Non-canonical nucleic acid structures and aging: the G4/R-loops connection

Silvia Onesti – Elettra Sincrotrone Trieste

Aging and cancer are intertwined phenomena, caused by the accumulation of DNA replication errors, mistakes in DNA damage repair, genomic and proteomic changes. A growing body of evidence indicates that non-canonical nucleic acid structures can play an important role in all of these processes. A complex network of proteins binds and/or unfolds these structures. We have been working on the biochemical, biophysical and structural characterization of proteins that interact with DNA and RNA G-quadruplexes, as well as with R-loops and D-loops.

Several helicases have been shown to target and regulate non-canonical structures. We have shown that R-loops are the preferred substrate of the RecQ4 helicase in vitro and in vivo. As part of CERIC-INTEGRA we have used CryoEM in SOLARIS to obtain a 3.5 Å structure of the DDX11 helicase and a medium resolution structure of the Regulator of Telomer Length 1 (RTEL1) helicase. Mutations in all these helicases cause rare genetic diseases characterized by cancer predisposition and premature aging.

We are studying two RNA binding proteins involved in splicing and strongly involved in neurodegenerative diseases: the interactions between TDP-43 and  RNA?DNA G4s, and the interaction of SFPQ with R-loops located at telomers and other repetitive region.

Towards Elettra 2.0: new challenges and opportunities for Life Sciences

Lisa Vaccari  – Elettra Sincrotrone Trieste

After 30 years of supporting scientific research into the study of hard, soft and biological materials, Elettra synchrotron (Triest, Italy) is undergoing a major upgrade, involving both the storage ring and its beamlines and laboratories.

The improved brightness and coherence of Elettra 2.0 will make it possible to tackle scientific problems that could not have been solved before, thus opening up new avenues also in the Life Sciences research filed.

The effort being made is to create an interconnected network of beamlines and offline facilities capable of looking at biomedical topics in a holistic manner, integrating structural, morphological, chemical and dynamical information from the atomic to the total body level.

Here the integration path and the status of its implementation will be presented, providing instrumental details and scientific examples in fields ranging from structural biology to X-ray computed tomography, via cell biology and biophysics of model systems.

In-Cell NMR as a Translational Bridge Between Structural Biology and Pharmacology

Lukas Trantirek – Central European Institute of Technology, Masaryk University

Modern drug discovery often relies on screening compounds for their ability to bind a target protein or nucleic acid under simplified in vitro conditions, followed by optimization to improve the interaction. However, many promising candidates fail in later development stages due to poor activity in living cells or unexpected side effects.

These issues often stem from ignoring structural changes in the target or reduced binding selectivity of the drug in the complex cellular environment, factors that cannot be assessed from in vitro data. In-cell NMR spectroscopy offers a powerful solution. By enabling direct, atomically resolved observation of target structures and drug–target interactions inside living cells, it provides critical insights into how drugs and their targets behave in a natural context.

In this presentation, I will highlight recent advances in applying in-cell NMR to drug screening, and how it helps bridge the gap between test-tube assays and cellular reality.

3D Micro/Nano synchrotron CT imaging in bone research

Francoise Peyrin – INSA‐Lyon, Univ. Claude Bernard Lyon 1

Osteoarticular diseases like osteoporosis are increasing with the aging of the population. However progresses in diagnosis and treatment are still necessary motivating research to have a better understanding of the biological mechanisms involved in bone fragility.

X-ray CT has always been a technique of choice to image bone at different scales. In this context, synchrotron CT imaging possesses additional advantages for bone imaging, since it may provide quantitative attenuation or phase contrast maps at very high spatial resolution. The combination of synchrotron CT image acquisition with the development of dedicated image processing algorithms allows to go beyond imaging and extract quantitative parameters on bone tissue at different scales.

We will review studies based on synchrotron micro and nano CT to assess bone microarchitecture, bone microcracks, micro-vascularization and the bone cell network.

Finally, we will also open perspectives with the promise of artificial intelligence, and particularly deep learning methods to improve X-ray CT imaging.

Advancing multidisciplinary research through cryo-EM at ESRF

Eaazhisai Kandiah – The European Synchrotron Radiation Facility

Cryo-electron microscopy (cryo-EM) has become a key technique in structural biology, allowing researchers to visualize macromolecular complexes at high resolution in near-native conditions. It has had a broad impact across many areas of biology, including aging, with important applications in understanding the underlying molecular mechanisms.

At the ESRF, our cryo-EM facility, CM01, is centered around a Titan Krios microscope and serves a diverse international user community. Work carried out at CM01 has led to significant findings across multiple disciplines, including several that connect closely with the main themes of this symposium. In this presentation, I’ll highlight a few examples of user projects that originated from data collected at CM01 and illustrate the kinds of scientific questions this technique can help address. I will also briefly outline the current facility setup and our future upgrade plans to keep CM01 at the forefront of research.

The ABC of TDP-43

Diana Arseni -Darwin College

Abnormal assemblies of TDP-43 in neurons and glia are the pathological hallmark of amyotrophic lateral sclerosis (ALS) and multiple types of frontotemporal lobar degeneration (FTLD). Mutations in the TDP-43 gene, TARDBP, can cause ALS and FTLD, and the temporospatial accumulation of TDP-43 assemblies correlates with neurodegeneration, indicating a causative role for TDP-43 assembly in disease. TDP-43 assemblies are also common co-pathologies in other diseases, including Alzheimer’s, Parkinson’s and Huntington’s.

The structural and molecular mechanisms of TDP-43 assembly in disease are poorly understood. We developed a protocol to isolate assembled TDP-43 from the brains of patients with ALS and FTLD and determined their structures using cryo-electron microscopy (cryo-EM). We found that TDP-43 assembles into amyloid filaments in these diseases. The ordered filament cores are comprised of the first half of the TDP-43 low-complexity domain and adopt distinct filament folds in different neurodegenerative conditions. These brain-derived filament folds show no similarity to TDP-43 filament folds formed in vitro. The structures, in combination with mass spectrometry, led to the identification of two new post-translational modifications of assembled TDP-43, citrullination and mono-methylation of R293, and suggest that they may facilitate filament formation and observed structural variation within individual filaments.

Unexpectedly, the structures also revealed that in specific cases TDP-43 can also co-assemble with Annexin A11 in heteromeric amyloid filaments. The structures of TDP-43 amyloid filaments from ALS and FTLD guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds for TDP-43 proteinopathies.