The deregulation of genome stability pathways caused by environmental agents is a major culprit of tumorigenesis in human cells; protein ubiquitination, a reversible posttranslational signaling process, is essential for maintaining this regulation. The ubiquitination status of a target protein is achieved via a delicate balance between two opposing forces: ubiquitin E3 ligases and deubiquitinating enzymes (DUBs). It has been postulated that the majority of proteins in a cell are regulated and modified by ubiquitin at some point; however, due to the multitude of DUBs present in cells, many of the modifications only exist transiently and are difficult to capture. There are nearly one hundred DUBs encoded in the human genome, many of which have uncharacterized function(s) and regulation. DUBs play an essential role in the maintenance of genomic stability, and their aberrant expression has been linked to tumorigenesis. The regulation of DUBs in mammalian cells and how this affects genome stability pathways remain unclear. DUBs constitute a large family of cysteine (Cys) proteases yet basic information on how the enzymatic activity of DUBs is modulated by oxidative stress caused by either environmental agents or intracellular signals is lacking. We recently discovered that bursts of reactive oxygen species (ROS) reversibly inactivate specific DUBs through the oxidation of the catalytic Cys residue. Importantly, the DUB USP1, a key regulator of the Fanconi Anemia (FA) genomic stability pathway and PCNA-mediated translesion synthesis (TLS), was reversibly inactivated upon oxidative stress. We hypothesize that DUBs of the cysteine protease family act as ROS sensors in human cells and that ROS-mediated DUB inactivation is a critical mechanism for fine-tuning genome stability pathways in both health and disease. Herein, we propose to dissect the different mechanisms of DUB regulation using USP1 as a model system, and then extend the analysis to other Cys- and metallo-based DUBs. We will elucidate the mechanisms and functions of DUB oxidation by using a set of unique biochemical and mass spectrometry-based assays to capture and characterize the reversibly oxidized form(s) of DUBs. We will investigate the role of DUB oxidation in DNA repair and cell growth pathways by generating DUB mutants that remain active, yet resistant to reversible oxidation. Finally, we will employ a unique approach to collect and study novel ubiquitinated species that are regulated by oxidative stress. Together, our studies will reveal core principles of deubiquitination and genome stability regulation, and will likely provide guidance for the development of novel classes of therapeutics that target DUBs.

Public Health Relevance

Many of the chemotherapeutics used to treat cancers cause an unintended elevation of reactive oxygen species (ROS) in both normal and tumor cells. DUBs are emerging as important regulators of many pathways germane to cancer. Thus, understanding how cancer-relevant DUBs are regulated by ROS in both normal and tumor cells could potentially impact the efficacy of drug treatments for cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES025166-01
Application #
8857706
Study Section
Molecular Oncogenesis Study Section (MONC)
Program Officer
Shaughnessy, Daniel
Project Start
2015-09-30
Project End
2020-08-31
Budget Start
2015-09-30
Budget End
2016-08-31
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Tonzi, Peter; Yin, Yandong; Lee, Chelsea Wei Ting et al. (2018) Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery. Elife 7:
Ma, Li; Lin, Kangyu; Chang, Guoqiang et al. (2018) Aberrant activation of ?-catenin signaling drives glioma tumorigenesis via USP1-mediated stabilization of EZH2. Cancer Res :
Suisse, Annabelle; Békés, Miklós; Huang, Tony T et al. (2018) The COP9 signalosome inhibits Cullin-RING E3 ubiquitin ligases independently of its deneddylase activity. Fly (Austin) :1-9
Coleman, Kate E; Békés, Miklós; Chapman, Jessica R et al. (2017) SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function. Elife 6:
Coleman, Kate E; Huang, Tony T (2016) HUWE1 comes to the rescue at stalled replication forks. EMBO Rep 17:781-2
Coleman, Kate E; Huang, Tony T (2016) How SUMOylation Fine-Tunes the Fanconi Anemia DNA Repair Pathway. Front Genet 7:61
Békés, Miklós; van der Heden van Noort, Gerbrand J; Ekkebus, Reggy et al. (2016) Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease. Mol Cell 62:572-85