Osipov, Stepan D

Osipov S.D., Ryzhykau Y.L., Zinovev E.V., Minaeva A.V., Ivashchenko S.D., Verteletskiy D.P., Sudarev V.V., Kuklina D.D., Nikolaev M.Y., Semenov Y.S., Zagryadskaya Y.A., Okhrimenko I.S., Gette M.S., Dronova E.A., Shishkin A.Y., et. al.

F-type ATP synthases play a key role in oxidative and photophosphorylation processes generating adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial FOF1-ATP synthases, those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light on the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized FOF1-ATP synthases from spinach chloroplasts at different ionic strengths. The structural data were obtained by SAXS and demonstrated dimerization in response to ionic strength. The best model describing SAXS data was two ATP-synthases connected through F1/F1′ parts, presumably via their δ-subunits, forming “I” shape dimers. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the FOF1 monomers comprising such dimers are embedded in parallel opposing stacked thylakoid membrane areas. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast FOF1-ATP synthase for preventing ATP hydrolysis in the dark, when ionic strength in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast FOF1 and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts.

Sudarev V.V., Dolotova S.M., Bukhalovich S.M., Bazhenov S.V., Ryzhykau Y.L., Uversky V.N., Bondarev N.A., Osipov S.D., Mikhailov A.E., Kuklina D.D., Murugova T.N., Manukhov I.V., Rogachev A.V., Gordeliy V.I., Gushchin I.Y., et. al.

Ferritin is a vital protein complex responsible for storing iron in almost all living organisms. It plays a crucial role in various metabolic pathways, inflammation processes, stress response, and pathogenesis of cancer and neurodegenerative diseases. In this review we discuss the role of ferritin in diseases, cellular iron regulation, its structural features, and its role in biotechnology. We also show that molecular mechanisms of ferritin self-assembly are key for a number of biotechnological and pharmaceutical applications. The assembly pathways strongly depend on the interface context of ferritin monomers and the stability of its different intermediate oligomers. To date, several schemes of self-assembly kinetics have been proposed. Here, we compare different self-assembly mechanisms and discuss the possibility of self-assembly control by switching between deadlock intermediate states.

Vlasov A.V., Osipov S.D., Bondarev N.A., Uversky V.N., Borshchevskiy V.I., Yanyushin M.F., Manukhov I.V., Rogachev A.V., Vlasova A.D., Ilyinsky N.S., Kuklin A.I., Dencher N.A., Gordeliy V.I.

ATP synthases are unique rotatory molecular machines that supply biochemical reactions with adenosine triphosphate (ATP)—the universal “currency”, which cells use for synthesis of vital molecules and sustaining life. ATP synthases of F-type (FOF1) are found embedded in bacterial cellular membrane, in thylakoid membranes of chloroplasts, and in mitochondrial inner membranes in eukaryotes. The main functions of ATP synthases are control of the ATP synthesis and transmembrane potential. Although the key subunits of the enzyme remain highly conserved, subunit composition and structural organization of ATP synthases and their assemblies are significantly different. In addition, there are hypotheses that the enzyme might be involved in the formation of the mitochondrial permeability transition pore and play a role in regulation of the cell death processes. Dysfunctions of this enzyme lead to numerous severe disorders with high fatality levels. In our review, we focus on FOF1-structure-based approach towards development of new therapies by using FOF1 structural features inherited by the representatives of this enzyme family from different taxonomy groups. We analyzed and systematized the most relevant information about the structural organization of FOF1 to discuss how this approach might help in the development of new therapies targeting ATP synthases and design tools for cellular bioenergetics control.

Vlasov A.V., Maliar N.L., Bazhenov S.V., Nikelshparg E.I., Brazhe N.A., Vlasova A.D., Osipov S.D., Sudarev V.V., Ryzhykau Y.L., Bogorodskiy A.O., Zinovev E.V., Rogachev A.V., Manukhov I.V., Borshchevskiy V.I., Kuklin A.I., et. al.

This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed.

Sudaraka Tennakoon M.S., Lee K., Shin H.

Yang R., Chen R., Li R., Liang L., Zhang Y.

Jiskani S.A.

Liao Y., Zeng T., Guo X., Li X.

Liu Y., Chen S., Xiong X., Wen Z., Zhao L., Xu B., Guo Q., Xia J., Pei J.

Gette M.S., Sudarev V.V., Osipov S.D., Laptenkova E.V., Bazhenov S.V., Zagryadskaya Y.A., Tilinova O.M., Dronova E.A., Kuklina D.D., Al Ebrahim R., Fedorov D.M., Kurkin T.S., Semenov Y.S., Bondarev N.A., Skoi V.V., et. al.

Wang Q., Zheng H., Yang Y., Chang X., Du Z., Hang Z., Li Z., Liao Z.

BackgroundRecurrent acute pancreatitis (RAP) poses significant clinical challenges, with 32.3% developing to chronic pancreatitis within 5 years. The underlying microbial factors contributing to RAP remain poorly understood. This study aims to identify blood microbial signatures associated with RAP and explore the potential microbial predictors for RAP.MethodsIn this prospective cohort, 90 acute pancreatitis patients are classified into non-recurrent acute pancreatitis (NRAP, n=68) and RAP (n=22) groups based on the number of pancreatitis episodes. Microbial composition of blood samples is analyzed using 5-region (5R) 16S rRNA gene sequencing. Key microbial taxa and functional predictions are made. A random forest model is used to assess the predictive value of microbial features for RAP. The impact of Staphylococcus hominis (S. hominis) on RAP is further evaluated in an experimental mouse model.ResultsLinear discriminant analysis effect size (LEfSe) analysis highlights significant microbial differences, with Paracoccus aminovorans, Corynebacterium glucuronolyticum and S. hominis being prominent in RAP. Functional predictions indicate enrichment of metabolic pathways in the RAP group. Random forest analysis identifies key microbial taxa with an AUC value of 0.759 for predicting RAP. Experimental validation shows that S. hominis exacerbates pancreatic inflammation in mice.ConclusionsThis study identifies distinct clinical and microbial features associated with RAP, emphasizing the role of specific bacterial taxa in pancreatitis recurrence. The findings suggest that microbial profiling could enhance the diagnosis and management of RAP, paving the way for personalized therapeutic approaches.

Xu W., Wen X., Fu Y., Yang J., Cui H., Fan R.

Nuclear receptor coactive 4 (NCOA4) is a specific receptor for ferritinophagy, transporting ferritin to lysosomal degradation, releasing free iron, and excessive iron levels may lead to cellular redox imbalance, contributing to cell death, predominantly ferroptosis. NCOA4 is regulated by a variety of transcriptional, post-transcriptional, translational, and post-translational modifications. Targeted modulation of NCOA4-mediated ferritinophagy has been successfully used as a therapeutic strategy in several disease models. Recent evidences have elucidated that ferritinophagy and ferroptosis played a major role in heavy metals toxicity. In this review, we explored the regulatory mechanism of NCOA4 as the sole receptor for ferritinophagy from multiple perspectives based on previous studies. The significant role of ferritinophagy-mediated ferroptosis in heavy metals toxicity was discussed in detail, emphasizing the great potential of NCOA4 as a target for heavy metals toxicity.

Anagnostou G., Ferragina A., Crofton E.C., Frias Celayeta J.M., Hamill R.M.

The sensory quality of meat, encompassing the traits of appearance, texture, and flavour, is essential to consumer acceptance. Conventional quality assessment techniques, such as instrumental methods and trained sensory panels, often face limitations due to their destructive and time-consuming nature. In recent years, optical sensing techniques have emerged as a fast, non-invasive, and non-destructive technique for the prediction of quality attributes in meat and meat products, achieving prediction accuracies of over 90%. This review critically examines the potential of optical sensing techniques, such as near-infrared spectroscopy (NIRS), Raman spectroscopy, and hyperspectral imaging (HSI), to inform about the sensory attributes of red meat, aligning with industrial demands for early information on the predicted sensory performance of inventory to support meeting consumer requirements. Recent trends and the remaining challenges associated with these techniques will be described. While technical issues related to spectral data acquisition and data processing are important challenges when considering industrial implementation, overall, optical sensing techniques, in tandem with recent developments in digitalisation and data analytics, provide potential for the online prediction of meat sensory quality in the meat processing industries. Establishing technologies for enhanced information on the product and improved possibilities for quality control will help the industry to meet consumer demands for a consistent quality of product.

Zhang B., Fan K.

Amrita, Chakraborti S., Dey S.

Xie Z., Peng B., Shi M., Yang G., Song Z.

In plants, Ferritin is the earliest discovered regulator of iron (Fe) metabolism and plays a critical role in maintaining Fe storage and sequestration, which contributes to cellular Fe homeostasis and tolerance to abiotic stresses. However, biological functions of Ferritin proteins in perennial fruit crops are largely rare. In this study, VvFerritin1 was isolated from ‘Irsay Oliver’ table grape, and it was mainly expressed in roots and induced under Fe toxicity, H2O2 stress, and abscisic acid (ABA) treatment. Complementation of VvFerritin2 in yeast mutant DEY1453 directly restored the mutant growth, and VvFerritin1 can transport Fe2+ in yeast. The heterologous expression of VvFerritin1 in fer1-2 mutant effectively rescued the dwarfed growth of Arabidopsis fer1-2 mutant, under the control condition, Fe toxicity, or H2O2 stress, embodied in enhanced fresh weight (126%, 81%, or 48%), total root length (140%, 98%, or 64%), total root surface (70%, 84%, or 120%), and total leaf chlorophyll (56%, 51%, or 53%), respectively. In particular, tissue Fe concentration and activities of nitrite reductase (NiR), aconitase (ACO), and succinate dehydrogenase (SDH) were significantly enhanced in fer1-2/35S::Ferritin1 lines, respectively, compared to that of fer1-2 mutant. This work contributes to the study of molecular mechanisms of Fe storage and homeostasis in ‘Irsay Oliver’ table grape.

Matveeva V.O., Grebennikova A.D., Sakharov D.I., Fomin V.V., Manukhov I.V., Bazhenov S.V.

AbstractFerritin FtnA is the main scavenger of Fe2+and storage of Fe3+in bacterial cells, together with Dps and Bfr preventing the Fenton reaction and thus protecting the cell from iron-induced oxidative stress. However, until now, it was not known how its expression is regulated under conditions of oxidative stress, and the available evidence was contradictory. To study the regulation ofE. coli ftnAexpression in response to oxidative stress, PftnA-luxCDABEtranscriptional fusion in different strains was used. It has been shown that PftnAis induced after the addition of oxidative stress inducers. The maximum amplitude of this activation did not depend on the presence of functional genesoxyRandsoxRin the cell, but completely disappeared in the absence offur. The response is amplified in theftnAmutant and is diminished in the FtnA-overproducing strain, which indicates that iron sequestration blocks the response. Exposure of a cell to H2O2initially inactivates Fur and a number of iron-utilizing proteins, and derepresses iron uptake. This results in an increase in the cellular iron content with the consequent Fur reactivation, which leads to the induction offtnAexpression. Thus, oxidative stress leads to PftnAactivation, which is mediated by Fur and time-delayed in comparison with OxyR-response.

Tang Y., Zhang Q., Chen H., Chen G., Li Z., Chen G., Lin L., Yu Z., Su M., Zhang B.

Li Y., Liu R., Zhao Z.

Brain diseases pose significant treatment challenges due to the restrictive nature of the blood–brain barrier (BBB). Recent advances in targeting macromolecules offer promising avenues for overcoming these obstacles through receptor-mediated transcytosis (RMT). We summarize the current progress in targeting brain drug delivery with macromolecules for brain diseases. This exploration details the transport mechanisms across the BBB, focusing on RMT and its use of natural ligands for drug delivery. Furthermore, the review examines macromolecular ligands such as antibodies, peptides, and aptamers that leverage RMT for effective BBB traversal. Advancements in macromolecules-based delivery systems for brain diseases are summarized, emphasizing their therapeutic potential and limitations. Finally, emerging RMT strategies, including viral vectors, exosomes, and boron neutron capture therapy, are discussed for their precision in brain-targeted treatments. This comprehensive overview underscores the potential of RMT-based approaches to revolutionize brain disease therapy.

Dayhoff G.W., Uversky V.N.

Protein intrinsic disorder is found in all kingdoms of life and is known to underpin numerous physiological and pathological processes. Computational methods play an important role in characterizing and identifying intrinsically disordered proteins and protein regions. Herein, we present a new high-efficiency web-based disorder predictor named Rapid Intrinsic Disorder Analysis Online (RIDAO) that is designed to facilitate the application of protein intrinsic disorder analysis in genome-scale structural bioinformatics and comparative genomics/proteomics. RIDAO integrates six established disorder predictors into a single, unified platform that reproduces the results of individual predictors with near-perfect fidelity. To demonstrate the potential applications, we construct a test set containing more than one million sequences from one hundred organisms comprising over 420 million residues. Using this test set, we compare the efficiency and accessibility (i.e. ease of use) of RIDAO to five well-known and popular disorder predictors, namely: AUCpreD, IUPred3, metapredict V2, flDPnn, and SPOT-Disorder2. We show that RIDAO yields per-residue predictions at a rate two to six orders of magnitude greater than the other predictors and completely processes the test set in under an hour. RIDAO can be accessed free of charge at https://ridao.app. This article is protected by copyright. All rights reserved.

Wang W., Liu X., Wang Y., Wang Y., Fu D., Xi H., Zhao Y., Wang H.

Ferritins can generally be divided into four subfamilies based on their structural characteristics, namely, the classic ferritins (Ftns), bacterioferritins (Bfrs), DNA-binding proteins from starved cells (Dps’), and encapsulated ferritins (EncFtns). However, the ferritin from Mycoplasma penetrans (Mpef) possesses a particular ferroxidase center with an extreme low activity and exhibits unusual characteristics, indicating that it could be a member of a quite different subfamily of ferritins. Hereby, the crystal structure of the ferritin from Ureaplasma urealyticum (Uurf) is presented, Mpef and Uurf have very similar properties, though they display very low sequence similarity. Thus, ferritins from Mycoplasma with these unique properties do not belong to any known subfamily, but they should rather be placed in a novel ferritin subfamily, which we term Mycoplasma Ferritin (Mfr). Ferritin from Mycoplasma represents some unique characteristics, including a unique ferroxidase center and much lower iron oxidation activity, as well as the channel for iron passthrough. Therefore, it should be classified into a new ferritin subfamily, Mycoplasma ferritin (Mfr).

Tsoraev G.V., Protasova E.A., Klimanova E.A., Ryzhykau Y.L., Kuklin A.I., Semenov Y.S., Ge B., Li W., Qin S., Friedrich T., Sluchanko N.N., Maksimov E.G.

The structural organization of natural pigment-protein complexes provides a specific environment for the chromophore groups. Yet, proteins are inherently dynamic and conformationally mobile. In this work, we demonstrate the heterogeneity of chromophores of C-phycocyanin (C-PC) from Arthrospira platensis. Part of the population of trimeric C-PC is subject to spontaneous disturbances of protein–protein interactions resulting in increased conformational mobility of the chromophores. Upon fluorescence excitation in the visible range, the spectral signatures of these poorly populated states are masked by bulk chromophore states, but the former could be clearly discriminated when the fluorescence is excited by near-infrared quanta. Such selective excitation of conformationally mobile C-PC chromophores is due to the structure of their S1 level, which is characterized by a significantly broadened spectral line. We demonstrate that the anti-Stokes C-PC fluorescence is the result of single-photon absorption. By combining spectral and structural methods, we characterize four distinct states of C-PC chromophores emitting at 620, 650, 665, and 720 nm and assigned the fast component in the anti-Stokes fluorescence decay kinetics in the range of 690–750 nm to the chromophores with increased conformational mobility. Our data suggest that the spectral and temporal characteristics of the anti-Stokes fluorescence can be used to study protein dynamics and develop methods to visualize local environment parameters such as temperature.

Kurakin S., Ivankov O., Skoi V., Kuklin A., Uhríková D., Kučerka N.

Combining small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and densitometric measurements, we have studied the interactions of the divalent cations Ca2+ and Mg2+ with the lipid vesicles prepared of a mixed-chain palmitoyl-oleoyl-phosphatidylcholine (POPC) at 25°C. The structural parameters of the POPC bilayer, such as the bilayer thickness, lateral area, and volume per lipid, displayed no changes upon the ion addition at concentrations up to 30 mM and minor changes at > 30 mM Ca2+ and Mg2+, while some decrease in the vesicle radius was observed over the entire concentration range studied. This examination allows us to validate the concept of lipid–ion interactions governed by the area per lipid suggested previously and to propose the mixed mode of those interactions that emerge in the POPC vesicles. We speculate that the average area per POPC lipid that corresponds to the cutoff length of lipid–ion interactions generates an equal but opposite impact on ion bridges and separate lipid–ion pairs. As a result of the dynamic equilibrium, the overall structural properties of bilayers are not affected. As the molecular mechanism proposed is affected by the structural properties of a particular lipid, it might help us to understand the fundamentals of processes occurring in complex multicomponent membrane systems.

Murugova T.N., Ivankov O.I., Ryzhykau Y.L., Soloviov D.V., Kovalev K.V., Skachkova D.V., Round A., Baeken C., Ishchenko A.V., Volkov O.A., Rogachev A.V., Vlasov A.V., Kuklin A.I., Gordeliy V.I.

AbstractDespite remarkable progress, mainly due to the development of LCP and ‘bicelle’ crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the “bicelle” crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of “bicelle” MP crystallization and will support rational design of crystallization.

Yin S., Davey K., Dai S., Liu Y., Bi J.

Ferritin stores and releases iron ions in mammals. It is globally important as a drug nanocarrier. This is because of its unique hollow-spherical structure, desirable stability and biological properties. Novel drug-loading approaches plus various functionalization approaches have been developed to improve ferritin in response to differing demands in disease treatments. Here, we critically review ferritin drug delivery and evaluate its diverse drug-loading and functionalization approaches, we: (1) Introduce basic structural and property information related to ferritin as a drug nanocarrier; (2) Contrast in detail the different means to load drugs and the selection of drug loading means; (3) Discuss multiple ferritin functionalization approaches, together with related advantages and potential risks; and, (4) Compare ferritin with alternative, commonly-used drug nanocarriers. We conclude that despite that no drugs based on ferritin are commercially available, the market potential for it is significant, and evaluate future research directions. Findings from this work will be of immediate benefit and interest to a wide range of researchers and manufacturers for drug delivery using ferritin.

Vlasov A.V., Osipov S.D., Bondarev N.A., Uversky V.N., Borshchevskiy V.I., Yanyushin M.F., Manukhov I.V., Rogachev A.V., Vlasova A.D., Ilyinsky N.S., Kuklin A.I., Dencher N.A., Gordeliy V.I.

ATP synthases are unique rotatory molecular machines that supply biochemical reactions with adenosine triphosphate (ATP)—the universal “currency”, which cells use for synthesis of vital molecules and sustaining life. ATP synthases of F-type (FOF1) are found embedded in bacterial cellular membrane, in thylakoid membranes of chloroplasts, and in mitochondrial inner membranes in eukaryotes. The main functions of ATP synthases are control of the ATP synthesis and transmembrane potential. Although the key subunits of the enzyme remain highly conserved, subunit composition and structural organization of ATP synthases and their assemblies are significantly different. In addition, there are hypotheses that the enzyme might be involved in the formation of the mitochondrial permeability transition pore and play a role in regulation of the cell death processes. Dysfunctions of this enzyme lead to numerous severe disorders with high fatality levels. In our review, we focus on FOF1-structure-based approach towards development of new therapies by using FOF1 structural features inherited by the representatives of this enzyme family from different taxonomy groups. We analyzed and systematized the most relevant information about the structural organization of FOF1 to discuss how this approach might help in the development of new therapies targeting ATP synthases and design tools for cellular bioenergetics control.

Joyce M.G., King H.A., Elakhal-Naouar I., Ahmed A., Peachman K.K., Macedo Cincotta C., Subra C., Chen R.E., Thomas P.V., Chen W., Sankhala R.S., Hajduczki A., Martinez E.J., Peterson C.E., Chang W.C., et. al.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019. We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50-μg) SpFN vaccine, given twice 28 days apart, induced a T helper cell 1 (T H 1)–biased CD4 T H response and elicited neutralizing antibodies against SARS-CoV-2 wild type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates after high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates support the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.

Golub M., Lokstein H., Soloviov D., Kuklin A., Wieland D.C., Pieper J.

The high-resolution crystal structure of the trimeric major light-harvesting complex of photosystem II (LHCII) is often perceived as the basis for understanding its light-harvesting and photoprotective functions. However, the LHCII solution structure and its oligomerization or aggregation state may generally differ from the crystal structure and, moreover, also depend on its functional state. In this regard, small-angle scattering experiments provide the missing link by offering structural information in aqueous solution at physiological temperatures. Herein, we use small-angle scattering to investigate the solution structures of two different preparations of solubilized LHCII employing the nonionic detergents n-octyl-β-d-glucoside (OG) and n-dodecyl-β-D-maltoside (β-DM). The data reveal that the LHCII-OG complex is equivalent to the trimeric crystal structure. Remarkably, however, we observe─for the first time─a stable oligomer composed of three LHCII trimers in the case of the LHCII-β-DM preparation, implying additional pigment-pigment interactions. The latter complex is assumed to mimic trimer-trimer interactions which play an important role in the context of photoprotective nonphotochemical quenching.

Wang B., Li S., Qiao Y., Fu Y., Nie J., Jiang S., Yao X., Pan Y., Zhao L., Wu C., Shi Y., Yin Y., Shan Y.

Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.

Joyce M.G., Chen W., Sankhala R.S., Hajduczki A., Thomas P.V., Choe M., Martinez E.J., Chang W.C., Peterson C.E., Morrison E.B., Smith C., Chen R.E., Ahmed A., Wieczorek L., Anderson A., et. al.

Summary The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

Varadi M., Anyango S., Deshpande M., Nair S., Natassia C., Yordanova G., Yuan D., Stroe O., Wood G., Laydon A., Žídek A., Green T., Tunyasuvunakool K., Petersen S., Jumper J., et. al.

Abstract The AlphaFold Protein Structure Database (AlphaFold DB, https://alphafold.ebi.ac.uk) is an openly accessible, extensive database of high-accuracy protein-structure predictions. Powered by AlphaFold v2.0 of DeepMind, it has enabled an unprecedented expansion of the structural coverage of the known protein-sequence space. AlphaFold DB provides programmatic access to and interactive visualization of predicted atomic coordinates, per-residue and pairwise model-confidence estimates and predicted aligned errors. The initial release of AlphaFold DB contains over 360,000 predicted structures across 21 model-organism proteomes, which will soon be expanded to cover most of the (over 100 million) representative sequences from the UniRef90 data set.

Wuertz K.M., Barkei E.K., Chen W., Martinez E.J., Lakhal-Naouar I., Jagodzinski L.L., Paquin-Proulx D., Gromowski G.D., Swafford I., Ganesh A., Dong M., Zeng X., Thomas P.V., Sankhala R.S., Hajduczki A., et. al.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

Ryzhykau Y.L., Vlasov A.V., Orekhov P.S., Rulev M.I., Rogachev A.V., Vlasova A.D., Kazantsev A.S., Verteletskiy D.P., Skoi V.V., Brennich M.E., Pernot P., Murugova T.N., Gordeliy V.I., Kuklin A.I.

Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is critical for the development of drugs. However, obtaining high-resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low-resolution methods small-angle X-ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small-angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane-protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII–NpHtrII) was performed. The influence of the polydispersity of NpSRII–NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.

Kryshtafovych A., Schwede T., Topf M., Fidelis K., Moult J.

Critical assessment of structure prediction (CASP) is a community experiment to advance methods of computing three-dimensional protein structure from amino acid sequence. Core components are rigorous blind testing of methods and evaluation of the results by independent assessors. In the most recent experiment (CASP14), deep-learning methods from one research group consistently delivered computed structures rivaling the corresponding experimental ones in accuracy. In this sense, the results represent a solution to the classical protein-folding problem, at least for single proteins. The models have already been shown to be capable of providing solutions for problematic crystal structures, and there are broad implications for the rest of structural biology. Other research groups also substantially improved performance. Here, we describe these results and outline some of the many implications. Other related areas of CASP, including modeling of protein complexes, structure refinement, estimation of model accuracy, and prediction of inter-residue contacts and distances, are also described.