A supramolecular assembly mediates lentiviral DNA integration

Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo–electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors

Molecular mechanism of double Holliday junction dissolution

Processing of homologous recombination intermediates is tightly coordinated to ensure that chromosomal integrity is maintained and tumorigenesis avoided. Decatenation of double Holliday junctions, for example, is catalysed by two enzymes that work in tight coordination and belong to the same 'dissolvasome' complex. Within the dissolvasome, the RecQ-like BLM helicase provides the translocase function for Holliday junction migration, while the topoisomerase III alpha-RMI1 subcomplex works as a proficient DNA decatenase, together resulting in double-Holliday-junction unlinking. Here, we review the available architectural and biochemical knowledge on the dissolvasome machinery, with a focus on the structural interplay between its components

A combined structural and biochemical approach reveals translocation and stalling of UvrB on the DNA lesion as a mechanism of damage verification in bacterial nucleotide excision repair

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 clamps a different DNA strand using its \u3b2-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps 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

Astrocytes-derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

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

Unravelling the regulation pathway of photosynthetic AB-GAPDH

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 A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4-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 (A2B2)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 A4B4 and A8B8 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

Intracerebral Injection of Extracellular Vesicles from Mesenchymal Stem Cells Exerts Reduced Aβ Plaque Burden in Early Stages of a Preclinical Model of Alzheimer's Disease

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 \u3b2-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 A\u3b2 plaque burden and the amount of dystrophic neurites in both the cortex and hippocampus. The presence of Neprilysin on BM-MSC-EVs, opens the possibility of a direct \u3b2-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

Identification of a small molecule that compromises the structural integrity of viroplasms and rotavirus double-layered particles

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 hamper 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

Cryo-EM structure of cardiac amyloid fibrils from an immunoglobulin light chain AL amyloidosis patient

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 \uc5 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 \uc5 stacking and cross-\u3b2 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

Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration : insights from DNA complex structures

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration