Search

Bacillus anthracis is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The entire surface of vegetative cells in exponential or stationary growth phase is covered in proteinaceous arrays called S-layers, composed of Sap or EA1 protein, respectively. The Sap S-layer represents an important virulence factor and cell envelope support structure whose paracrystalline nature is essential for its function. However, the spatial organization of Sap in its lattice state remains elusive. Here, we employed cryoelectron tomography and subtomogram averaging to obtain a map of the Sap S-layer from tubular polymers that revealed a conformational switch between the postassembly protomers and the previously available X-ray structure of the condensed monomers. To build and validate an atomic model of the lattice within this map, we used a combination of molecular dynamics simulations, X-ray crystallography, cross-linking mass spectrometry, and biophysics in an integrative structural biology approach. The Sap lattice model produced recapitulates a close-to-physiological arrangement, reveals high-resolution details of lattice contacts, and sheds light on the mechanisms underlying the stability of the Sap layer., Competing Interests: Competing interests statement:H.R. and A.F. are named as inventors on patent WO/2019/068677 describing the use of S-layer disrupting nanobodies as antibacterial agents. The other authors declare no competing interests.

Enveloped viruses enter cells by fusing their envelopes to host cell membranes. Vesicular stomatitis virus (VSV) glycoprotein (G) is a prototype for class III fusion proteins. Although structures of the stable pre- and postfusion ectodomain of G are known, its fusogenic intermediates are insufficiently characterized. Here, we incubated VSV virions with late endosome-mimicking liposomes at pH 5.5 and used cryo-electron tomography (cryo-ET) to visualize stages of VSV's membrane fusion pathway, capture refolding intermediates of G, and reconstruct a sequence of G conformational changes. We observe that the G trimer disassembles into monomers and parallel dimers that explore a broad conformational space. Extended intermediates engage target membranes and mediate fusion, resulting in viral uncoating and linearization of the ribonucleoprotein genome. These viral fusion intermediates provide mechanistic insights into class III viral fusion processes, opening avenues for future research and structure-based design of fusion inhibition-based antiviral therapeutics.

Epilepsy is a common neurological disorder characterized by abnormal activity of neuronal networks, leading to seizures. The racetam class of anti-seizure medications bind specifically to a membrane protein found in the synaptic vesicles of neurons called synaptic vesicle protein 2 (SV2) A (SV2A). SV2A belongs to an orphan subfamily of the solute carrier 22 organic ion transporter family that also includes SV2B and SV2C. The molecular basis for how anti-seizure medications act on SV2s remains unknown. Here we report cryo-electron microscopy structures of SV2A and SV2B captured in a luminal-occluded conformation complexed with anticonvulsant ligands. The conformation bound by anticonvulsants resembles an inhibited transporter with closed luminal and intracellular gates. Anticonvulsants bind to a highly conserved central site in SV2s. These structures provide blueprints for future drug design and will facilitate future investigations into the biological function of SV2s., Competing Interests: Competing interests: E.L., C.A., M.Y., L.P., A. Hall, M.P., M.L., A. Hillisch, C.W., M.G. and P.S.H. are employees of UCB Pharma. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Lipopolysaccharide binding proteins (LBPs) and bactericidal permeability increasing proteins (BPIs) play significant roles in the immune response of vertebrates against bacterial pathogens. These soluble proteins produced by immune cells, specifically interact with and bind to bacterial lipopolysaccharides (LPS), with BPIs also displaying antibacterial activity. In Argopecten purpuratus scallop larvae resistant to Vibrio bivalvicida VPAP30, we identified a significant overexpression of a transcript displaying molecular features of an LBP/BPI protein, both before and after infection. Therefore, in the present work we aimed to understand the role of this novel LBP/BPI, named ApLBP/BPI3, in the scallop resistance to this Vibrio. The ApLBP/BPI3 open reading frame encodes a putative protein of 506 amino acids, with a molecular weight 56.78 kDa. The protein contains a C-terminal domain of 403-amino acid that, after theorical cleavage, displays a soluble form of 44.99 kDa, featuring two BPI/LBP/CETP domains, an apolar binding pocket, a single disulfide bond and a BPI dimerization interface. Phylogenetic analysis reveals high similarity between ApLBP/BPI3 and other mollusk LBP/BPI proteins. Aplbp/bpi3 transcripts were constitutively and highly expressed in hemocytes, gills, mantle, and digestive gland tissues, and were induced following VPAP30 infection in scallop larvae and adult hemocytes. We characterized ApLBP/BPI3 protein using a polyclonal antibody against a synthetic peptide. ApLBP/BPI3 was secreted to the media by infected cultured hemocytes, detected by ELISA. ApLBP/BPI3 was spotted inside non-infected hemocytes, bound to the cell wall of V. bivalvicida after in vitro hemocyte infection, and coating the gills and mantle epithelial barriers before and after an in vivo immune challenge, with stronger detection after VPAP30 injection, detected by immunofluorescence. Infected scallop larvae showed increased ApLBP/BPI3 levels, with slightly higher production in resistant larvae, determined by Western blot. Finally, silencing the Aplbp/bpi3 transcript through RNA interference and and subsequently infecting scallop juveniles with an LD50 of V. bivalvicida resulted in 100 % mortality. Altogether, results demonstrate the essential role of this immune effector in the resistance of A. purpuratus., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To rich the research for mannoproteins (MPs) suppressive effect on the bitterness of wine, this study distinguished bitterness into initial bitterness and bitter aftertaste. By utilizing the thermal alkali extraction method, MPs were extracted from three different yeast species: Saccharomyces cerevisiae (CECA), Lachancea thermotolerans (A38), and Torulaspora delbrueckii (2082). Their basic structures, addition concentrations, and correlation with bitter suppression ability were characterized. CECA exhibited stronger initial bitterness suppression ability, may attributed to its more branches and lack of a triple-helix structure. 2082 showed greater bitter aftertaste suppression and might due to smaller particle aggregation, fewer branches, and triple-helix structure. Additionally, it was noteworthy that due to the unique structure of 2082, it may bound more monomer and oligomeric proanthocyanidins (MOPC) on MPs surface, reducing its initial bitterness suppression ability. Concerning concentration, the increase in polysaccharide chain polymerization hindered further interaction with MOPC, leading to a decrease in its initial bitterness suppression ability. Bitter aftertaste exhibited different behaviors. As the concentration of CECA increased, there was an increase in oral adhesion instead., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein-polysaccharide-tannin interactions are important in every aspect of red wine production from physical stability to color, astringency, and body. For this model study, bovine serum albumin (BSA) was selected as the protein, while carboxymethyl cellulose (CMC), mannoproteins, and pectin were the model polysaccharides. Each protein-polysaccharide combination was analyzed for zeta (ζ) potential and particle size at neutral pH and within the wine-like solution. Mixtures were assessed regarding their protective, affinitive, and aggregative behaviors. Based on their individual ζ-potentials, pectin and mannoprotein were most stable at lower concentrations. At higher concentrations, they reduced the suspension's stability and increased the aggregate sizes. CMC consistently increased the stability of any solution under neutral pH conditions. However, with increasing concentrations, these large aggregates are expected to precipitate. Fruit pectin (FP) and BSA interactions seemed to be the main factors in the formation of visible precipitates at neutral pH. FP and the mannoprotein decreased stability enough to cause precipitation without haze formation. The mannoprotein decreased particle sizes, in both the suspension and precipitation, which may indicate greater selectivity toward proteins. FP also decreased the suspended particle sizes under wine conditions. These findings demonstrate the use of ζ-potential and particle size values to characterize macromolecular interactions in model systems and can also be used to indicate effective fining agents. PRACTICAL APPLICATION: This work demonstrates the capabilities of ζ-potential analysis paired with size particle measurements to predict and characterize the interactions between macromolecules in complex systems. The interactions between model wine macromolecules can be evaluated with this technology at a level that cannot be reached with any other analytical technique., (© 2024 Institute of Food Technologists.)

Glucose-regulated protein 94 (Grp94) is an isoform of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Inhibiting Grp94 has been implicated for many diseases. Co-crystal structures of two generations of Grp94 inhibitors revealed the importance of investigating the ester group, which is projected into the site 2 pocket unique to Grp94. Therefore, a series of KUNG65 benzamide analogs was designed and synthesized to evaluate their impact on the affinity and selectivity for Grp94. The data demonstrated that substituents with small and saturated ring systems that contain hydrogen bond acceptors exhibited increased affinity for Grp94, whereas larger saturated ring system manifested increased selectivity for Grp94 over Hsp90α., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Post-translational modifications such as protein N-glycosylation, significantly influence cellular processes. Dysregulated N-glycosylation, exemplified in Grp94, a member of the Hsp90 family, leads to structural changes and the formation of epichaperomes, contributing to pathologies. Targeting N-glycosylation-induced conformations offers opportunities for developing selective chemical tools and drugs for these pathologic forms of chaperones. We here demonstrate how a specific Grp94 conformation induced by N-glycosylation, identified previously via molecular dynamics simulations, rationalizes the distinct behavior of similar ligands. Integrating dynamic ligand unbinding information with SAR development, we differentiate ligands productively engaging the pathologic Grp94 conformers from those that are not. Additionally, analyzing binding site stereoelectronic properties and QSAR models using cytotoxicity data unveils relationships between chemical, conformational properties, and biological activities. These findings facilitate the design of ligands targeting specific Grp94 conformations induced by abnormal glycosylation, selectively disrupting pathogenic protein networks while sparing normal mechanisms., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Ltd.)

The triggering receptor expressed on myeloid cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer's disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2's binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including surface plasmon resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2's robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer's disease., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ara h1 was the highest content of peanut allergen protein, identified as a biomarker of peanut allergen. In this study, Ara h1 was covalently complexed with caffeic acid (CA) to research the effects of covalent conjugation on the antigenicity and protein structural properties of Ara h1. After the covalent complexing of Ara h1 and CA, the IgG-binding capacity of Ara h1 was reduced compared with that of control Ara h1. Moreover, the structure of Ara h1 changed from ordered to disordered, the number of intermolecular hydrogen bonds decreased, and some hydrophobic groups were exposed or hydrophobic peptides were released. The carboxyl group in CA reacted with the amino group in Ara h1. The digestibility of Ara h1-CA was increased. The antigenicity of Ara h1-CA was undetectable after 30 min of digestion in vitro. These findings can serve as a reference for further research on hypoallergenic peanut products., (© 2024 Institute of Food Technologists.)

Tropomyosin kinase receptor B (TrkB) has been explored as a therapeutic target for neurological and psychiatric disorders. However, the development of TrkB agonists was hindered by our poor understanding of the TrkB agonist binding location and affinity (both affect the regulation of disorder types). This motivated us to develop a combined computational and experimental approach to study TrkB binders. First, we developed a docking method to simulate the binding affinity of TrkB and binders identified by our magnetic drug screening platform from Gotu kola extracts. The Fred Docking scores from the docking computation showed strong agreement with the experimental results. Subsequently, using this screening platform, we identified a list of compounds from the NIH clinical collection library and applied the same docking studies. From the Fred Docking scores, we selected two compounds for TrkB activation tests. Interestingly, the ability of the compounds to increase dendritic arborization in hippocampal neurons matched well with the computational results. Finally, we performed a detailed binding analysis of the top candidates and compared them with the best-characterized TrkB agonist, 7,8-dyhydroxyflavon. The screening platform directly identifies TrkB binders, and the computational approach allows for the quick selection of top candidates with potential biological activities based on the docking scores.

The taurine transporter (TauT, SLC6A6) is a member of the solute carrier 6 (SLC6) family, which plays multiple physiological roles. The SLC6 family is divided into four subfamilies: GABA (γ-aminobutyric acid), monoamine, glycine and neutral amino acid transporters. Proteins from the GABA group, including the taurine transporter, are primarily considered therapeutic targets for treating central nervous system disorders. However, recent studies have suggested that inhibitors of SLC6A6 could also serve as anticancer agents. Overexpression of TauT has been associated with the progression of colon and gastric cancer. The pool of known ligands of this transporter is limited and the exact spatial structure of taurine transporter remains unsolved. Understanding its structure could aid in the development of novel inhibitors. Therefore, we utilized homology modelling techniques to create models of TauT. Docking studies and molecular dynamics simulations were conducted to describe protein-ligand interactions. We compared the obtained information for TauT with literature data on other members of the GABA transporter group. Our in silico analysis allowed us to characterize the transporter structure and point out amino acids crucial for ligand binding: Glu406, Gly62 and Tyr138. The significance of selected residues was confirmed through structural studies of mutants. These results will aid in the development of novel taurine transporter inhibitors, which can be explored as anticancer agents.

Background : NTRK1, NTRK2, and NTRK3 are members of the neurotrophic receptor tyrosine kinases (NTRK) family, which encode TrkA, TrkB, and TrkC receptors, respectively. Hematologic cancers are also linked to point mutations in the NTRK gene's kinase domain. Trk fusions are the most common genetic change associated with oncogenic activity in Trk-driven liquid tumors. Thus, point mutations in NTRK genes may also play a role in tumorigenesis. The structural and functional effect of mutations in Trk-B & Trk-C proteins remains unclear. Methods : In this research, Homology (threading-based approach) modeling and the all-atom molecular dynamics simulations approaches are applied to examine the structural and functional behavior of native and mutant Trk-B and Trk-C proteins at the molecular level. Results: The result of this study reveals how the mutations in Trk-B (A203T & R458G) and Trk-C (E176D & L449F) proteins lost their stability and native conformations. The Trk-B mutant A203T became more flexible than the native protein, whereas the R458G mutation became more rigid than the native conformation of the Trk-B protein. Also, the Trk-C mutations (E176D & L449F) become more rigid compared to the native structure. Conclusions: This structural transition may interrupt the function of Trk-B and Trk-C proteins. Observing the impact of NTRK-2/3 gene alterations at the atomic level could aid in discovering a viable treatment for Trk-related leukemias., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 M Al-Subaie A et al.)

S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer's pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general., Competing Interests: Competing interests statement:The authors declare no competing interest.

In silico modeling was applied to study the efficiency of two ligands, namely, UCB-J and UCB-F , to bind to isoforms of the synaptic vesicle glycoprotein 2 (SV2) that are involved in the regulation of synaptic function in the nerve terminals, with the ultimate goal to understand the selectivity of the interaction between UCB-J and UCB-F to different isoforms of SV2. Docking and large-scale molecular dynamics simulations were carried out to unravel various binding patterns, types of interactions, and binding free energies, covering hydrogen bonding and nonspecific hydrophobic interactions, water bridge, π-π, and cation-π interactions. The overall preference for bonding types of UCB-J and UCB-F with particular residues in the protein pockets can be disclosed in detail. A unique interaction fingerprint, namely, hydrogen bonding with additional cation-π interaction with the pyridine moiety of UCB-J , could be established as an explanation for its high selectivity over the SV2 isoform A (SV2A). Other molecular details, primarily referring to the presence of π-π interactions and hydrogen bonding, could also be analyzed as sources of selectivity of the UCB-F tracer for the three isoforms. The simulations provide atomic details to support future development of new selective tracers targeting synaptic vesicle glycoproteins and their associated diseases.

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 10 6  cells/mL, a plasmid concentration of 2 µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3 days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2 mM of sodium butyrate added 18 h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32 nm with 91.4% of protein similarity to exosomes., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Previous studies have demonstrated that early intervention was the best plan to inhibit the progression of Alzheimer's disease (AD), which relied on the discovery of early diagnostic biomarkers. In this study, synaptic vesicle glycoprotein 2 A (SV2A) was examined to improve the early diagnostic efficiency in AD., Methods: In this study, biomarker testing was performed through the single-molecule array (Simoa). A total of 121 subjects including cognitively unimpaired controls, amnestic mild cognitive impairment (aMCI), AD and other types of dementia underwent cerebrospinal fluid (CSF) SV2A testing; 430 subjects including health controls, aMCI, AD and other types of dementia underwent serum SV2A, glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL) and p-tau217 testing; 92 subjects including aMCI and AD underwent both CSF SV2A and serum SV2A testing; 115 cognitively unimpaired subjects including APOE ε4 carriers and APOE ε4 non-carriers were tested for serum SV2A, GFAP, NfL and p-tau217. Then, the efficacy of SV2A for the early diagnosis of AD and its ability to identify those at high risk of AD from a cognitively unimpaired population were further analyzed., Results: Both CSF and serum SV2A significantly and positively correlated with cognitive performance in patients with AD, and their levels gradually decreased with the progression of AD. Serum SV2A demonstrated excellent diagnostic efficacy for aMCI, with a sensitivity of 97.8%, which was significantly higher than those of NfL, GFAP, and p-tau217. The SV2A-positive rates ranged from 92.86 to 100% in aMCI cases that were negative for the above three biomarkers. Importantly, of all the biomarkers tested, serum SV2A had the highest positivity rate (81.82%) in individuals at risk for AD., Conclusions: Serum SV2A was demonstrated to be a novel and ideal biomarker for the early diagnosis of AD, which can effectively distinguish those at high risk of AD in cognitively unimpaired populations., (© 2024. The Author(s).)

We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin-like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function., (© 2023 The Protein Society.)

The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N -glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.

Cell surface proteins (CSPs) are often involved in various biological processes such as cell-cell interactions, immune responses, and molecular transport. The abnormal expression of CSP usually indicates the occurrence and development of human diseases. Most CSPs are glycosylated and have been explored as potential drug targets and disease biomarkers; however, efficient isolation of CSPs from intracellular proteins is difficult due to their low abundance and strong hydrophobicity. The comprehensive characterization of surface glycoproteins remains a great challenge and is often underrepresented in proteomics. In recent years, unprecedented progress has been made in the mass spectrometry analysis of surface proteins, and CSP capture methods and mass spectrometry have been greatly developed. In this article, we aim to give a comprehensive overview of innovative analytical methods that can enrich CSPs, including centrifugation-based separation, phase partitioning, adhesion-based capture of surface proteins, antibody or lectin affinity, and biotin-based chemical labeling. Surface glycoproteins are captured by chemical oxidation of glycans or click chemistry for carbohydrate metabolic labeling. These techniques offer a wide range of applications for studying the function of cell surface receptors and identifying markers for diagnostic and therapeutic development.

The S-layer or surface layer protein (SLP) is the most ancient biological envelope, highly conserved in several Bacteria and Archaea. In lactic acid bacteria (LAB), SLP is only found in species belonging to the Lactobacillaceae family, many of them considered probiotic microorganisms. New reclassification of members within the Lactobacillaceae family (International Journal of Systematic and Evolutionary Microbiology, 2020, 70, 2782) and newly sequenced genomes demands an updated revision on SLP genes and domain organization. There is growing information concerning SLP occurrence, molecular biology, biophysical properties, and applications. Here, we focus on the prediction of slp genes within the Lactobacillaceae family, and specifically, on the neat interconnection between the two different modular SLP domain organizations and the new reclassified genera. We summarize the results in a concise tabulated manner to review the present knowledge on SLPs and discuss the most relevant and updated concepts regarding SLP sequence clustering. Our assessment is based on sequence alignments considering the new genera classification and protein domain definition with post-translational modifications. We analyse the difficulties encountered to resolve the SLPs 3D structure, describing the need for structure prediction approaches and the relation between protein structure and its anchorage mechanism to the cell wall. Finally, we enumerate new SLP applications regarding heterologous display, pathogen exclusion, immunostimulation, and metal binding., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Hepatocyte growth factor activator inhibitor (HAI)-2 is an integral membrane Kunitz-type serine protease inhibitor that regulates the proteolysis of matriptase and prostasin in a cell-type selective manner. The cell-type selective nature of HAI-2 function depends largely on whether the inhibitor and potential target enzymes are targeted to locations in close proximity. The N-glycan moiety of HAI-2 can function as a subcellular targeting signal. HAI-2 is synthesized with 1 of 2 different N-glycan modifications: one of oligomannose-type, which largely remains in the endoplasmic reticulum/GA, and another of complex-type, which is targeted toward the apical surface in vesicle-like structures, and could function as an inhibitor of matriptase and prostasin. HAI-2 contains 2 putative N-glycosylation sites, Asn-57 and Asn-94, point mutations of which were generated and characterized in this study. The protein expression profile of the HAI-2 mutants indicates that Asn-57, and not Asn-94, is responsible for the N-glycosylation of both HAI-2 species, suggesting that the form with oligomannose-type N-glycan is the precursor of the form with complex-type N-glycan. Unexpectedly, the vast majority of non-glycosylated HAI-2 is synthesized into multiple disulfide-linked oligomers, which lack protease inhibitory function, likely due to distorted conformations caused by the disarrayed disulfide linkages. Although forced expression of HAI-2 in HAI-2 knockout cells artificially enhances HAI-2 oligomerization, disulfide-linked HAI-2 oligomers can also be observed in unmodified cells. These results suggest that N-glycosylation on Asn-57 is required for folding into a functional HAI-2 with full protease suppressive activity and correct subcellular targeting signal., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Niemann-Pick C1 protein (NPC1) is a membrane protein that primarily resides in late endosomes and lysosomes, and plays an important role in cholesterol homeostasis in the cell. The second luminal domain of NPC1 (NPC1-C) serves as the intracellular receptor for Ebola and Marburg viruses. Here, the recombinant production of nonglycosylated and glycosylated NPC1-C and a new crystal form of the nonglycosylated protein are reported. The crystals belonged to space group P2 1 and diffracted to 2.3 Å resolution. The structure is similar to other reported structures of NPC1-C, with differences observed in the protruding loops when compared with NPC1-C in complex with Ebola virus glycoprotein or NPC2., (open access.)

Botulinum neurotoxins are the deadliest microbial neurotoxins in humans, with a lethal dose of 1 ng/kg. Incidentally, these neurotoxins are also widely used for medical and cosmetic purposes. However, little is known about the molecular mechanisms that control binding of botulinum neurotoxin type F1 (BoNT/F1) to its membrane receptor, glycosylated human synaptic vesicle glycoprotein A (hSV2Ag). To elucidate these mechanisms, we performed a molecular dynamics simulation (MDS) study of initial binding kinetics of BoNT/F1 to SV2A. Since this toxin also interacts with gangliosides, the simulations were performed at the periphery of a lipid raft in the presence of both SV2A and gangliosides. Our study suggested that interaction of BoNT/F1 with SV2A is exclusively mediated by N-glycan moiety of SV2A, which interacts with aromatic residues Y898, Y910, F946, Y1059 and H1273 of this toxin. Thus, in contrast with botulinum neurotoxin A1 (BoNT/A1), BoNT/F1 does not interact with protein content of SV2A. We attributed this incapability to a barrage effect exerted by neurotoxin residues Y1132, Q1133 and K1134, which prevent formation of long-lasting intermolecular hydrogen bonds. We also provided structural elements that suggest that BoNT/F1 uses the strategy of BoNT/A1 combined with the strategy of botulinum neurotoxin type E to bind N-glycan of its glycoprotein receptor. Overall, our study opened a gate for design of a universal inhibitor aimed at disrupting N-glycan-toxin interactions and for bioengineering of a BoNT/F1 protein that may be able to bind protein content of synaptic vesicle glycoprotein for therapeutic purposes.

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies., Competing Interests: Declaration of competing interest The University of Granada, the University of Strasbourg and the Institut National de la Santé et de la Recherche Médicale (INSERM) have filed a patent application (P202230188) on the chimeric proteins described in this article. Inventors: F.C-L., M.C-M. and C.M., (Copyright © 2022. Published by Elsevier B.V.)

The effectiveness of therapeutic monoclonal antibodies (mAbs) against variants of the SARS-CoV-2 virus is highly variable. As target recognition of mAbs relies on tight binding affinity, we assessed the affinities of five therapeutic mAbs to the receptor binding domain (RBD) of wild type (A), Delta (B.1.617.2), and Omicron BA.1 SARS-CoV-2 (B.1.1.529.1) spike using microfluidic diffusional sizing (MDS). Four therapeutic mAbs showed strongly reduced affinity to Omicron BA.1 RBD, whereas one (sotrovimab) was less impacted. These affinity reductions correlate with reduced antiviral activities suggesting that affinity could serve as a rapid indicator for activity before time-consuming virus neutralization assays are performed. We also compared the same mAbs to serological fingerprints (affinity and concentration) obtained by MDS of antibodies in sera of 65 convalescent individuals. The affinities of the therapeutic mAbs to wild type and Delta RBD were similar to the serum antibody response, indicating high antiviral activities. For Omicron BA.1 RBD, only sotrovimab retained affinities within the range of the serum antibody response, in agreement with high antiviral activity. These results suggest that serological fingerprints provide a route to evaluating affinity and antiviral activity of mAb drugs and could guide the development of new therapeutics., (© 2022. The Author(s).)

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are the major pathogens of the spore-forming genus Bacillus and possess an outer spore layer, the exosporium, not found in many of the nonpathogenic species. The exosporium consists of a basal layer with the ExsY, CotY, and BxpB proteins being the major structural components and an exterior nap layer containing the BclA glycoprotein. During the assembly process, the nascent exosporium basal layer is attached to the spore coat by a protein linker that includes the CotO and CotE proteins. Using transmission electron microscopy, Western blotting, immunofluorescence, and fluorescent fusion protein approaches, we examined the impact of single, double, and triple mutants of the major exosporium proteins on exosporium protein content and distribution. Plasmid-based expression of exsY and cotE resulted in increased production of exosporium lacking spores, and the former also resulted in outer spore coat disruptions. The exosporium bottlecap produced by exsY null spores was found to be more stable than previously reported, and its spore association was partially dependent on CotE. Deletion mutants of five putative spore genes ( bas1131 , bas1142 , bas1143 , bas2277 , and bas3594 ) were created and shown not to have obvious effects on spore morphology or BclA and BxpB content. The BclC collagen-like glycoprotein was found to be present in the spore and possibly localized to the interspace region. IMPORTANCE B. anthracis is an important zoonotic animal pathogen causing sporadic outbreaks of anthrax worldwide. Spores are the infectious form of the bacterium and can persist in soil for prolonged periods of time. The outermost B. anthracis spore layer is the exosporium, a protein shell that is the site of interactions with both the soil and with the innate immune system of infected hosts. Although much is known regarding the sporulation process among members of the genus Bacillus , significant gaps in our understanding of the exosporium assembly process exist. This study provides evidence for the properties of key exosporium basal layer structural proteins. The results of this work will guide future studies on exosporium protein-protein interactions during the assembly process.

The 94 kDa molecular chaperone, glucose-regulated protein 94 (Grp94), has garnered interest during the last decade due to its direct association with endoplasmic reticulum (ER) stress and disease. Grp94 belongs to the Hsp90 family of molecular chaperones and is a master regulator of ER homeostasis due to its ability to fold and stabilize proteins/receptors, and to chaperone misfolded proteins for degradation. Multiple studies have demonstrated that Grp94 knockdown or inhibition leads to the degradation of client protein substrates, which leads to disruption of disease-dependent signaling pathways. As a result, small molecule inhibitors of Grp94 have become a promising therapeutic approach to target a variety of disease states. Specifically, Grp94 has proven to be a promising target for cancer, glaucoma, immune-mediated inflammation, and viral infection. Moreover, Grp94-peptide complexes have been utilized effectively as adjuvants for vaccines against a variety of disease states. This work highlights the significance of Grp94 biology and the development of therapeutics that target this molecular chaperone in multiple disease states., (© 2022 Wiley Periodicals LLC.)

The SARS-CoV-2 Omicron variant is characterized by substantial changes in the antigenic structure of the Spike (S) protein. Therefore, antibodies induced by primary Omicron infection lack neutralizing activity against earlier variants. In this study, we analyzed whether these antigenic changes impact the sensitivity of commercial anti-SARS-CoV-2 antibody assays. Sera from 37 unvaccinated, convalescent individuals after putative primary Omicron infection were tested with a panel of 20 commercial anti-SARS-CoV-2 immunoassays. As controls, we used samples from 43 individuals after primary infection with the SARS-CoV-2 ancestral wild-type strain. In addition, variant-specific live-virus neutralization assays were used as a reference for the presence of SARS-CoV-2-specific antibodies in the samples. Notably, in Omicron convalescents, there was a statistically significant reduction in the sensitivity of all antibody assays containing S or its receptor-binding-domain (RBD) as antigens. Furthermore, antibody levels quantified by these assays displayed a weaker correlation with Omicron-specific neutralizing antibody titers than with those against the wild type. In contrast, the sensitivity of nucleocapsid-protein-specific immunoassays was similar in wild-type and Omicron-infected subjects. In summary, the antigenic changes in the Omicron S lead to reduced immunoreactivity in the current commercial S- and RBD-specific antibody assays, impairing their diagnostic performance. IMPORTANCE This study demonstrates that the antigenic changes of the SARS-CoV-2 Omicron variant affect test results from commercial Spike- and RBD-specific antibody assays, significantly diminishing their sensitivities and diagnostic abilities to assess neutralizing antibodies.

A new detection strategy was developed to improve the sensitivity of a lateral flow immunoassay platform utilizing a delayed hydrophobic barrier fabricated with trimethylsilyl cellulose (TMSC). The SARS-CoV-2 spike receptor-binding domain (SARS-CoV-2 SP RBD) antigen was chosen as a model analyte to demonstrate the superior detectability of this scheme. The novel device consists of 2 separate layers, so-called delayed lateral flow immunoassay (d-LFIA). The upper layer is intended for the analyte or sample flow path, where the test solution flows freely straight to the detection zone to bind with the primary antibody. The lower layer, located just underneath, is designed for the SARS-CoV-2 spike receptor-binding domain-conjugated gold nanoparticles (SARS-CoV-2 SP RBD-AuNPs) used for producing a colorimetric signal. This layer is fabricated with a TMSC barrier to time-delay the movement of SARS-CoV-2 SP RBD-AuNPs, thus allowing the antigen to bind with the primary antibody more efficiently. This platform exhibited a 2.6-fold enhancement in the sensitivity and 9.1-fold improvement in the limit of detection (LOD) as compared with the conventional LFIA. In addition, this d-LFIA device was satisfactorily applied to accurate screening of COVID-19 patients., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Given that COVID-19 continues to wreak havoc around the world, it is imperative to search for a conserved region involved in viral infection so that effective vaccines can be developed to prevent the virus from rapid mutations. We have established a twelve-fragment library of recombinant proteins covering the entire region of spike protein of both SARS-CoV-2 and SARS-CoV from Escherichia coli. IgGs from murine antisera specifically against 6 spike protein fragments of SARS-CoV-2 were produced, purified, and characterized. We found that one specific IgG against the fusion process region, named COVID19-SF5, serologically cross-reacted with all twelve S-protein fragments. COVID19-SF5, with amino acid sequences from 880 to 1084, specifically bound to VERO-E6 and BEAS-2B cells, with K d values of 449.1 ± 21.41 and 381.9 ± 31.53 nM, and IC 50 values of 761.2 ± 28.2 nM and 862.4 ± 32.1 nM, respectively. In addition, COVID19-SF5 greatly enhanced binding of the full-length CHO cell-derived spike protein to the host cells in a concentration-dependent manner. Furthermore, COVID19-SF5 and its IgGs inhibited the infection of the host cells by pseudovirus. The combined data from our studies reveal that COVID19-SF5, a novel cell-binding fragment, may contain a common region(s) for mediating viral binding during infection. Our studies also provide valuable insights into how virus variants may evade host immune recognition. Significantly, the observation that the IgGs against COVID19-SF5 possesses cross reactivity to all other fragments of S protein, suggesting that it is possible to develop universal neutralizing monoclonal antibodies to curb rapid mutations of COVID-19., (© 2022. The Author(s).)

To orchestrate cell mechanics, trafficking, and motility, cytoskeletal filaments must assemble into higher-order networks whose local subcellular architecture and composition specify their functions. Cross-linking proteins bridge filaments at the nanoscale to control a network's μm-scale geometry, thereby conferring its mechanical properties and functional dynamics. While these interfilament linkages are key determinants of cytoskeletal function, their structural mechanisms remain poorly understood. Plastins/fimbrins are an evolutionarily ancient family of tandem calponin-homology domain (CHD) proteins required to construct multiple classes of actin networks, which feature diverse geometries specialized to power cytokinesis, microvilli and stereocilia biogenesis, and persistent cell migration. Here, we focus on the structural basis of actin network assembly by human T-plastin, a ubiquitously expressed isoform necessary for the maintenance of stable cellular protrusions generated by actin polymerization forces. By implementing a machine-learning-enabled cryo-electron microscopy pipeline for visualizing cross-linkers bridging multiple filaments, we uncover a sequential bundling mechanism enabling T-plastin to bridge pairs of actin filaments in both parallel and antiparallel orientations. T-plastin populates distinct structural landscapes in these two bridging orientations that are selectively compatible with actin networks featuring divergent architectures and functions. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable cross-linking that are likely to be disrupted by T-plastin mutations that cause hereditary bone diseases.

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X 3 -Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The nanoscale structure and dynamics of proteins on surfaces has been extensively studied using various imaging techniques, such as transmission electron microscopy and atomic force microscopy (AFM) in liquid environments. These powerful imaging techniques, however, can potentially damage or perturb delicate biological material and do not provide chemical information, which prevents a fundamental understanding of the dynamic processes underlying their evolution under physiological conditions. Here, we use a platform developed in our laboratory that enables acquisition of infrared (IR) spectroscopy and AFM images of biological material in physiological liquids with nanometer resolution in a cell closed by atomically thin graphene membranes transparent to IR photons. In this work, we studied the self-assembly process of S-layer proteins at the graphene-aqueous solution interface. The graphene acts also as the membrane separating the solution containing the proteins and Ca 2+ ions from the AFM tip, thus eliminating sample damage and contamination effects. The formation of S-layer protein lattices and their structural evolution was monitored by AFM and by recording the amide I and II IR absorption bands, which reveal the noncovalent interaction between proteins and their response to the environment, including ionic strength and solvation. Our measurement platform opens unique opportunities to study biological material and soft materials in general.

Epstein-Barr virus is a tumor-associated, enveloped virus with glycoprotein receptor gHgL on its surface. gH attaches to epithelial or B cells and mediates internalization. Till date, no specific anti-EBV FDA approved drug is available. Targeting gH may aid in designing virus-specific therapeutics and reducing the drug induced complications in host. We investigated the influence of antiviral phytochemicals on gH using computational approaches. Through molecular docking, we performed binding energy analysis of cellocidin, bruceantin, EGCG, formononetin and sesquiterpene lactones with gH DII/DIII interface, crucial for gH functions. Further, to cause any perturbations in the protein function, the molecules must bind stably to gH. Bruceantin and EGCG interacted with high affinities to gH. Simulation of these two molecules revealed stable binding with gH throughout 100 ns moreover, van der Waal interactions stabilized overall binding. Mutation of amino acids like V265, L269, L315, I423, I459, L474 and F475 involved in stable binding to gH was predicted deleterious to protein function. We obtained no difference in RMSD between these two ligands and minor deviations in the RMSF were noticed compared to gH. Conclusively, our study provided insights into the potential of bruceantin and EGCG to target gH. Different amino acids are involved in binding of each ligand to gH, engagement of certain amino acids may affect the virus binding with epithelial or B cells. The interaction of the ligand with gH may trap it in its native conformation or induce structural flexibility thereby inhibiting the interaction with host receptors or other glycoproteins.Communicated by Ramaswamy H. Sarma.

SARS-CoV-2 variants may threaten the effectiveness of vaccines and antivirals to mitigate serious COVID-19 disease. This is of most concern in clinically vulnerable groups such as older adults. We analysed 72 sera samples from 37 individuals, aged 70-89 years, vaccinated with two doses of BNT162b2 (Pfizer-BioNTech) 3 weeks apart, for neutralizing antibody responses to wildtype SARS-CoV-2. Between 3 and 20 weeks after the second vaccine dose, neutralizing antibody titres fell 4.9-fold to a median titre of 21.3 (neutralization dose 80%), with 21.6% of individuals having no detectable neutralizing antibodies at the later time point. Next, we examined neutralization of 21 distinct SARS-CoV-2 variant spike proteins with these sera, and confirmed substantial antigenic escape, especially for the Omicron (B.1.1.529, BA.1/BA.2), Beta (B.1.351), Delta (B.1.617.2), Theta (P.3), C.1.2 and B.1.638 spike variants. By combining pseudotype neutralization with specific receptor-binding domain (RBD) enzyme-linked immunosorbent assays, we showed that changes to position 484 in the spike RBD were mainly responsible for SARS-CoV-2 neutralizing antibody escape. Nineteen sera from the same individuals boosted with a third dose of BNT162b2 contained higher neutralizing antibody titres, providing cross-protection against Omicron BA.1 and BA.2. Despite SARS-CoV-2 immunity waning over time in older adults, booster vaccines can elicit broad neutralizing antibodies against a large number of SARS-CoV-2 variants in this clinically vulnerable cohort., (© 2022. The Author(s).)

The Niemann-Pick type C1 (NPC1) protein is one of the key players of cholesterol trafficking from the lysosome and its function is closely coupled with the Niemann-Pick type C2 (NPC2) protein. The dysfunction of one of these proteins can cause problems in the overall cholesterol homeostasis and leads to a disease, which is called the Niemann-Pick type C (NPC) disease. The parts of the cholesterol transport mechanism by NPC1 have begun to recently emerge, especially after the full-length NPC1 structure was determined from a cryo-EM study. However, many details about the overall cholesterol trafficking process by NPC1 still remain to be elucidated. Notably, the NPC1 could act as one of the target proteins for the control of infectious diseases due to its role as the virus entry point into the cells as well as for cancer treatment due to the inhibitory effect of tumor growth. A mutation of NPC1 can leads to dysfunctions and understanding this process can provide valuable insights into the mechanisms of the corresponding protein and the therapeutic strategies against the disease that are caused by the mutation. It has been found that patients with the point mutation R518W (or R518Q) on the NPC1 show the accumulation of lipids within the lysosomal lumen. In this paper, we report how the corresponding mutation can affect the cholesterol transport process by NPC1 in the different stages by the molecular dynamics simulations. The simulation results show that the point mutation intervenes at least at two different steps during the cholesterol transport by NPC1 and NPC2 in combination, which includes the association step of NPC2 with the NPC1, the cholesterol transfer step from NPC2 to NPC1-NTD while the cholesterol passage within the NPC1 via a channel is relatively unaffected by R518W mutation. The detailed analysis of the resulting simulation trajectories reveals the important structural features that are essential for the proper functioning of the NPC1 for the cholesterol transport, and it shows how the overall structure, which thereby includes the function, can be affected by a single mutation., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high-affinity and cost-effective ACE2 mimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2-5 kDa) and highly stable proteins containing solvent-exposed alpha-helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha-helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS-CoV-2 spike protein. The engineered proteins (h-deface2, p-deface2, and p-deface2-MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high-affinity binding to the spike protein with apparent K d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h-deface2, p-deface2, and p-deface2-MUT, respectively, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing of high-affinity ligands., (© 2022 The Protein Society.)

Tests for SARS-CoV-2 are crucial for the mass surveillance of the incidence of infection. The long waiting time for classic nucleic acid test results highlights the importance of developing alternative rapid biosensing methods. Herein, we propose a fiber-optic biolayer interferometry-based biosensor (FO-BLI) to detect SARS-CoV-2 spike proteins, extracellular domain (ECD), and receptor-binding domain (RBD) in artificial samples in 13 min. The FO-BLI biosensor utilized an antibody pair to capture and detect the spike proteins. The secondary antibody conjugated with horseradish peroxidase (HRP) reacted with the enzyme substrate for signal amplification. Two types of substrates, 3,3'-diaminobenzidine (DAB) and an advanced 3-Amino-9-ethylcarbazole (i.e., AMEC), were applied to evaluate their capabilities in enhancing signals and reaching high sensitivity. After careful comparison, the AMEC-based FO-BLI biosensor showed better assay performance, which detected ECD at a concentration of 32-720 pM and RBD of 12.5-400 pM in artificial saliva and serum, respectively. The limit of detection (LoD) for SARS-CoV-2 ECD and RBD was defined to be 36 pM and 12.5 pM, respectively. Morphology of the metal precipitates generated by the AMEC-HRP reaction in the fiber tips was observed using field emission scanning electron microscopy (SEM). Collectively, the developed FO-BLI biosensor has the potential to rapidly detect SARS-CoV-2 antigens and provide guidance for "sample-collect and result-out on-site" mode.

Various chimeric receptors have been developed and used for biological experiments. In the present study, we constructed three types of chimeric receptor activator of nuclear factor-kappa B (RANK) with the glutathione S-transferase (GST) protein in the extracellular domain, and stimulated them using newly synthesized chemical trimerizers with three glutathiones. Although this stimulation did not activate these proteins, we unexpectedly found that the chimera named RANK-GST-SC, in which GST replaced a major part of the RANK extracellular domain, activated nuclear factor-kappa B (NF-κB) signaling approximately sixfold more strongly than wild-type RANK without the ligand. The dimerization of extracellular GST is considered to function as a switch outside the cell, and signal transduction then occurs. GST has been widely employed as a tag for protein purification; GST-fusion protein can be conveniently captured by glutathione-conjugated beads and easily purified from impurity. The present study is a pioneering example of the novel utility of GST and provides information for the development of new chemical biology systems., (© 2021 John Wiley & Sons A/S.)

In this study, the surface layer protein (SLP) from Lactobacillus kefiri HBA20 was characterized. The SLP was extracted by 5M LiCl. The molecular mass of the SLP was approximately 64 kDa as analyzed via SDS-PAGE. The surface morphology and the adhesion potential of L. kefiri HBA20 in the absence and presence of SLP were measured by AFM. Moreover, the protein secondary structure was evaluated by using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR), respectively. SLP had high β-sheet contents and low content of α-helix. Thermal analysis of SLP of Lactobacillus kefiri HBA20 exhibited one transition peak at 129.64 °C. Furthermore, SEM measurements were showed that after the SLP were removed from the cell surface, the coaggregation ability with Saccharomyces cerevisiae Y8 of the strain was significantly reduced. In conclusion, the SLP of Lactobacillus kefiri HBA20 has a stable structure and the ability of adhesion to yeast. Molecular docking study revealed that mannan bind with the hydrophobic residues of SLP. Our results will help further understanding of the new surface layer protein and the interaction between L. kefiri and S. cerevisiae., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

While they have many properties of interest in enology, the structure-function relationships of mannoproteins and the part played by their polysaccharide moiety are not yet well understood. Mannoproteins (MP) extracted with β-glucanase from a laboratory yeast strain (WT), two of its mutants (Mnn2 with unbranched N-glycosylated chains and Mnn4 without mannosyl-phosphorylation), and an enological strain (Com) were purified and thoroughly characterized. The protein moiety of the four MPs had the same amino acid composition. Glycosyl-linkage and net charge analyses confirmed the expected differences in mutant strain MPs. MP-Com had the highest mannose/glucose ratio followed by MP-WT/MP-Mnn4, and MP-Mnn2 (13.5 > 5.6 ≈ 5.2 > 2.2). The molar mass dependencies of R g , R h , and [η], determined through HPSEC-MALLS-QELS-Viscosimetry, revealed specific conformational properties of mannoproteins related to their nature of highly branched copolymers with two branching levels. It also clearly showed structural differences between MP-Com, MP-WT/Mnn4, and MP Mnn2, and differences between two populations within the four mannoproteins., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: To study the cyclic fertilin peptide effects on preimplantation human embryogenesis. Cyclic fertilin peptide reproduces the structure of the binding site of the sperm Fertilin β (also named A Disintegrin and Metalloprotease 2: ADAM2) disintegrin domain. It binds to the oocyte membrane and increases sperm-oocyte fusion index in human and fertilization rate in mouse, providing healthy pups. It also improves human oocyte maturation and chromosome segregation in meiosis I and binds to human embryo blastomeres, suggesting that it has a membrane receptor., Design: Thawed human embryos at the 3 to 4 cells stage were randomly included in a dose-response study with cyclic fertilin peptide. Inner cell mass (ICM), trophectoderm (TE), and total cell numbers were evaluated in top- and good-quality blastocysts., Setting: The study was performed in an academic hospital and research laboratory., Patient(s): Human embryos donated for research. This project was approved by the French "Agence de la Biomédecine.", Intervention(s): Immunofluorescence and tissue-specific gene expression analysis, using Clariom D microarrays, were performed to study its mechanism of action., Main Outcome Measure(s): Cyclic fertilin peptide improves blastocyst formation by almost 20%, the concentration of 1 μM being the lowest most efficient concentration. It significantly increases twice the TE cell number, without modifying the ICM. It increases the in vitro hatching rate from 14% to 45%., Result(s): Cyclic fertilin peptide stimulates TE growth. In the ICM, it induces transcriptional activation of intracellular protein and vesicle-mediated transport., Conclusion(s): Cyclic fertilin peptide dramatically improves human embryo development potential. It could be used to supplement culture medium and improve the in vitro human embryo development. Starting supplementation immediately after fertilization, instead of day 2, could significantly upgrade assisted reproductive technology outcome., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic inflammatory diseases and transplant rejection represent major challenges for modern health care. Thus, identification of immune checkpoints that contribute to resolution of inflammation is key to developing novel therapeutic agents for those conditions. In recent years, the CD83 (cluster of differentiation 83) protein has emerged as an interesting potential candidate for such a "pro-resolution" therapy. This molecule occurs in a membrane-bound and a soluble isoform (mCD83 and sCD83, respectively), both of which are involved in resolution of inflammation. Originally described as a maturation marker on dendritic cells (DCs), mCD83 is also expressed by activated B and T cells as well as regulatory T cells (Tregs) and controls turnover of MHC II molecules in the thymus, and thereby positive selection of CD4 + T cells. Additionally, it serves to confine overshooting (auto-)immune responses. Consequently, animals with a conditional deletion of CD83 in DCs or regulatory T cells suffer from impaired resolution of inflammation. Pro-resolving effects of sCD83 became evident in pre-clinical autoimmune and transplantation models, where application of sCD83 reduced disease symptoms and enhanced allograft survival, respectively. Here, we summarize recent advances regarding CD83-mediated resolution of inflammatory responses, its binding partners as well as induced signaling pathways, and emphasize its therapeutic potential for future clinical trials.

Disorders in bile acid transport and metabolism have been related to a number of metabolic disease states, atherosclerosis, type-II diabetes, and cancer. Bile acid-binding proteins (BABPs), a subfamily of intracellular lipid-binding proteins (iLBPs), have a key role in the cellular trafficking and metabolic targeting of bile salts. Within the family of iLBPs, BABPs exhibit unique binding properties including positive binding cooperativity and site-selectivity, which in different tissues and organisms appears to be tailored to the local bile salt pool. Structural and biophysical studies of the past two decades have shed light on the mechanism of bile salt binding at the atomic level, providing us with a mechanistic picture of ligand entry and release, and the communication between the binding sites. In this review, we discuss the emerging view of bile salt recognition in intestinal- and liver-BABPs, with examples from both mammalian and non-mammalian species. The structural and dynamic determinants of the BABP-bile-salt interaction reviewed herein set the basis for the design and development of drug candidates targeting the transcellular traffic of bile salts in enterocytes and hepatocytes.

TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its overexpression being associated with familial frontotemporal lobar degeneration, and point mutation linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV-2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a nonstandard way to show that the lumenal portion of TMEM106B is a member of the late embryogenesis abundant-2 (LEA-2) domain superfamily. More sensitive tools (HMMER, HHpred, and trRosetta) extended this to predict LEA-2 domains in two yeast proteins. One is Vac7, a regulator of PI(3,5)P 2 production in the degradative vacuole, equivalent to the lysosome, which has a LEA-2 domain in its lumenal domain. The other is Tag1, another vacuolar protein, which signals to terminate autophagy and has three LEA-2 domains in its lumenal domain. Further analysis of LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid-binding groove. This implies that TMEM106B, Vac7, and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles., (© 2021 The Author. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

The purpose of this study was to screen the method with the highest yield of mannoprotein (MP) without damaging their functional structure. The recovery rates of the extracts, protein, and mannose were determined along with the mannose/protein ratio, molecular weight and distribution, monosaccharide and amino acid compositions, and secondary structures of the three MP extracts to compare the extraction methods. The MP extracts recovery rate prepared by the thermal method was significantly higher (35.89%) than those obtained using fermentation (31.66%) and SDS treatment (19.77%). Three protein bands with similar molecular weights of 59, 47, and 34 kDa were detected in the MPs obtained via the different extraction methods. The thermally extracted MP has a broader molecular weight distribution. After purification, the proportion of mannose in the polysaccharide parts of the MPs increased from 6-7 to 90.4-91.3%. The essential amino acid content of the hot-extracted MP (170.07 mg/g) was the highest. The thermally extracted MP had similar secondary structural characteristics to that isolated at room temperature, and had a higher protein characteristic peak intensity. In general, the heating method to extract yeast mannoprotein is time-saving and efficient., (Copyright © 2021 Elsevier B.V. All rights reserved.)