Seminari Napolitano – Paoletti
Structural and functional characterization of human RecQ and iron-sulfur (Fe-S) cluster DNA helicases.
Helicases are molecular motors that play central roles in nucleic acid metabolism. Mutations in genes encoding DNA helicases of the RecQ and iron–sulfur (Fe–S) helicase families are linked to hereditary disorders characterized by chromosomal instabilities and premature aging highlighting the importance of these enzymes. Interestingly, RecQ and Fe-S helicase mutations are also strongly associated to a broad spectrum of cancers. In the last years, I have been working on different members of both the DNA helicase families in order to carry out a comprehensive structural and functional characterization. In this seminar, I will focus on RecQ4, a member of the RecQ family of helicase which are highly conserved throughout the evolution and play an essential role in maintaining genomic stability due to their involvement in DNA replication, recombination and repair. In the last few years, I was able to carry out a detailed biochemical analysis of the human RecQ4 showing that, although the protein binds a variety of nucleic acids, it has a preference for R-loops, DNA:RNA hybrids, which are unwound with a higher efficiency that the other substrates. The biochemical data are in accordance with the in vivo analysis showing that cells from Rothmund-Thompson Syndrome (RTS) patients display a high frequency of DNA:RNA hybrids which is correlating with an increase of replication stress markers providing a functional role for RecQ4 helicase in maintaining genomic stability. Moreover, in order to achieve a structural characterization, I was also able to express and purify different fragments of human RecQ4 that will be tested in Cryo-Electron Microscopy experiments. I will also present data on the structural and biochemical characterization of the C-terminus of RTEL1 (Regulator of Telomere length 1), an essential DNA helicase which prevents the chromosome ends instability by disassembling telomere loops and suppressing telomere fragility. In this case, together with a PhD student, we were able to select, express and purify a variety of deletion fragments encompassing the main C-terminal domain. By combining X-ray crystallography and SAXS, we obtained structural information on the architecture of all the fragments tested. Finally, by performing a comprehensive biochemical analysis, we were able to provide a useful framework to elucidate the central role of the RTEL1 C-terminal domain at the interface of the DNA recombination, repair and DNA replication processes.
Dr. Luisa Maria Rosaria Napolitano
An integrative structural biophysics approach to study the interactions between ATP and neurotrophins.
I will present a typical flowchart of the experiments needed to characterize a protein/small molecules interactions system from a molecular point of view. The techniques employed will be described and the results will be presented.
This molecular approach will be esemplified through the recent results obtained by our group on a set of neurotrophic factors proteins. The prototype of the neurotrophin family, Nerve Growth Factor (NGF), is essential for the development and maintenance of neurons and is crucial in immune and endocrine systems and in the pain pathway. NGF precursor, proNGF, whose pro-peptide is an intrinsically unstructured domain (IUD), is endowed with different biological properties. The binding to TrkA, p75NTR and sortilin receptors activates the NGF/proNGF signaling pathways. Much is known about NGF in neuronal physiology. However few reports described essential endogenous ligands as modulators of NGF biology. Recently, the binding of ATP to NGF was identified. To determine the molecular elements of this binding, we used integrative structural biology to unveil for the first time the binding cartography of ATP to NGF . Isothermal Titration Calorimetry (ITC), 1H Saturation Transfer Difference NMR (1H STD-NMR), coupled to the determination of the 3D solution NMR structure of NGF and MD simulations, helped identifying the likely binding mode of ATP on NGF. ATP/NGF binding to the receptors was investigated through Surface Plasmon Resonance (SPR). We also undertook a complementary biophysical study on the binding of ATP to proNGF. Our results reveal a different binding profile for mature and precursor proteins. A combination of Small Angle X-ray Scattering (SAXS), Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) and limited proteolysis showed that ATP binding induces a change in the conformation and/or dynamics of proNGF, predominantly in the IUD pro-peptide . Combined, these results suggest a functional role for ATP in modulating the biological role of proNGF/NGF in health and disease states.
Dr. Francesca Paoletti