as targets of ROS. Thus, we suggest that ROS-mediated signalling can play a major role in cell cycle-decision making in physiological and pathological conditions.
A multitude of cancer cells are not only characterized by continuous proliferation and the inability to enter quiescence but also by increased levels of reactive oxygen species (ROS). On the molecular level, intracellular ROS are by-products of the aerobic metabolism, hence their occurrence is strongly correlated with the metabolic state of the cell. ROS include e.g. the superoxide anion, hydrogen peroxide, and hydroxyl radicals, and are a crucial part of the intracellular redox system that is crucial for cellular and thus also organismal homeostasis.
In recent years, emerging evidence indicates that ROS can positively influence the proliferation potential of cells, e.g. by affected the decision between proliferation and quiescence. However thus far, we only have a limited understanding of how cell cycle and redox regulation are coordinated. An excellent example why we need to fill this gap is illustrated by our failure to target the redox system in cancer treatment using antioxidants. While cancer cells fully take advantage of elevated levels of ROS to constitutively activate proliferative pathways, they counter adverse effects of ROS by increasing the level of antioxidants. Thus, the use of antioxidants in treatment can have undesirable consequences. Another good example is the emerging evidence that ROS also function as signalling molecules in normal physiological conditions targeting cysteine residues on cell cycle proteins. This indicates that much more subtle changes in redox signalling can affect proliferation and create an intracellular environment that promotes tumorigenesis.
To reveal how the decision between proliferation and quiescence and proliferation in general influenced by the redox system, in particular by ROS, REDOXCYCLE aims to answer three key questions at the edge of cell cycle and redox research:
i) How do changes of the intracellular redox potential initiate, reinforce, and maintain cell cycle decisions?
ii) Which components of the cell cycle machinery are targeted by the redox system on the molecular level?
iii)What distinguishes the physiological cell cycle-redox response from pathological responses that promote cancer?
Our work on REDOXCYCLE revealed that the levels of ROS increase during the progression from G1 to S and G2/M phases as a result of increased mitochondrial activity. A main target of mitochondrial ROS is cyclin-dependent kinase 2 (CDK2), whose oxidation is crucial for full CDK2 activity and genome replication in S phase. Thus, cancer cells that often display elevated levels of ROS could exploit CDK2 activity regulation to promote cell proliferation. Beyond CDK2 our work revealed a multitude of further cell cycle regulators involved in tumorigenesis as targets of ROS. Thus, we suggest that ROS-mediated signalling can play a major role in cell cycle-decision making in physiological and pathological conditions.
To understand ”how changes of the intracellular redox potential initiate, reinforce, and maintain cell cycle decisions” we first set out to determine the spatial-temporal framework of cell cycle and redox regulation by developing a new endogenous all-in-one cell cycle reporter. The reporter is able to distinguish all cell cycle phases and quiescence using a single fluorescent protein in non-transformed cells, primary cells and cancer cells. Using the reporter and complementary approaches we established that the levels of ROS oscillate during the cell cycle and continuously increase when cell progress from G1 to S and G2 phases. To reveal which cell cycle regulatory proteins are modified by ROS we performed mass spectrometry analyses that indicated that a multitude of cell cycle proteins become oxidized during the cell cycle. We focused on the main S phase kinase CDK2, which is crucial for DNA replication, to understand the mechanism by which oxidation by ROS can influence proliferation in human cells. Our results show that oxidation of a conserved cysteine residue close to the activation domain of CDK2 promotes full CDK2 activity and thus efficient replication of the genome. In collaboration with scientists, we further showed that ROS also promotes proliferation in otherwise only slowly proliferating cell types such as beta cells and neuronal stem cells. Furthermore, we uncovered and characterized interaction surfaces of a cell cycle ubiquitin enzyme that is prime candidate for regulation by ROS. Thus, the pro-proliferative role of ROS appears to be conserved in different tissues and organisms indicative of typical cell cycle regulatory signaling. Since several of ROS modified proteins we identified have been linked to cancer and will be in the focus of subsequent studies in the laboratory REDOXCYCLE provided an excellent basis to further unravel the interplay between cell cycle regulation and ROS signaling in the future.
In total our work on REDOXCYCLE including the development of technologies to pursue the action and associated collaborations resulted in 12 publication in well-recognized scientific journals. Results from REDOXCYCLE have been presented in oral and poster presentations at national and international conferences, and in invited seminars.
In total our work on REDOXCYCLE including the development of technologies to pursue the action and associated collaborations resulted in 12 publication in well-recognized scientific journals. Results from REDOXCYCLE have been presented in oral and poster presentations at national and international conferences, and in invited seminars.
The work of REDOXCYCLE established that the levels of cellular ROS, primarily derived from respiration in the mitochondria, oscillate during the cell cycle. Our work established that a multitude of cell cycle enzymes are targets of ROS and by virtue S phase kinase CDK2, we elucidated a mechanism by which the oxidation of a single cysteine residue can regulate the activity of a key cell cycle enzymes involved in the decision "to proliferate or not to proliferate". REDOXCYCLE also revealed further cancer-relevant cell cycle targets of ROS that we will continue investigating in the future. Thereby, REDOXCYCLE has provided an excellent foundation for future cutting edge research at the intersection of cell cycle and redox biology.