Molecular, Structural, and Functional Studies of Leukemia-Associated Transcription Factor-Nucleoporin Fusion Proteins

Project Information

TFNup

Grant agreement ID: 894106

DOI

10.3030/894106

Project closed

EC signature date 1 April 2020

Start date 1 September 2021

End date 31 August 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 174 806,40

EU contribution

€ 174 806,40

Coordinated by

JOHANNES GUTENBERG-UNIVERSITAT MAINZ
Germany

Periodic Reporting for period 1 - TFNup (Molecular, Structural, and Functional Studies of Leukemia-Associated Transcription Factor-Nucleoporin Fusion Proteins)

Reporting period: 2021-09-01 to 2023-08-31

Summary of the context and overall objectives of the project

The TFNup project aimed to tackle a fundamental challenge in biology—the study of intrinsically disordered proteins (IDPs). IDPs lack a fixed 3D structure, making them elusive subjects for scientific inquiry. However, their roles in cellular processes, particularly in health and disease, are of immense importance. The overarching goal was to develop innovative biochemical and biomolecular imaging tools that provide high-resolution insights into the plasticity of IDPs, particularly in living cells. In addition, the project aimed to shed light on the phase separation of IDPs that lead to the formation of larger protein assemblies often implicated in diseases.
The project specifically focused on the molecular behaviors of an intrinsically disordered FG-rich nucleoporin (NUP98). Using the newly developed tools, the researchers measured the conformations and dynamics of NUP98 in live cells, even within functional nuclear pore complexes. This study pioneered the measurement of IDP conformations and dynamics in a nanosized object or biomolecular condensates inside cells.
The project's significance lies in its potential to transform our understanding of molecular and cell biology. With over 30% of human proteins consisting of IDPs, this project has opened new avenues to unravel the complex relationships between disorder and function within cells.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In working package 1 (WP1), a site-specific labeling scheme was developed utilizing genetic code expansion facilitated by synthetic organelles for NUP98 in mammalian cells. A custom-designed FLIM setup was employed to investigate the conformations of the labeled proteins within live cells. By measuring the distance distribution of the disordered chain segments of NUP98, we demonstrated that the FG domain adopts a more expanded conformation inside the nuclear pore complex (NPC) during its functional state. This was probably the first time the dimensions of an IDP were probed inside the cell, but for sure, the first time inside functional nuclear pore complexes. The results on NUP98 published in Nature (Yu et al., 2023) have received wide international attention. The pipeline developed for NUP98 can also be used to study other IDPs in the cell.
In WP2, an in vitro reconstitute assay was performed to understand the conformational dynamics of NUP98 during phase separation. The formed NUP98 condensates undergo a liquid-to-gel transition after about five minutes. The liquid-like condensates during the initial phase transition could mimic the physiological nuclear transport pathway seen in intact NPCs within cells. The conformational dynamics of the FG domain were monitored using fluorescence anisotropy, revealing the slowing of segmental rotation as the molecular aging process continued. This outcome correlated with macroscopic observations of the condensates transitioning from a liquid-like to a gel-like state.
In WP3, the focus was on the dynamics of NUP98 in live cells utilizing high-resolution fluorescence tools. A pipeline was developed to examine the dynamics of IDPs within the cell using fluorescence anisotropy.
The results found in this project provide novel insights into the behavior and conformational dynamics of intrinsically disordered proteins both in vitro and within live cells. These findings pave the way for a deeper understanding of biological processes. In particular, the results on NUP98 have been presented at several conferences, published open access in peer-reviewed journals, and shared on various social media channels. The knowledge gained from this project has the potential to revolutionize structural biology, phase separation studies, and our comprehension of IDPs' roles in cellular processes and diseases, ultimately benefiting society through the advancement of medical research and treatments.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project introduced a remarkable capability to measure the conformational distributions of IDPs within nanosized objects and biomolecular condensates inside living cells, revolutionizing the field of in situ structural biology. The pioneering work showcased the potential of directly probing protein conformational dynamics and phase separation within living cells, offering unprecedented insights into the behavior of IDPs and their role in cellular processes. Moreover, the developed tools and techniques are not limited to specific proteins but can be extended to investigate a wide range of IDPs within cells, expanding the scope of research possibilities in the study of diseases associated with IDPs.
The societal impact of the project is substantial, particularly in the context of healthcare and biotechnology. By shedding light on the molecular mechanisms of NUP98, the project paves the way for innovative therapeutic strategies targeting diseases related to malfunction of NUP98. The developed tools to detect molecular dynamics in aberrant phase transitions open avenues for drug design against diseases characterized by protein misaggregation, as often observed in neurodegenerative diseases. Furthermore, the project's findings provide valuable insights into fundamental cellular processes, enhancing our knowledge of biology and potentially leading to future medical breakthroughs. The dissemination of these results, both within the scientific community and to the broader public, ensures that the societal implications of the project are far-reaching, with the potential to improve healthcare outcomes and drive advancements in biotechnology.

Art impression of disordered proteins inside the nuclear pore complex