190 research outputs found
Role of the XPA protein in the NER pathway: A perspective on the function of structural disorder in macromolecular assembly
No full textAbstractLack of structure is often an essential functional feature of protein domains. The coordination of macromolecular assemblies in DNA repair pathways is yet another task disordered protein regions are highly implicated in. Here I review the available experimental and computational data and within this context discuss the functional role of structure and disorder in one of the essential scaffolding proteins in the nucleotide excision repair (NER) pathway, namely Xeroderma pigmentosum complementation group A (XPA). From the analysis of the current knowledge, in addition to protein–protein docking and secondary structure prediction results presented for the first time herein, a mechanistic framework emerges, where XPA builds the NER pre-incision complex in a modular fashion, as “beads on a string”, where the protein–protein interaction “beads”, or modules, are interconnected by disordered link regions. This architecture is ideal to avoid the expected steric hindrance constraints of the DNA expanded bubble. Finally, the role of the XPA structural disorder in binding affinity modulation and in the sequential binding of NER core factors in the pre-incision complex is also discussed
Understanding the Structure and Function of Viral Glycosylation by Molecular Simulations: State-of-the-Art and Recent Case Studies
No full textThe chemical nature and heterogeneity of most complex carbohydrates makes their structural characterization very difficult, if not impossible, through experimental structural biology. This limits our understanding of glycan-mediated recognition processes and their contribution to protein dynamics, function and shielding, all aspects of great importance in understanding viral activity. Because glycans cannot be “seen” with standard structural biology techniques, their role is often disregarded, preventing our understanding of the biological function of glycoproteins and causing delays to the development of therapies. This is concerning in view of the urgency for new approaches to detect and block viral infection against COVID-19. High-performance computing (HPC)-based molecular simulations can now provide the missing atomistic-detailed description of fully glycosylated viral envelope proteins, delivering knowledge both alternative and complementary to experiment structural biology. In this article I discuss the basic principles of biomolecular simulations, focusing primarily on glycan-specific topics and research cases concerning viral fusion glycoproteins, namely the SARS-CoV-2 S, the influenza A hemagglutinin (HA) and the HIV-1 Env trimer, where HPC provided crucial missing information about key roles of viral glycosylation
Conformational Determinants for the Recruitment of ERCC1 by XPA in the Nucleotide Excision Repair (NER) Pathway: Structure and Dynamics of the XPA Binding Motif
No full textAbstractXPA is an essential protein in the nucleotide excision repair (NER) pathway, in charge of recruiting the ERCC1-XPF endonuclease complex to the DNA damage site. The only currently available structural insight into the binding of XPA to ERCC1 derives from the solution NMR structure of a complex between the ERCC1 central fragment and a 14-residue peptide, corresponding to the highly conserved binding motif of the XPA N-terminus, XPA67-80. The extensive all-atom molecular-dynamics simulation study of the XPA67-80 peptide both bound to the ERCC1 central fragment and free in solution presented here completes the profile of the structural determinants responsible for the ERCC1/XPA67-80 complex stability. In addition to the wild-type, this study also looks at specific XPA67-80 mutants in complex with the ERCC1 central domain and thus contributes to defining the conformational determinants for binding, as well as all of the essential structural elements necessary for the rational design of an XPA-based, ERCC1-specific inhibitor
The role of conformational selection in the molecular recognition of the wild type and mutants XPA67‐80 peptides by ERCC1
No full textMolecular recognition is a fundamental step in the coordination of biomolecular pathways. Understanding how recognition and binding occur between highly flexible protein domains is a complex task. The conformational selection theory provides an elegant rationalization of the recognition mechanism, especially valid in cases when unstructured protein regions are involved. The recognition of a poorly structured peptide, namely XPA67‐80, by its target receptor ERCC1, falls in this challenging study category. The microsecond molecular dynamics (MD) simulations, discussed in this work, show that the conformational propensity of the wild type XPA67‐80 peptide in solution supports conformational selection as the key mechanism driving its molecular recognition by ERCC1. Moreover, all the mutations of the XPA67‐80 peptide studied here cause a significant increase of its conformational disorder, relative to the wild type. Comparison to experimental data suggests that the loss of the recognized structural motifs at the microscopic time scale can contribute to the critical decrease in binding observed for one of the mutants, further substantiating the key role of conformational selection in recognition. Ultimately, because of the high sequence identity and analogy in binding, it is conceivable that the conclusions of this study on the XPA67‐80 peptide also apply to the ERCC1‐binding domain of the XPA protein
Molecular simulations of complex carbohydrates and glycoconjugates
No full textComplex carbohydrates (glycans) are the most abundant and versatile biopolymers in nature. The broad diversity of biochemical functions that carbohydrates cover is a direct consequence of the variety of 3D architectures they can adopt, displaying branched or linear arrangements, widely ranging in sizes, and with the highest diversity of building blocks of any other natural biopolymer. Despite this unparalleled complexity, a common denominator can be found in the glycans’ inherent flexibility, which hinders experimental characterization, but that can be addressed by high-performance computing (HPC)-based molecular simulations. In this short review, I present and discuss the state-of-the-art of molecular simulations of complex carbohydrates and glycoconjugates, highlighting methodological strengths and weaknesses, important insights through emblematic case studies, and suggesting perspectives for future developments
Isolation, characterization and analysis of the osmotic behaviour of hMSCs from UCB for optimal cryopreservation
No full textDemographic studies have shown that population is ageing. As a consequence, degenerative events are increasing and the regenerative medicine market is growing rapidly. Within this context, stem cells possess an enormous therapeutic potential for regeneration and replacement of degenerated tissues. In particular, the ability to readily expand in culture, while maintaining a self-renewing phenotype, has made human Mesenchymal Stem Cells (hMSCs) a candidate for many cell-based therapies (Pittenger et al., 1999; Parekkadan et al., 2007). Unlike induced pluripotent stem cells and embryonic stem cells, adult hMSCs do not raise ethical and legislative issues, so that their use takes advantage of an increased likelihood for authority approval and public acceptance.
Even if bone marrow has been established as the primary source of adult hMSCs, due to the invasive nature of bone marrow aspiration, the identification of other abundant and reliable sources has nowadays become a priority. Regarding this, the successful isolation based on adherence capability to tissue culture plastic of hMSCs from peripheral sources, such as Umbilical Cord Blood (UCB), has been reported, , even if, according to other contradictory studies, hMSCs in these biological samples were not found. In this framework, the ability to preserve these rare cells with high efficiency represents an even more crucial step in the regenerative medicine supply chain, since preservation now represents a core technology to bring cell-based products to market, on demand (Karlsson and Toner, 2000).
The principal preservation method consists of freezing the bio-specimens to cryogenic temperature in order to take advantage of the preservative power of the cold. If compared to the other preservation methods like maintaining the bio samples in continuous culture, cryopreservation has the benefits of affording long shelf lives, genetic stability, reduced microbial contamination risks, and cost effectiveness. The other side of the coin is that cryopreserved biological material can be damaged by the cryopreservation process itself. This damage ultimately translates into a reduced number of viable or functional cells, a loss that can be as high as 50% (Wang et al., 2011). This can be tolerated for some cell lineages, but it’s unacceptable for others, as the hMSCs from UCB, whose collection and isolation is known to be difficult (Bieback et al., 2004). Even if, in principle, cell expansion/proliferation may solve the problem, an increased number of passages will inexorably lead these cells to lose their peculiar characteristics, and should be avoided (Lee et al., 2004).
Cryopreservation consists of cooling to sub-zero temperatures with or without a Cryo-Protectant Agent (CPA), storage, thawing and return to physiological environment for specific usages. Moreover, the steps of addition and removal of a cytotoxic CPA as DiMethyl SulfOxide (DMSO) which permeates through cells membrane into cytoplasm, needs to be taken into account when looking for optimal cryopreservation. A part from storage, all these different stages are potentially able to damage the cells due to the physical and/or chemical phenomena involved such as intra-cellular ice formation, excessive solutes concentrations and cell shrinkage or swelling. In general, due to the high number of trials actually required for experimental optimization of the cryopreservation process, mathematical modelling is considered a practical solution (Karlsson and Toner, 2000). To this aim, the osmotic behaviour needs to be first investigated in order to be able to predict the volume of residual intra-cellular water left by osmosis to form lethal ice or glass during the cryopreservation process, as well as to limit excessive cell volume excursions and solutes concentrations that might lead to the so-called solution injury (Fadda et al., 2010; 2011).
In this work, the hMSCs from UCB of three different donors were isolated by a density gradient centrifugation method, followed by plastic adherence of mononuclear cells. The isolation (20% success rate) was verified through phenotypic cytofluorimetric analysis, and adipogenesis/osteogenesis capability differentiations. The osmotic properties, namely inactive cell volume, water and CPA (DMSO) permeabilities, were determined by means of experimental runs carried out under hypertonic conditions (obtained with the addition of sucrose or DMSO to PBS, isotonic solution), at three different temperatures. To the best of authors’ knowledge, these osmotic transport parameters have never been studied before for the hMSCs from UCB. Cell size was determined using an impedance measurement device (Coulter Counter), under equilibrium and dynamic conditions. Since the impedance measuring device does not discriminate single cells by debris or cells agglomerates, the measured cell volume distributions were filtered out from the data originally provided by the Coulter Counter through a novel data treatment, proposed in this work. Linear and non-linear regression analyses were carried out to determine the adjustable parameters by means of the salt-water sack model in the 2-parameters bi-compartimental version by Kleinahns (1998), as applied to a single-sized cell population (i.e. identical cells, with size equal to the average), as classically proposed for numerous cell lineages in the technical literature addressing cryopreservation. Basically, this model addresses a suspension of cells in a liquid, ideal solution, characterized by a given osmolality; the cells are supposed to act as a perfect osmometer in response of the ruling driving force, i.e. the difference between intra- and extra-cellular solute osmolalities, where only water and DMSO are assumed to permeate through cell membrane, while neglecting ionic transfer.
According to this model, in this work a rational parameter estimation was attempted by carrying out an ideal fitting procedure. It was found that the adopted model is not capable to simulate entirely the osmotic response for the cells under investigation. Specifically, only during swelling an apparent dependence of the so-called inactive cell volume from temperature and CPA concentration needs to be considered for hMSCs from UCB. Thus, these cells do not behave as a perfect osmometer, and show a peculiar osmotic response.
It may be concluded that, a regulatory volume system is activated for these cells, albeit only during swelling. This control system is presumably related to the action of ion pumps and transport channels, which are well-known in the literature for conditioning cells to return to their isotonic size, thus contrasting the lethal effect produced by osmosis (Hoffmann et al., 2009). In such a case, a more complex mathematical model than the standard salt-water sack model needs to be taken into account for capturing system behaviour. Specifically, resorting to the complex Goldman-Hodgkin-Katz model of permeant ions where quantities as membrane potential and ion permeabilities are introduced (Fernandez et al., 2013) may be considered. But, this demands a complex validation through direct comparison to data measured in well-designed experiments, which may be difficult to achieve. As an alternative, or in conjunction with this ionic transport mechanism for controlling cell swelling, the extrusion of permeant osmolites/solutes (produced inside the cells to the detriment of the inactive cell volume to osmosis) may be hypothesised. This would result in a smaller deviation from the standard salt-water sack model, simpler than taking into account the electro-diffusion of ions through cell membrane.
References
Bieback, K., Kern, S., Kluter, H., Eichler, H., 2004. Critical Parameters for the Isolation of Mesenchymal Stem Cells from Umbilical Cord Blood. Stem Cells 22, 625–634.
Fadda, S., Cincotti, A., Cao, G., 2010. The Effect of Cell Size Distribution During the Cooling Stage of Cryopreservation without CPA. AIChE Journal 56 (8), 2173-2185.
Fadda, S., Cincotti, A., Cao, G., 2011. Rationalizing the Equilibration and Cooling Stages of Cryopreservation: The Effect, of Cell Size Distribution. AIChE Journal 57 (4), 1075-1095.
Fernandez, J.M., Di Giusto, G., Kalstein, M., Melamud, L., Rivarola, V., Ford, P., Capurro, C., 2013. Cell Volume Regulation in Cultured Human Retinal Muller Cells Is associated with Changes in trasmembrane potential. Plos One 8 (2), e57268.
Hoffmann, E.K., Lambert, I.H., Pedersen, S.F., 2009. Physiology of Cell Volume Regulation in Vertebrates. Physiological Reviews 89, 193–277.
Karlsson, J.O.M., Toner, M., 2000. Cryopreservation, in: Lanza, R.P., Langer, R., Vacanti, J., (Eds.), Principles of Tissue Engineering, 2nd Ed. Academic press, San Diego, pp. 293-307.
Kleinhans, F.W., 1998. Membrane Permeability Modeling: Kedem-Katchalsky vs a Two Parameter Formalism. Cryobiology 37, 271-289.
Lee, M.W., Choi, J., Yang, M.S., Moon, Y.J., Park, J.S., Kim, H.C., Kim, Y.J., 2004. Mesenchymal stem cells from cryopreserved human umbilical cord blood. Biochemical and Biophysical Research Communications 320, 273–278.
Parekkadan, B., Sethu, P. Van Poll, D., Yarmush, M.L., Toner, M., 2007. Osmotic selection of human mesenchymal stem/progenitor cells from umbilical cord blood. Tissue Engineering 13, 2465-2473.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411), 143– 147.
Wang, H.Y., Lun, Z.R., Lu, S.S., 2011. Cryopreservation of Umbilical Cord Blood-Derived Mesenchymal Stem Cells Without Dimethyl Sulfoxide. CryoLetters 32 (1), 81-88.Demographic studies have shown that population is ageing. As a consequence, degenerative events are increasing and the regenerative medicine market is growing rapidly. Within this context, stem cells possess an enormous therapeutic potential for regeneration and replacement of degenerated tissues. In particular, the ability to readily expand in culture, while maintaining a self-renewing phenotype, has made human Mesenchymal Stem Cells (hMSCs) a candidate for many cell-based therapies (Pittenger et al., 1999; Parekkadan et al., 2007). Unlike induced pluripotent stem cells and embryonic stem cells, adult hMSCs do not raise ethical and legislative issues, so that their use takes advantage of an increased likelihood for authority approval and public acceptance
NMR shieldings from sum-over-states density-functional-perturbation theory: Further testing of the ‘‘Loc.3’’ approximation
No full textThe development and implementation of sum-over-states density-functional-perturbation theory (SOS-DFPT) [V.G. Malkin, O.L. Malkina, M.E. Casida, and D.R. Salahub, J. Am. Chem. Soc. 116, 5898 (1994)] has allowed a significant improvement in the accuracy of nuclear magnetic resonance (NMR) chemical shift values over the Hartree–Fock approximation. Furthermore, due to its computational efficiency, SOS-DFPT has opened the way to the study of systems of increased size compared to those that may be approached by more sophisticated but also computationally more intensive methods, such as Møller–Plesset perturbation theory or coupled-cluster theory. The success of SOS-DFPT relies on the introduction of an ad hoc correction to the excitation energy that improves the calculation of the paramagnetic component of the NMR shielding tensor. The lack of a clear physical basis for this approximation has left the SOS-DFPT open to some criticism. We have shown in a previous article [E. Fadda, M.E. Casida, and D.R. Salahub, Int. J. Quantum Chem. 91, 67 (2003)] that the electric field and magnetic field responses are given by equivalent expressions within the Tamm–Dancoff approximation of time-dependent density-functional theory (TD-DFT). This provides an SOS-DFPT expression which, upon restriction to diagonal contributions, yields a new rigorous “Loc.3” approximation. In this article, we more than double our original test set of 10 molecules for 13C, 15N, and 17O chemical shifts to a set of 25 molecules. In addition, we compare the results of “Loc.3” SOS-DFPT with the results of promising recent functionals for DFT calculations of chemical shifts. The results show not only that the “Loc.3” approximation represents the rigorous physical connection between SOS-DFPT and TD-DFT, but also that it has very good potential for the prediction of NMR shielding constants, opening the way to further developments in DFT-based NMR parameter calculations
On the molecular basis of uracil recognition in DNA: comparative study of T-A versus U-A structure, dynamics and open base pair kinetics
No full textUracil (U) can be found in DNA as a mismatch paired
either to adenine (A) or to guanine (G). Removal of U
from DNA is performed by a class of enzymes
known as uracil–DNA–glycosylases (UDG). Recent
studies suggest that recognition of U–A and U–G
mismatches by UDG takes place via an extra-helical
mechanism. In this work, we use molecular
dynamics simulations to analyze the structure,
dynamics and open base pair kinetics of U–A base
pairs relative to their natural T–A counterpart in 12
dodecamers. Our results show that the presence of
U does not alter the local conformation of B-DNA.
Breathing dynamics and base pair closing kinetics
are only weakly dependent on the presence of U
versus T, with open T–A and U–A pairs lifetimes in
the nanosecond timescale. Additionally, we
observed spontaneous base flipping in U–A pairs.
We analyze the structure and dynamics for this
event and compare the results to available crystallographic
data of open base pair conformations. Our
results are in agreement with both structural and
kinetic data derived from NMR imino proton
exchange measurements, providing the first
detailed description at the molecular level of
elusive events such as spontaneous base pair
opening and flipping in mismatched U–A sequences
in DNA. Based on these results, we propose that
base pair flipping can occur spontaneously at room
temperature via a 3-step mechanism with an open
base pair intermediate. Implications for the molecular
basis of U recognition by UDG are discussed
Molecular simulations of carbohydrates and protein–carbohydrate interactions: motivation, issues and prospects
No full textThe characterization of the 3D structure of oligosaccharides, their conjugates and analogs is particularly challenging for traditional experimental methods. Molecular simulation methods provide a basis for interpreting sparse experimental data and for independently predicting conformational and dynamic properties of glycans. Here, we summarize and analyze the issues associated with modeling carbohydrates, with a detailed discussion of four of the most recently developed carbohydrate force fields, reviewed in terms of applicability to natural glycans, carbohydrate–protein complexes and the emerging area of glycomimetic drugs. In addition, we discuss prospectives and new applications of carbohydrate modeling in drug discovery
An atomistic perspective on antibody-dependent cellular cytotoxicity quenching by core-fucosylation of IgG1 Fc N-glycans from enhanced sampling molecular dynamics
No full textThe immunoglobulin type G (IgG) Fc N-glycans are known to modulate the interaction with membrane-bound Fc γreceptors (FcγRs), fine-tuning the antibody's effector function in a sequence-dependent manner. Particularly interesting in this respect are the roles of galactosylation, which levels are linked to autoimmune conditions and aging, of core fucosylation, which is known to reduce significantly the antibody-dependent cellular cytotoxicity (ADCC), and of sialylation, which also reduces antibody-dependent cellular cytotoxicity (ADCC) but only in the context of core-fucosylation. In this article, we provide an atomistic level perspective through enhanced sampling computer simulations, based on replica exchange molecular dynamics (REMD), to understand the molecular determinants linking the Fc N-glycans sequence to the observed IgG1 function. Our results indicate that the two symmetrically opposed N-glycans interact extensively through their core trimannose residues. At room temperature, the terminal galactose on the α (1-6) arm is restrained to the protein through a network of interactions that keep the arm outstretched; meanwhile, the α (1-3) arm extends toward the solvent where a terminal sialic acid remains fully accessible. We also find that the presence of core fucose interferes with the extended sialylated α (1-3) arm, altering its conformational propensity and as a consequence of steric hindrance, significantly enhancing the Fc dynamics. Furthermore, structural analysis shows that the core-fucose position within the Fc core obstructs the access of N162 glycosylated FcγRs very much like a "door-stop,"potentially decreasing the IgG/FcγR binding free energy. These results provide an atomistic level-of-detail framework for the design of high potency IgG1 Fc N-glycoforms.</p
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