The Cdv proteins constitute the cell division system of the Crenarchaea, a machinery closely related to the ESCRT system of eukaryotes. Using a combination of TEM imaging and biochemical assays, we ...here present an in vitro study of Metallosphaera sedula CdvB1, the Cdv protein that is believed to play a major role in the constricting ring that drives cell division in the Crenarchaea. We show that CdvB1 self‐assembles into filaments that are depolymerized by the Vps4‐homolog ATPase CdvC. Furthermore, we find that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.
This study shows that the archaeal cell division protein CdvB1 can form filaments in vitro, and how these filaments are depolymerized by the AAA ATPase CdvC. We show that CdvB1 can bind to lipid membranes and can be detached from them by CdvC, providing new insights on the protein interactions of the archaeal Cdv system.
ABSTRACT
Among human species B adenovirus, HAdV-11, has the particular feature of exploiting two primary receptors, desmoglein 2 (DSG2) and CD46, to mediate cell attachment. This serotype represents ...a particularly promising class of oncolytic viral vaccines. For instance, Enadenotucirev, a species B HAdV-11/HAdV-3 chimeric adenoviral vector, demonstrated preclinical tumor-selective cytotoxicity. Understanding the mechanism of HAdV-11 entry is a major challenge for the development of powerful vectors. Interactions between HAdV-11 and CD46 are well-characterized and clearly depend on the fiber protein, but the mechanism whereby HAdV-11 engages DSG2 has been still unresolved. In this study, we described a new cryo-EM structure of the HAdV-11 fiber in complex with DSG2. Kinetic interaction studies demonstrated that the binding stability of the HAdV-11 in complex with DSG2 was significantly lower than with CD46. Furthermore, competition assays with pseudotyped viruses suggested that the interaction of the HAdV-11 fiber with DSG2 was not required for infection but that DSG2 could serve as a receptor in the absence of CD46. Unexpectedly, we succeeded to isolate a HAdV-11 fiber/DSG2/CD46 “super complex” showing that these two receptors could simultaneously bind to the same trimeric HAdV-11K. Our work deciphers the use of two independent receptors by HAdV-11 from a biochemical and structural point of view, which can have an impact for future vectors design.
IMPORTANCE
The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there’s still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.
The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there’s still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.
In addressing the challenges faced by laboratories and universities with limited (or no) cryo‐electron microscopy (cryo‐EM) infrastructure, the ESRF, in collaboration with the Grenoble Institute for ...Structural Biology (IBS), has implemented the cryo‐EM Solution‐to‐Structure (SOS) pipeline. This inclusive process, spanning grid preparation to high‐resolution data collection, covers single‐particle analysis and cryo‐electron tomography (cryo‐ET). Accessible through a rolling access route, proposals undergo scientific merit and technical feasibility evaluations. Stringent feasibility criteria demand robust evidence of sample homogeneity. Two distinct entry points are offered: users can either submit purified protein samples for comprehensive processing or initiate the pipeline with already vitrified cryo‐EM grids. The SOS pipeline integrates negative stain imaging (exclusive to protein samples) as a first quality step, followed by cryo‐EM grid preparation, grid screening and preliminary data collection for single‐particle analysis, or only the first two steps for cryo‐ET. In both cases, if the screening steps are successfully completed, high‐resolution data collection will be carried out using a Titan Krios microscope equipped with a latest‐generation direct electron counting detector coupled to an energy filter. The SOS pipeline thus emerges as a comprehensive and efficient solution, further democratizing access to cryo‐EM research.
The European Synchrotron's Solution‐to‐Structure (SOS) pipeline streamlines cryo‐EM studies, providing an inclusive process from grid preparation to high‐resolution data collection for single‐particle analysis and cryo‐electron tomography. This service is especially tailored for users with restricted or no access to cryo‐EM infrastructure, enabling them to conduct screening experiments and acquire samples suitable for high‐resolution data collection.
Virus-like particles (VLPs) are highly suited platforms for protein-based vaccines. In the present work, we adapted a previously designed non-infectious adenovirus-inspired 60-mer dodecahedric VLP ...(ADDomer) to display a multimeric array of large antigens through a SpyTag/SpyCatcher system. To validate the platform as a potential COVID-19 vaccine approach, we decorated the newly designed VLP with the glycosylated receptor binding domain (RBD) of SARS-CoV-2. Cryoelectron microscopy structure revealed that up to 60 copies of this antigenic domain could be bound on a single ADDomer particle, with the symmetrical arrangements of a dodecahedron. Mouse immunization with the RBD decorated VLPs already showed a significant specific humoral response following prime vaccination, greatly reinforced by a single boost. Neutralization assays with SARS-CoV-2 spike pseudo-typed virus demonstrated the elicitation of strong neutralization titers, superior to those of COVID-19 convalescent patients. Notably, the presence of pre-existing immunity against the adenoviral-derived particles did not hamper the immune response against the antigen displayed on its surface. This plug and play vaccine platform represents a promising new highly versatile tool to combat emergent pathogens.
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In this study, Chevillard and colleagues report a non-infectious adenovirus-inspired self-assembling particle designed to spontaneously and covalently display up to 60 SARS-CoV-2 glycoantigens. Immunization of mice with this pseudoviral mimic generates a strong neutralizing antibody response capable of neutralizing SARS-CoV-2 in the long term. A new tool for pandemic preparedness.
Pigments are among the oldest nanoparticulate products known to mankind, and theiruse in tattoos is also very old. Nowadays, 25% of American people aged 18 to 50 aretattooed, which poses the question ...of the delayed effects of tattoos. In this article, weinvestigated three cobalt Pigment Violet 14 (purple color) or cobalt alloy pigmentsPigment Blue 28 (blue color), Pigment Green 14 (green color), and one zinc pigmentPigment White 4 (white color) which constitute a wide range of colors found in tattoos.These pigments contain microparticles and a significant proportion of submicroparticlesor nanoparticles (in either aggregate or free form). Because of the key role of macrophagesin the scavenging of particulate materials, we tested the effects of cobalt- and zinc-basedpigments on the J774A.1 macrophage cell line. In order to detect delayed effects, wecompared two exposure schemes: acute exposure for 24 hours and an exposure for 24hours followed by a 3-day post-exposure recovery period. The conjunction of these twoschemes allowed for the investigation of the delayed or sustained effects of pigments. Allpigments induced functional effects on macrophages, most of which were pigmentdependent.For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigmentstested induced a slight but significant increase in tumor necrosis factor secretion. Thiseffect, however, was transitory. Conversely, only Pigment Blue 28 induced both a shortand sustained increase in interleukin 6 secretion. Results showed that in response tobacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declinedafter exposure to pigments followed by a recovery period. For chemoattractant cytokines(MCP-1 or MIP-1a), delayed effects were observed with a secretion decreased inFrontiers in presence of Pigment Blue 28 and Pigment violet 14, both with or without LPS stimuli. Thepigments also induced persisting changes in some important macrophage membranemarkers such as CD11b, an integrin contributing to cell adhesion and immunologicaltolerance. In conclusion, the pigments induced functional disorders in macrophages,which, in some cases, persist long after exposure, even at non-toxic doses.
The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with ...the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
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•The sporulation/germination protein YhcN contains a RBM domain.•The RBM core of SpoIIIAG, SpoIIIAH and YhcN is not sufficient to trigger oligomerization in vitro.•The β-barrel ...forming region in SpoIIIAG forms rings on its own.
Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for “Ring-Building Motifs”. During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the β-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.
The efficient spread of SARS-CoV-2 resulted in a pandemic that is unique in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood ...about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLR S ) of antigen-presenting cells, widely present in air mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. Thus, we described a mechanism potentiating viral capture and spreading of infection. Early involvement of APCs opens new avenues for understanding and treating the imbalanced innate immune response observed in COVID-19 pathogenesis
The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with ...the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull‐down experiments using the proteasome regulatory complex (proteasome‐activating nucleotidase PAN) as bait. X‐ray crystallography and small‐angle X‐ray scattering experiments revealed that the protein is monomeric and adopts a β‐barrel core structure with an oligonucleotide/oligosaccharide‐binding (OB)‐fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)‐binding properties. Pull‐downs, co‐immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull‐downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN–20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome‐Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA‐processing enzymes and exosome subunits dependent on Pbp11's N‐terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.
Partner of the archaeal proteasome PAN:20S complex in Thermococcales, Pbp11 directly interacts with the unfoldase PAN. From the cellular extract, Pbp11 pulls down the proteasome system and other macromolecular assemblies related to RNA processes. These last interactions are dependent on the presence of the flexible N‐terminal tail of Pbp11, a key feature of Pbp11 to bind transfer ribonucleic acids. Pbp11 becomes an interesting candidate to study tight connections between these nanomachines in the context of extremophilic Archaea.
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