JCJC SVSE 8 - JCJC - SVSE 8 - Biochimie, biologie moléculaire et structurale

Nuclear Ubiquitylation – NucUb

Nuclear Ubiquitylation

Ubiquitylation is a posttranslational modification of proteins that regulates a wide variety of cellular functions and is involved in diverse human pathologies. The main objective of this project is to better characterize the nuclear functions of protein ubiquitylation, especially focusing on ubiquitylation events that occur in response to DNA damage.

Nuclear ubiquitylation events are difficult to identify

The primary function of protein ubiquitylation is to maintain the health of the cellular proteome and to target defective and non-functional proteins for proteasomal degradation. This function is achieved through a variety of protein quality control mechanisms that are continuously catalyzing the ubiquitylation of numerous defective proteins in the cell. In comparison, nuclear ubiquitylation events that occur in response to a particular situation such as DNA damage are sparse. Those events are thus difficult to identify and still poorly studied. The main objective of our project is thus to set up a method that will enable us to more easily identify nuclear ubiquitylation events. We will then aim to understand how those ubiquitylation events contribute to cell survival after DNA damage.

To identify nuclear ubiquitylation events, we propose to use the baker’s yeast as a model organism because it can be easily manipulated genetically. We will modify their genome to be able to selectively inhibit the nuclear proteasomes in an inducible manner. This approach will enable to specifically enrich nuclear ubiquitylation events compared to the other cellular ubiquitylation events. We will then identify those events using mass spectrometry and characterize them using both genetic and biochemical methods.

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Ubiquitin and ubiquitin-like proteins (UBLs) are key regulators of a wide array of biological events. They function as posttranslational protein modifiers that trigger highly versatile molecular signals in eukaryotic cells. In particular, the attachment of K48-linked polyubiquitin chains to any cellular protein causes its rapid degradation by the proteasome. This form of ubiquitylation is widely used by the cell to tightly control its most important processes, such as DNA transcription or DNA replication.
While many of the basic principles of posttranslational protein modification by ubiquitin and UBLs are now understood, we are far from completely comprehending their full functional spectrum. In particular, transient and local polyubiquitylation events remain particularly difficult to study, especially under physiological conditions, and will require new or more sensitive methods to be functionally characterized.
The overall theme of this project is to investigate ubiquitylation events that occur in the nucleus in response to DNA damage as well as their spatiotemporal regulation. To achieve this we will use a combination of biochemical, genetic and imaging approaches in a tractable model organism. We will manipulate this organism to enable controlled inactivation of the proteasome in the nucleus. I expect this to be a powerful strategy to detect low-abundance and/or local proteasome-targeting ubiquitylation events in response to DNA damage. We will also analyze specific molecular mechanisms that control protein ubiquitylation in the nucleus. Altogether, I expect that our studies will contribute to a better understanding of the spatiotemporal organization of nuclear ubiquitylation and its contribution to the cellular response to DNA damage. Furthermore, the methods we will implement will be transposable to analyze nuclear ubiquitylation in other physiological conditions.

Project coordinator

Monsieur Gwenaël RABUT (Institut de Génétique et Développement de Rennes)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IGDR Institut de Génétique et Développement de Rennes

Help of the ANR 199,992 euros
Beginning and duration of the scientific project: December 2012 - 36 Months

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