BRCA2-HSF2BP oligomeric ring disassembly by BRME1 promotes homologous recombination

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSBs). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here, we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to interlock into a large ring-shaped 24-nucleotide oligomer. Addition of BRME1 leads to dissociation of both of these ring structures and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during interstrand DNA crosslink repair in Xenopus egg extracts. We propose that, during meiosis, the control of HSF2BP-BRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting HR.</p

Low-boiling-point perfluorocarbon nanodroplets for adaptable ultrasound-induced blood-brain barrier opening.

Low-boiling point perfluorocarbon nanodroplets (NDs) are valued as effective sonosensitive agents, encapsulating a liquid perfluorocarbon that would instantaneously vaporize at body temperature without the NDs shell. Those NDs have been explored for both therapeutic and diagnostic purposes. Here, phospholipid-shelled nanodroplets containing octafluoropropane (C3F8) or decafluorobutane (C4F10) formed by condensation of microbubbles were thoroughly characterized before blood-brain (BBB) permeabilization. Transmission electron microscopy (TEM) and cryo-TEM were employed to confirm droplet formation while providing high-resolution insights into the droplet surface and lipid arrangement assessed from electron density observation after condensation. The vaporization threshold of NDs was determined with a high-speed camera, and the frequency signal emitted by the freshly vaporized bubbles was analyzed using cavitation detection. C3F8 NDs exhibited vaporization at 0.3 MPa (f0 = 1.5 MHz, 50 cycles), and emitted signals at 2 f0 and 1.5 f0 from 0.45 MPa onwards (f0 = 1.5 MHz, 50 cycles), while broadband noise was measured starting from 0.55 MPa. NDs with the higher boiling point C4F10 vaporized at 1.15 MPa and emitted signals at 2 f0 from 0.65 MPa and 1.5 f0 from 0.9 MPa, while broadband noise was detected starting from 0.95 MPa. Both ND formulations were used to permeabilize the BBB in healthy mice using tailored ultrasound sequences, allowing for the identification of optimal applications for each NDs type. C3F8 NDs proved suitable and safe for permeabilizing a large area, potentially the entire brain, at low acoustic pressure. Meanwhile, C4F10 droplets facilitated very localized (400 μm isotropic) permeabilization at higher pressure. This study prompts a closer examination of the structural rearrangements occurring during the condensation of microbubbles into NDs and highlight the potential to tailor solutions for different brain pathologies by choosing the composition of the NDs and adjusting the ultrasound sequence

BRCA2-HSF2BP oligomeric ring disassembly by BRME1 promotes homologous recombination

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSBs). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here, we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to interlock into a large ring-shaped 24-nucleotide oligomer. Addition of BRME1 leads to dissociation of both of these ring structures and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during interstrand DNA crosslink repair in Xenopus egg extracts. We propose that, during meiosis, the control of HSF2BP-BRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting HR

Unraveling the structure of the photosynthetic membrane using electron microscopy approach

Cyanobacteriën of blauwalgen zijn foto-autotrofe bacteriën die leven van fotosynthese. Cyanobacterien hebben in tegenstelling tot hogere planten geen chloroplasten maar phycobilisomen, supramoleculaire aggregaten van fotosynthesemembranen. Fotosynthese vindt plaats door middel van pigment bevattende eiwitten die geassocieerd zijn met de fotosynthesemembranen. Fotosysteem I (PSI) en fotosysteem II (PSII) zijn beide belangrijke schakels in het omzetten van zonne-energie naar chemisch vastgelegde energie. Elektronenmicroscopie en beeldverwerking van losse deeltjes is gebruikt om de structuur van PSI en PSII nader te bestuderen. Door een systematische vergelijking te maken tussen op twee verschillende manieren gezuiverd PSII kon een 6.8 kDa kleine subeenheid (PsbZ) in het eiwit worden gelokaliseerd. Nieuw was de combinatie tussen massaspectrometrie en elektronenmicroscopie. NAD(P)H dehydrogenase type 1 (NDH-1) van cyanobacteriën werd bestudeerd na groei onder verschillende CO2 condities. Hierdoor konden twee kleinere eiwit complexen (NDH-1M en NDH-1I) worden geïdentificeerd zonder de NdhD1 en NdhF1 subeenheid. Verder werden eiwitcomplexen van NDH-1 gevonden met extra massa vergelijkbaar met complex-1 van E. coli en andere organisme. Cyanobacterie PSII ondergaat een structurele verandering als het organisme groeit bij gebrek aan ijzer. Onder deze condities bindt het IsiA (iron-stress-induced protein A) uitsluitend aan PSII dimeren en niet aan PSII monomeren. Mutanten van phycobilisomen werden gebruikt om de posities van een aantal eiwitten te bepalen. Door vergelijking van de projectiemappen van de wild-type en de mutanten die één of meerdere eiwitten missen kon de bindingspositie bepaald worden.

Self-assembly of amphiphilic copolymers containing polysaccharide: PISA versus nanoprecipitation, and the temperature effect

International audienceFor many decades, the self-assembly of amphiphilic copolymers containing polysaccharide, called glycopolymers, has been induced in water via nanoprecipitation, whereas the polymerization-induced self-assembly (PISA) approach has been less reported. Using advanced characterization techniques including static/dynamic light scattering and negative-stain/cryo TEM analyses, we studied the impact of the experimental conditions/process on the morphology of formed glyco-nanostructures. A model amphiphilic glycopolymer (Dex-g12-PHPMA400) based on dextran as hydrophilic polysaccharide backbone and poly(2-hydroxypropyl methacrylate) (PHPMA) as hydrophobic grafts was prepared in water using photo-induced RAFT polymerization at 405 nm. On the one hand, our findings revealed that photo-initiated PISA (photo-PISA) at room temperature (RT) led to a mixture of spheres with a significant population of worm-like micelles (WLM), whereas the nanoprecipitation of purified Dex-g12-PHPMA400 led only to spherical micelles. On the other hand, we demonstrate that the morphology of glyco-nanostructures is affected by temperature since performing photo-PISA at 60 °C led to spheres instead of WLM. Finally, our findings revealed that pre-synthesized WLM at RT underwent irreversibly an unusual morphological transition to spheres by heating to 60 °

Multicompartment Vesicles: A Key Intermediate Structure in Polymerization-Induced Self-Assembly of Graft Copolymers

International audienceThe morphological evolution of graft copolymer-based nano-objects was monitored by light scattering and electron microscopy during their formulation in water by polymerization induced self-assembly using a photo-mediated reversible addition–fragmentation chain transfer mechanism. The copolymer models used were composed of a dextran backbone bearing poly(2-hydroxypropyl methacrylate) grafts of two degrees of polymerization (X). At a full monomer conversion, unilamellar vesicles (ULVs) and large compound nano-objects (LCNs) were formed when targeting X = 100 and 500, respectively. For X = 100, some spherical, worm-like, then jellyfish-like structures were progressively observed before the ULVs formation. For X = 500, electron cryotomography revealed an unprecedented intermediate morphology formed from the onset of self-assembly called a multicompartment vesicle (MCV) that fused to form LCN. The formation of MCV was attributed to a local phase separation between dextran and the residual 2-hydroxypropyl methacrylate inducing the appearance of multiple hydrophilic core

Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment.

Human retinal macular pigment (MP) is formed by the carotenoids lutein and zeaxanthin (including the isomer meso-zeaxanthin). MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health-specifically, to predict the likelihood of developing age-related macular degeneration. While the roles of these carotenoids in retinal health have been the object of intense study in recent years, precise mechanistic details of their protective action remain elusive. We have measured the Raman signals originating from MP carotenoids in ex vivo human retinal tissue, in order to assess their structure and conformation. We show that it is possible to distinguish between lutein and zeaxanthin, by their excitation profile (related to their absorption spectra) and the position of their ν1 Raman mode. In addition, analysis of the ν4 Raman band indicates that these carotenoids are present in a specific, constrained conformation in situ, consistent with their binding to specific proteins as postulated in the literature. We discuss how these conclusions relate to the function of these pigments in macular protection. We also address the possibilities for a more accurate, consistent measurement of MP levels by Raman spectroscopy

Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex

International audienceSingle particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images