ORCID Profile
0000-0001-9712-2757
Current Organisation
Human Technopole
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Publisher: MDPI AG
Date: 16-11-2020
DOI: 10.3390/IJMS21228643
Abstract: In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at erse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.
Publisher: Elsevier BV
Date: 12-2019
DOI: 10.1016/J.MOLCEL.2019.09.012
Abstract: In the arms race against bacteria, bacteriophages have evolved erse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular ersification of Cas9 proteins.
Publisher: Cold Spring Harbor Laboratory
Date: 17-03-2023
DOI: 10.1101/2023.03.17.532973
Abstract: RNA can directly control protein activity in a process called riboregulation only a few mechanisms of riboregulation have been described in detail, none of these being characterized on structural grounds. Here we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme’s reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in the evolution of eukaryotic SHMT1 and the compartmentalization of one-carbon metabolism. The findings also provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.MOLCEL.2016.11.005
Abstract: Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.
Publisher: Springer Science and Business Media LLC
Date: 20-03-2019
DOI: 10.1038/S41467-019-09133-W
Abstract: Systemic light chain amyloidosis (AL) is a life-threatening disease caused by aggregation and deposition of monoclonal immunoglobulin light chains (LC) in target organs. Severity of heart involvement is the most important factor determining prognosis. Here, we report the 4.0 Å resolution cryo-electron microscopy map and molecular model of amyloid fibrils extracted from the heart of an AL amyloidosis patient with severe amyloid cardiomyopathy. The helical fibrils are composed of a single protofilament, showing typical 4.9 Å stacking and cross-β architecture. Two distinct polypeptide stretches (total of 77 residues) from the LC variable domain (V l ) fit the fibril density. Despite V l high sequence variability, residues stabilizing the fibril core are conserved through different cardiotoxic V l , highlighting structural motifs that may be common to misfolding-prone LCs. Our data shed light on the architecture of LC amyloids, correlate amino acid sequences with fibril assembly, providing the grounds for development of innovative medicines.
Publisher: Springer Science and Business Media LLC
Date: 2014
Publisher: American Society for Microbiology
Date: 02-2018
DOI: 10.1128/JVI.01943-17
Abstract: Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to h er the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells. IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.
Publisher: Elsevier BV
Date: 12-2021
Publisher: Cold Spring Harbor Laboratory
Date: 14-05-2023
DOI: 10.1101/2023.05.11.540146
Abstract: Cryo-focused ion beam milling has enabled groundbreaking structural discoveries in native cells. Progress toward medically relevant applications, however, has been slow. We here present an adaptation of the cryo-lift out procedure for Serialized On-grid Lift-In Sectioning for Tomography (SOLIST), which increases throughput, reduces ice contamination, and enhances s le stability. With these improvements, new specimens, ranging from high-pressure frozen reconstituted LLPS droplets to human forebrain organoids, are accessible to cryo-electron tomography.
Publisher: eLife Sciences Publications, Ltd
Date: 12-08-2014
DOI: 10.7554/ELIFE.03273
Abstract: The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.
Publisher: Elsevier BV
Date: 2017
Publisher: Elsevier BV
Date: 2012
DOI: 10.1016/J.BMC.2011.10.069
Abstract: Here, we report on the development of a novel methodology to aid in design of Hsp90 inhibitors, using molecular docking combined with artificial neural network (ANN) modelling. Inhibitors are first docked into the ATPase site of the Human Hsp90α crystal structures and the thermodynamic properties of the complexes together with various physical-chemical properties of the ligands are used as input to train a simple feed-forward, back propagation ANN, to predict the inhibitors' pIC(50)s. For an objective test set of 60 known Hsp90 inhibitors for which there are no crystallographic data available, the trained ANN is shown to give pIC(50)s accurate to within ±1 log unit, and the predictions are sufficiently good as to allow the majority of the inhibitors to be ranked correctly according to their potency.
Publisher: Elsevier BV
Date: 2020
DOI: 10.1016/J.DNAREP.2019.102746
Abstract: Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules cl s a different DNA strand using its β-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that cl s the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway.
Publisher: Elsevier BV
Date: 11-2019
DOI: 10.1016/J.JMB.2019.08.011
Abstract: Bacterial NADPH-dependent glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive synthesis of two L-Glu molecules from L-Gln and 2-oxo-glutarate. GltS functional unit hosts an α-subunit (αGltS) and a β-subunit (βGltS) that assemble in different αβ oligomers in solution. Here, we present the cryo-electron microscopy structures of Azospirillum brasilense GltS in four different oligomeric states (α
Publisher: Elsevier BV
Date: 09-2013
Publisher: Elsevier BV
Date: 06-2017
Publisher: MDPI AG
Date: 10-09-2019
DOI: 10.3390/CELLS8091059
Abstract: Bone marrow Mesenchymal Stem Cells (BM-MSCs), due to their strong protective and anti-inflammatory abilities, have been widely investigated in the context of several diseases for their possible therapeutic role, based on the release of a highly proactive secretome composed of soluble factors and Extracellular Vesicles (EVs). BM-MSC-EVs, in particular, convey many of the beneficial features of parental cells, including direct and indirect β-amyloid degrading-activities, immunoregulatory and neurotrophic abilities. Therefore, EVs represent an extremely attractive tool for therapeutic purposes in neurodegenerative diseases, including Alzheimer’s disease (AD). We examined the therapeutic potential of BM-MSC-EVs injected intracerebrally into the neocortex of APPswe/PS1dE9 AD mice at 3 and 5 months of age, a time window in which the cognitive behavioral phenotype is not yet detectable or has just started to appear. We demonstrate that BM-MSC-EVs are effective at reducing the Aβ plaque burden and the amount of dystrophic neurites in both the cortex and hippoc us. The presence of Neprilysin on BM-MSC-EVs, opens the possibility of a direct β-amyloid degrading action. Our results indicate a potential role for BM-MSC-EVs already in the early stages of AD, suggesting the possibility of intervening before overt clinical manifestations.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 06-01-2017
Abstract: An essential step in the life cycle of lentiviruses such as HIV-1 is when viral DNA integrates into the host genome, establishing a permanent infection of the host cell. The viral integrase enzyme catalyzes this process and is a major drug target. During viral integration, integrase binds the ends of viral DNA, forming a higher-order structure called the intasome. Passos et al. and Ballandras-Colas et al. used cryo—electron microscopy to solve the structures of the intasomes from HIV-1 and maedi-visna virus (ovine lentivirus), respectively. These structures reveal how integrase self-associates to form a functional intasome and help resolve previous conflicting models of intasome assembly. Science , this issue p. 89 , p. 93
Publisher: Proceedings of the National Academy of Sciences
Date: 23-03-2015
Abstract: RecQ DNA helicases are critical enzymes for the maintenance of genome integrity. Here, we determined the first DNA complex structures, to our knowledge, of the human RECQ1 helicase. These structures provide new insight into the RecQ helicase mechanism of DNA tracking, strand separation, strand annealing, and Holliday junction (HJ) branch migration. We identified a surface region in the winged-helix domain of RECQ1 that is important for both dsDNA recognition and HJ resolution, and we used a combination of biochemical, analytical ultracentrifugation, and EM experiments to begin elucidating the molecular basis of the distinct HJ resolution activities of human RecQ helicases.
Publisher: Wiley
Date: 07-2021
DOI: 10.1002/JEV2.12114
Abstract: Astrocytes‐derived extracellular vesicles (EVs) are key players in glia‐neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV‐neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)‐coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin‐mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface.
Publisher: International Union of Crystallography (IUCr)
Date: 27-10-2022
DOI: 10.1107/S2059798322010014
Abstract: Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A 2 B 2 heterotetramers and the least abundant by A 4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A 4 -GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC–SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A 2 B 2 ) n -GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A 4 B 4 and A 8 B 8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.
Publisher: AIP Publishing
Date: 10-05-2010
DOI: 10.1063/1.3428576
Abstract: Electromagnetic metamaterials attract much attention since they can be engineered to exhibit optical properties not found in nature. Their fabrication, however, is challenging, especially in volume. We introduce drawing as a means of fabricating metamaterials, thus demonstrating a terahertz metamaterial. We codraw polymethyl-methacrylate and indium, producing several meters of metamaterial with wire diameters down to ∼10 μm, and lattice constants of ∼100 μm. We experimentally characterize the transmission properties of different s les, observing high-pass filtering between 0.3–0.4 THz, in good agreement with simulations.
Publisher: Cold Spring Harbor Laboratory
Date: 21-11-2021
DOI: 10.1101/2021.11.21.469434
Abstract: Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme of the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant formed by hetero-tetramers of A and B-subunits, and the homo-tetramer A 4 . Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and for this reason is strictly regulated. While A 4 -GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of B-subunits. Reversible inactivation of AB-GAPDH occurs via oxidation of a cysteine pair located in the CTE, and substitution of NADP(H) with NAD(H) in the cofactor binding domain. These combined conditions lead to a change in the oligomerization state and enzyme inactivation. SEC-SAXS and single-particle cryoEM analysis disclosed the structural basis of this regulatory mechanism. Both approaches revealed that (A 2 B 2 ) n -GAPDH oligomers with n=1, 2, 4 and 5 co-exist in a dynamic system. B-subunits mediate the contacts between adjacent A 2 B 2 tetramers in A 4 B 4 and A 8 B 8 oligomers. The CTE of each B-subunit penetrates into the active site of a B-subunit of the adjacent tetramer, while the CTE of this subunit moves in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B-subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H) by removing NADP(H) from A-subunits allows the entrance of the CTE in B-subunits active sites and hence inactive oligomer stabilization. In land plants, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) unique sink of reducing power of the entire Calvin-Benson cycle, is finely regulated. Based on the redox state and substrates concentration, its heteromeric form AB-GAPDH oscillates between a fully active heterotetramer (A 2 B 2 ) and inactive oligomers. Experimental evidence demonstrates that GAPDH inactivation depends on the formation of dimers, tetramers or pentamers of A 2 B 2 -modules, linked together by C-terminal extensions (CTE) of B-subunits that extrude from one modular tetramer and occupy two active sites of the adjacent one. This molecular mechanism along with the unexpected observed dynamism of the system, shed light on how the Calvin-Benson cycle is modulated in function of the light environmental changes.
Publisher: Elsevier BV
Date: 12-2020
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Paolo Swuec.