A novel mechanism of post-translational modulation of HMGA functions by the histone chaperone nucleophosmin

High Mobility Group A are non-histone nuclear proteins that regulate chromatin plasticity and accessibility, playing an important role both in physiology and pathology. Their activity is controlled by transcriptional, post-transcriptional, and post-translational mechanisms. In this study we provide evidence for a novel modulatory mechanism for HMGA functions. We show that HMGAs are complexed in vivo with the histone chaperone nucleophosmin (NPM1), that this interaction requires the histone-binding domain of NPM1, and that NPM1 modulates both DNA-binding affinity and specificity of HMGAs. By focusing on two human genes whose expression is directly regulated by HMGA1, the Insulin receptor (INSR) and the Insulin-like growth factor-binding protein 1 (IGFBP1) genes, we demonstrated that occupancy of their promoters by HMGA1 was NPM1-dependent, reflecting a mechanism in which the activity of these cis-regulatory elements is directly modulated by NPM1 leading to changes in gene expression. HMGAs need short stretches of AT-rich nucleosome-free regions to bind to DNA. Therefore, many putative HMGA binding sites are present within the genome. Our findings indicate that NPM1, by exerting a chaperoning activity towards HMGAs, may act as a master regulator in the control of DNA occupancy by these proteins and hence in HMGA-mediated gene expression

HMGA1 positively regulates the microtubule-destabilizing protein stathmin promoting motility in TNBC cells and decreasing tumour sensitivity to paclitaxel

High Mobility Group A1 (HMGA1) is an architectural chromatin factor involved in the regulation of gene expression and a master regulator in Triple Negative Breast Cancer (TNBC). In TNBC, HMGA1 is overexpressed and coordinates a gene network that controls cellular processes involved in tumour development, progression, and metastasis formation. Here, we find that the expression of HMGA1 and of the microtubule-destabilizing protein stathmin correlates in breast cancer (BC) patients. We demonstrate that HMGA1 depletion leads to a downregulation of stathmin expression and activity on microtubules resulting in decreased TNBC cell motility. We show that this pathway is mediated by the cyclin-dependent kinase inhibitor p27(kip1) (p27). Indeed, the silencing of HMGA1 expression in TNBC cells results both in an increased p27 protein stability and p27-stathmin binding. When the expression of both HMGA1 and p27 is silenced, we observe a significant rescue in cell motility. These data, obtained in cellular models, were validated in BC patients. In fact, we find that patients with high levels of both HMGA1 and stathmin and low levels of p27 have a statistically significant lower survival probability in terms of relapse-free survival (RFS) and distant metastasis-free survival (DMFS) with respect to the patient group with low HMGA1, low stathmin, and high p27 expression levels. Finally, we show in an in vivo xenograft model that depletion of HMGA1 chemo-sensitizes tumour cells to paclitaxel, a drug that is commonly used in TNBC treatments. This study unveils a new interaction among HMGA1, p27, and stathmin that is critical in BC cell migration. Moreover, our data suggest that taxol-based treatments may be more effective in reducing the tumour burden when tumour cells express low levels of HMGA1

Molecular landscape of the ribosome pre-initiation complex during mRNA scanning: structural role for eIF3c and its control by eIF5

During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1 and 3c2. The N-terminal part, 3c0, binds eIF5 strongly, but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. Using X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core, together with cross-linking coupled to mass spectrometry, we were able to use IMP to position and orient all eIF3 components on the 40S•eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. For more information about how to reproduce this modeling, see https://salilab.org/40S-eIF1-eIF3 or the README file

Complete Phenotypic Recovery of an Alzheimer's Disease Model by a Quinone-Tryptophan Hybrid Aggregation Inhibitor

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease

Multiscale modelling of protein aggregation, peptide/membrane interactions and hydrophobic core stabilization

Biochemie und Molekularbiologie stehen im direkten Zusammenhang mit dem täglichen Leben: das Wissen von biologischen Mechanismen hat eine grosse Bedeutung für die Gesundheit, die Umwelt und die technologische Entwicklung. Die vorliegende Doktorarbeit befasst sich mit computergestützten Beiträgen zur Molekularbiologie. Eine Vielzahl von komplexen molekularen Phänomenen wurde dank der inhärenten Flexibilität numerischer Simulationen untersucht. Ein zentrales Problem von Simulationen der Moleküldynamik ist die Frage, wie der Konformationsraum eines komplexen Systems mit einem genügend genauen Kraftfeld abgetastet werden soll. Das Verhältnis zwischen Genauigkeit und Effizienz ergibt sich hierbei aus den Anforderungen des zu untersuchenden Systems. In dieser Arbeit wurden drei Themen untersucht: Amyloid Aggregation, Stabilisierung des gefalteten Zustands eines Proteins und Membran- Protein Interaktionen. Alle Projekte dieser Arbeit wurden mit verschiedenen computergestützten Methoden mit unterschiedlichem Grad an Vereinfachung und Genauigkeit behandelt. Die Aggregation von Amyloid Proteinen wird mit vielen degenerativen Krankheiten wie Alzheimer, Parkinson und Typ II Diabetes, in Verbindung gebracht. Aus diesem Grund kann das Verständnis der Vorgänge auf molekularer Ebene dazu genutzt werden, Strategien gegen die Giftigkeit der Amyloidablagerung zu entwickeln. Die Vorhersage der Aggregationsgeschwindigkeit von Amyloidproteinen wurde mit Hilfe einer phänomenologischen Formel untersucht, welche Aufschluss gab über die Abhängigkeit der Bildung von amyloiden Strukturen aufgrund der Polypeptidesequenz. Danach wurde ein grobkörniges Modell von Amyloidpeptiden entwickelt. Die eingeführte Vereinfachung erlaubte die Erforschung von Phänomenen wie Oligomerbildung, Keimbildung und Verlängerung von Fibrillen, die mit genauen Kraftfeldern nicht zugänglich sind. Des Weiteren liess die Änderung eines einzigen energetischen Parameters, welcher die relative Verteilung amyloidophiler or amyloidophober Zustände des Monomers bestimmt, die Nachahmung vieler Phänomologien zu, was Aufschluss über die Kinetik der Aggregation geben kann.Proteinfaltung ist ein Phänomen dem die Funktionalität von molekularen Komponenten zugrundeliegt. Vorhersagen welche Sequenzmodifikationen eine stabilere Strukur und möglicherweise ein funktionaleres Protein zur Folge haben, haben eine enorme Auswirkung auf molekülbasierte Technologien. Eine Zusammenarbeit mit Experimentalisten führte zur Entdeckung von Mutationen, die den gefalteten Zustand eines Proteins, bestehend aus mehreren Armadilloeinheiten, stabilisieren, das vorgängig Charakteristika eines geschmolzenen Kügelchens (molten globule) aufwies. Die rechenbetonten Beiträge halfen den Sequenzraum einzuschränken und die aussichtsreichsten Mutanten auszuwählen. Membran-Protein Interaktionen bilden die Basis der Zellkommunikation und des Kerntransports. Diese Mechanismen, obwohl in einer Vielzahl von entscheidenden Prozessen involviert, sind nach wie vor auf molekularer Ebene schlecht verstanden. Die Erforschung von Peptiden mittels Simulationen der Moleküldynamik, die sowohl mit Membranen als auch mit Mizellen interagieren, verhalf zu neuen Einsichten dieser molekularen Mechanismen. Im ersten System konnte die spontane Faltung von Melittin an der Lipidoberfläche einer Mizelle reproduziert werden, währenddem die Gleichgewichtseigenschaften des Mizellen-Melittin-Komplexes ebenfalls erhalten blieb. In einer zweiten Arbeit wurden das in einer Membrandoppelschicht eingebettete, Lipid-modifizierte C-terminale Heptapeptid des menschlichen N-ras Proteins untersucht. Die Ergebnisse der Simulation bestätigten ein vorgängiges strukturelles Modell, basierend auf spektroskopischen Daten und schlugen einen Mechanismus des Einschubs der Peptids in die Membran vor. Biochemistry and molecular biology are directly related to everyday life: the knowledge of the biological mechanisms at the molecular level has a strong impact on health, environment and technological development. In the present thesis, the contributions of computational methods to molecular biology were investigated. Thanks to the intrinsic flexibility of numerical simulations, a variety of complex molecular phenomena have been addressed. One of the major issues of molecular dynamics simulations is how to efficiently sample the conformational space of complex systems with a sufficiently accurate force field. The relationship between accuracy and efficiency must be established by the requirements of the investigated system. In this work three subjects were treated: amyloid aggregation, stabilization of protein folded state, and membrane-protein interactions. All the projects exposed in this thesis were approached using different computational methods with different levels of simplification and accuracy. Amyloid protein aggregation is related to many degenerative diseases, such as Alzheimer’s disease, Parkinson and type II Diabetes. Therefore the understanding of this process at the molecular level can help to devise strategies against the toxicity induced by amyloid deposits. The prediction of the aggregation rate of amyloid peptides was addressed using a phenomenological formula that gave insights into the dependence of amyloidogenesis on the polypeptide sequence. Thereafter a coarse-grained model of amyloid peptides was developed. Here, the simplification introduced allowed the exploration of phenomena such as oligomer formation, fibril nucleation and fibril elongation, which are not accessible by accurate force fields. Furthermore, the change of a single energetic parameter, which determines the relative population of the amyloid-prone and amyloid-protected states of the monomer, allowed to reproduce a vast phenomenology, which is useful to shed light into the kinetics of aggregation. Protein folding is the phenomenon that lies behind the functionality of molecular components of the cell. Predicting which sequence modifications results in a more stable fold, and possibly more functional protein, has an enormous influence on molecular-based technologies. Here, a collaboration with experimentalists has led to the discovery of the mutations that stabilize the folded state of an Armadillo repeat protein, which previously displayed molten globule-like features. The computational contribution helped to restrict the sequence space, and to select the most promising mutants. Membrane-protein interactions are the basis for cell signalling and cellular transport. These mechanisms, although involved in a number of vital processes, are still poorly understood at the molecular level. In this work, the investigation of peptides that interact with both micelles and membrane by means of molecular dynamics simulations has provided new hints on the underlying molecular mechanisms. In the first system investigated the spontaneous folding of melittin on the lipid micelle surface was reproduced, together with equilibrium properties of the micelle-melittin complex. In the second work the lipid modified C-terminal heptapeptide of the human N-ras protein embedded into a membrane bilayer was studied. The simulation results validated a previous structural model based on spectroscopic data, and propose a mechanism for peptide insertion into the membrane

How Does a Simplified-Sequence Protein Fold?

To investigate a putatively primordial protein we have simplified the sequence of a 56-residue α/β fold (the immunoglobulin-binding domain of protein G) by replacing it with polyalanine, polythreonine, and diglycine segments at regions of the sequence that in the folded structure are α-helical, β-strand, and turns, respectively. Remarkably, multiple folding and unfolding events are observed in a 15-μs molecular dynamics simulation at 330 K. The most stable state (populated at ∼20%) of the simplified-sequence variant of protein G has the same α/β topology as the wild-type but shows the characteristics of a molten globule, i.e., loose contacts among side chains and lack of a specific hydrophobic core. The unfolded state is heterogeneous and includes a variety of α/β topologies but also fully α-helical and fully β-sheet structures. Transitions within the denatured state are very fast, and the molten-globule state is reached in <1 μs by a framework mechanism of folding with multiple pathways. The native structure of the wild-type is more rigid than the molten-globule conformation of the simplified-sequence variant. The difference in structural stability and the very fast folding of the simplified protein suggest that evolution has enriched the primordial alphabet of amino acids mainly to optimize protein function by stabilization of a unique structure with specific tertiary interactions

Role of integrative structural biology in understanding transcriptional initiation

International audienceIntegrative structural biology combines data from multiple experimental techniques to generate complete structural models for the biological system of interest. Most commonly cross-linking data sets are employed alongside electron microscopy maps, crystallographic structures, and other data by computational methods that integrate all known information and produce structural models at a level of resolution that is appropriate to the input data. The precision of these modelled solutions is limited by the sparseness of cross-links observed, the length of the cross-linking reagent, the ambiguity arisen from the presence of multiple copies of the same protein, and structural and compositional heterogeneity. In recent years integrative structural biology approaches have been successfully applied to a range of RNA polymerase II complexes. Here we will provide a general background to integrative structural biology, a description of how it should be practically implemented and how it has furthered our understanding of the biology of large transcriptional assemblies. Finally, in the context of recent breakthroughs in microscope and direct electron detector technology, where increasingly EM is capable of resolving structural features directly without the aid of other structural techniques, we will discuss the future role of integrative structural techniques