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Structural and functional investigations into the FAT10 conjugation pathway – STRUFAT10

Structural and functional characterization of the FAT10 conjugation pathway

Post-translational modifications modulate the function of proteins, hence contributing to the fine-tuning of critical cellular processes. This project aims at better understanding mechanisms involved in the conjugation of FAT10, a ubiquitin-like modifier , onto target proteins.

Understanding the regulation of fatylation

Fatylation is involved in the immune response and proteasomal degradation. FAT10 conjugation shares enzymes also used in ubiquitination, suggesting a balance between the fatylation and ubiquitination in cells. Disruption of such a balance is likely to yield pathological conditions. This project aims at uncovering mechanisms, through which fatylation is regulated.

We use biochemical, biophysical and structural approaches to gain insight into the FAT10 conjugation pathway.
- Crystallography, SAXS, NMR
- Calorimetry, fluorescence anisotropy, FRET, SPR
- Blots etc.

The expression of the different protein components of hte project has been established. Currently, biophysical and biochemical methods towards the characterization of the fatylation pathway are being set up.

With this study, we aim at better understanding the regulation of FAT10 conjugation and to uncover the role of FAT10 in pathophysiological processes.

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Post-translational modifications modulate the function of proteins, hence contributing to the fine-tuning of critical cellular processes. This project aims at better understanding mechanisms involved in the conjugation of FAT10, a ubiquitin-like modifier (UBL), onto target proteins. Whereas the conjugation pathways of ubiquitin and other UBLs such as SUMO or Nedd8 are relatively well described, mechanisms involved in protein fatylation (FAT10 conjugation) still remain elusive. The recent characterization of Uba6 and Ube2z, a ubiquitin activating enzyme (E1) and a ubiquitin conjugating enzyme (E2) respectively, has contributed to partially uncover mechanisms involved in fatylation. Indeed, these proteins can activate and conjugate both ubiquitin and FAT10, hence displaying an unusual dual modifier specificity. Furthermore, Ube2z can only be loaded with activated ubiquitin by Uba6 but not by Ube1, the prototypical E1 in the ubiquitin conjugation pathway, indicative of selective E2 charging by Uba6. Whereas partially lifting the veil over the fatylation process, these unique features of Uba6 and Ube2z also raise more questions concerning the complexity of regulatory mechanisms involved in UBL conjugation. In this project, structural, biophysical and biochemical approaches will be used to investigate how dual modifier specificity arises in Uba6 and how these UBLs are recognized by, and transferred to E2s, for example Ube2z. Similarly, the selective loading of Ube2z by Uba6 as opposed to Ube1 will be characterized. A second aspect of this project will be the identification of potential E3 ligases and targets of the Uba6-Ube2z couple. Altogether, the work achieved will provide novel insight into the ubiquitin and ubiquitin-like modifier conjugation pathways, hence contributing towards a better understanding of patho-physiological conditions arising from altered ubiquitin-mediated signaling events.

Project coordination

Prakash RUCKTOOA (Centre National de la Recherche Scientifique - Unité de glycobiologie structurale et fonctionnelle)

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

CNRS - UGSF Centre National de la Recherche Scientifique - Unité de glycobiologie structurale et fonctionnelle

Help of the ANR 385,216 euros
Beginning and duration of the scientific project: November 2012 - 36 Months

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