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Comparison of ex vivo and in vitro human fibroblast ageing models.
Federica Boraldi, Giulia Annovi, Roberta Tiozzo, Pascal Sommer, Daniela Quaglino, 2010, original scientific article

Abstract: Several studies have analyzed modulation of gene expression during physiological ageing with interesting, but often contradictory results, depending on the model used. In the present report we compare age-related metabolic and synthetic parameters in human dermal fibroblasts (HDF) isolated from young and old subjects (ex vivo ageing model) and cultured from early up to late cumulative population doublings (CPD) (in vitro ageing model) in order to distinguish changes induced in vivo by the aged environment and maintained in vitro, from those associated with cell senescence and progressive CPD. Results demonstrate that fibroblasts from aged donors, already at early CPD, exhibit an impaired redox balance, highlighting the importance of this parameter during ageing, even in the presence of standard environmental conditions, which are considered optimal for cell growth. By contrast, several proteins, as those related to heat shock response, or involved in endoplasmic reticulum and membrane trafficking, appeared differentially expressed only during in vitro ageing, suggesting that, at high CPD, the whole cell machinery becomes permanently altered. Finally, given the importance of the elastic component for a long-lasting connective tissue structural and functional compliance, this study focuses also on elastin and fibulin-5 synthesis and deposition, demonstrating a close relationship between fibulin-5 and ageing.
Keywords: Ageing Fibroblast Connective tissue Oxidative stress Protein expression Elastin
Published in RUNG: 23.08.2019; Views: 3525; Downloads: 0
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Structural and functional determinants of TDP-43 aggregation : Dissertation
Sanja Škaro, 2019, doctoral dissertation

Abstract: TDP-43 (TAR DNA-binding protein) is an hnRNP that was identified as the main component of the brain inclusions characteristically found in patients suffering of Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. As an hnRNP protein, TDP-43 fulfills diverse roles in mRNA metabolism, localization and transport. Structurally, TDP-43 is composed of a well conserved N terminal domain (NTD), two RRM domains of which RRM1 is necessary for recognizing and binding to its target, UG rich RNA sequences, and the C-terminal domain (CTD) which is a Glycine rich domain. The CTD also contains a Q/N rich region that plays a key role in protein aggregation and interaction with another hnRNP proteins and polyglutamine repeats. This thesis focus on the structural determinants involved in the different TDP-43 interactions with itself and with other hnRNPs. Both the carboxyl and amino terminal domains are involved in these interactions. We have mapped the regions more relevant for the function of TDP-43 and for the aggregation process characteristic of the pathological pathway leading to neurodegeneration. We have started to further study the N-terminal domain. Previous results in our laboratory using a cellular aggregation model have shown that the N-terminal domain is also necessary for sequestering the endogenous TDP-43 into the aggregates. In particular, the intact NTD, specifically residues 1 to 77, have been shown to be needed to efficiently recruit TDP-43 monomers into these aggregates. We have extended our knowledge of NTD structure and function, by assessing the behavior of a series of proteins in which key structural features (α-helix and β-sheets) were modified and TDP-43 splicing function together with structure via NMR were analyzed. It was found that by disrupting protein secondary structure in the NTD (mutation in α-helix NTD-31V/R-32T/R) the capacity of the aggregates to sequester enough TDP-43 to induce loss of function was lost.In fact, this protein is also unable to recovery TDP-43 functionality when it is disrupted due to sequestration of the endogenous TDP-43 in add back experiments. Disturbing protein stability through substitution of residues in α-helix also affects its ability to form an active conformation. On the other hand, synthesis of hybrid peptides containing certain NTD and CTD segments was performed in order to see if they are capable to bind to the TDP-43 aggregates. However, it has been shown that these synthetic peptides have a greater ability to induce TDP-43 aggregation than to bind to them, probably due to specific functional characteristics of NTD and CTD segments used for their synthesis.The main focus of the thesis was on the C-terminal domain sequences involved in protein-protein interaction, misfolding and aggregation.A comparison of human, mouse, zebrafish, Annelida, flatworms and Drosophila showed a very strong conservation of the NTD and RRMs, but the C terminal regions of human and other TDP-43 orthologues are very different.I have studied Human and Drosophila melanogaster orthologues, because Drosophila orthologue contains different paralogs of TDP-43.Through a series of deletions and mutations it was shown that the shorter paralog of Drosophila TDP-43 (TBPH-RA) is more active than the longer one (TBPH-RC), and that this is due to a combination of two factors: 1. TBPH-RC by itself aggregates more than TBPH-RA, 2. The functionality of TBPH-RC is downregulated by intramolecular interactions in the C terminal domain. Apparently there is a cation-π interaction involving Tryptophan and Arginine in TBPH-RC that has a high relevance to the protein function and is lacking in the TBPH-RA.Overall this data has identified structural features essential for the proper function of TDP-43.In addition, we have also identified sequences that are critical in the pathological aggregation process of TDP-43 that lead to the characteristic brain inclusions in ALS and FTLD and to the loss of functionality
Keywords: TDP-43 structural determinants, hybrid peptides, protein-protein interactions, intramolecular interaction, cation-π interaction, Drosophila orthologues.
Published in RUNG: 22.07.2019; Views: 3611; Downloads: 193
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Phosphorylation of HPV-16 L2 Contributes To Efficient Virus Infectious Entry
Justyna Broniarczyk, Paola Massimi, David Pim, Martina Bergant Marušič, Michael P. Myers, Robert L. Garcea, Lawrence Banks, 2019, original scientific article

Abstract: The Human Papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are post-translationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and BPV-1 L2 proteins. The phospho-modification of L2, and its presence in HPV pseudovirions (PsVs), was confirmed using anti-phospho L2-specific antibodies. Mutation of the phospho-acceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiency of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry.
Keywords: HPV, L2, infection, protein phosphorylation
Published in RUNG: 05.06.2019; Views: 3735; Downloads: 0
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Naked protein nanoparticles
Ario De Marco, 2017, published scientific conference contribution abstract (invited lecture)

Keywords: Protein nanoparticles, targeted delivery, cell uptake
Published in RUNG: 15.12.2017; Views: 3686; Downloads: 415
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Acting of folding effectors to improve recombinant protein yields and functional quality
Ario De Marco, 2017, independent scientific component part or a chapter in a monograph

Keywords: recombinant proteins, chaperones, osmolytes, protein aggregation
Published in RUNG: 24.05.2017; Views: 4684; Downloads: 0
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Physics behind the Conformational Transitions in Biopolymers. Demystification of DNA melting and Protein Folding
Artem Badasyan, invited lecture at foreign university

Abstract: Biophysics is the area of research, devoted to the studies of physical problems related to living systems. Animal cell is the smallest unit of an organism and mainly contains water solutions of structurally inhomogeneous polymers of biological origin: polypeptides (proteins) and polynucleotides (DNA, RNA). Statistical physics of macromolecules allows to describe the conformations of both synthetic and bio-polymers and constitutes the basis of Biophysics. During the talk I will report on the biophysical problems I have solved with numerical simulations (Langevin-based Molecular Dynamics of Go-like protein folding model and Monte Carlo with Wang-Landau sampling) and analytical studies of spin models (formula evaluation by hand, enforced with computer algebra systems). The direct connections with the theory of phase transitions, algebra of non-commutative operators and decorated spin models will be elucidated.
Keywords: Biophysics, protein folding, helix-coil transition, spin models
Published in RUNG: 13.12.2016; Views: 6152; Downloads: 0
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Distance-based configurational entropy of proteins from molecular dynamics simulations
Federico Fogolari, Alessandra Corazza, Sara Fortuna, Miguel Angel Soler, Bryan VanSchouwen, Giorgia Brancolini, Stefano Corni, Giuseppe Melacini, Gennaro Esposito, 2015, original scientific article

Abstract: Estimation of configurational entropy from molecular dynamics trajectories is a difficult task which is often performed using quasi-harmonic or histogram analysis. An entirely different approach, proposed recently, estimates local density distribution around each conformational sample by measuring the distance from its nearest neighbors. In this work we show this theoretically well grounded the method can be easily applied to estimate the entropy from conformational sampling. We consider a set of systems that are representative of important biomolecular processes. In particular: reference entropies for amino acids in unfolded proteins are obtained from a database of residues not participating in secondary structure elements; the conformational entropy of folding of β2-microglobulin is computed from molecular dynamics simulations using reference entropies for the unfolded state; backbone conformational entropy is computed from molecular dynamics simulations of four different states of the EPAC protein and compared with order parameters (often used as a measure of entropy); the conformational and rototranslational entropy of binding is computed from simulations of 20 tripeptides bound to the peptide binding protein OppA and of β2-microglobulin bound to a citrate coated gold surface. This work shows the potential of the method in the most representative biological processes involving proteins, and provides a valuable alternative, principally in the shown cases, where other approaches are problematic.
Keywords: entropy, protein, molecular dynamics, simulations, MD
Published in RUNG: 12.10.2016; Views: 4675; Downloads: 221
.pdf Full text (2,40 MB)

In silico generation of peptides by replica exchange Monte Carlo: Docking-based optimization of maltose-binding-protein ligands
Anna Russo, Pasqualina Liana Scognamiglio, Rolando Pablo Hong Enriquez, Carlo Santambrogio, Rita Grandori, Daniela Marasco, Antonio Giordano, Giacinto Scoles, Sara Fortuna, 2015, original scientific article

Abstract: Short peptides can be designed in silico and synthesized through automated techniques, making them advantageous and versatile protein binders. A number of docking-based algorithms allow for a computational screening of peptides as binders. Here we developed ex-novo peptides targeting the maltose site of the Maltose Binding Protein, the prototypical system for the study of protein ligand recognition. We used a Monte Carlo based protocol, to computationally evolve a set of octapeptides starting from a polialanine sequence. We screened in silico the candidate peptides and characterized their binding abilities by surface plasmon resonance, fluorescence and electrospray ionization mass spectrometry assays. These experiments showed the designed binders to recognize their target with micromolar affinity. We finally discuss the obtained results in the light of further improvement in the ex-novo optimization of peptide based binders.
Keywords: peptides, docking, optimisation, computation, maltose binding protein, probe, ligand
Published in RUNG: 12.10.2016; Views: 4281; Downloads: 140
.pdf Full text (4,27 MB)

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