BACKGROUND:Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1.OBJECTIVES:To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction.METHODS:A series of recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen-deuterium exchange mass spectrometry, and in silico.RESULTS:Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII-binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII.CONCLUSIONS:FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non-canonical binding combinations. We propose that FVIII-LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low-density lipoprotein receptor family members including LRP1.

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.

Marakasova and Baranova make a good, incremental step trying to explain the results of our paper, "Analysis of Measles-Mumps-Rubella (MMR) Titers of Recovered COVID-19 Patients" (1). We believe the predictable waning of mumps IgG titers over time allows mumps titers to serve as a proxy measure of overall MMR II vaccine persistence. Even though we observed a significant inverse correlation only with mumps virus titers, any of the components of MMR II alone or in tandem, including measles as Marakasova and Baranova suggest, could be responsible for what we observed. They present a conventional approach to matching protein sequence and structure to structures recognized by antibodies generated during and after vaccination or disease.

Background Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated. Objective To isolate and characterize the FVIIIFT fraction in rFVIII products. Methods Protein fractions unable (FVIIIFT) and able (FVIIIEL) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results and Conclusions A robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%-21.5%) in all products. Compared with FVIIIEL, FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low-density lipoprotein receptor-related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.

Francisella tularensis is a Gram-negative bacterium that causes the zoonotic disease tularemia. The historical development of tularemia as a biological weapon has led to it being characterized by the CDC as a category A biothreat agent. Neither posttranslational modification (PTM) of proteins, in particular lysine acetylation, in Francisella nor its subsequent regulation of the protein activity has been well studied. In this work, we analyze N-ε-lysine acetylation of the F. tularensis ssp. novicida proteome by mass spectrometry for the first time. To create a comprehensive acetylation profile, we enriched protein acetylation using two approaches: (1) the addition of glucose or acetate into the culture medium and (2) direct chemical acetylation of N-ε-lysines with acetyl phosphate. We discovered 280 acetylated proteins with 1178 acetylation sites in the F. tularensis ssp. novicida strain U112. Lysine acetylation is an important PTM that regulates multiple cellular processes in bacteria, including metabolism, transcription, translation, stress response, and protein folding. We discovered that Francisella chitinases A and B are acetylated naturally and when chemically induced by acetyl phosphate. Moreover, chemical overacetylation of chitinases results in silencing of the enzymatic activity. Our findings suggest a novel mechanism of posttranslational regulation of the chitinase activity and that acetylation may play a role in Francisella's regulation of the protein activity.

Intracellular bacteria were recently shown to employ eukaryotic prenylation system for modifying activity and ensuring proper intracellular localization of their own proteins. Following the same logic, the proteins of viruses may also serve as prenylation substrates. Using extensively validated high-confidence prenylation predictions by PrePS with a cut-off for experimentally confirmed farnesylation of hepatitis delta virus antigen, we compiled in silico evidence for several new prenylation candidates, including IRL9 (CMV) and few other proteins encoded by Herpesviridae, Nef (HIV-1), E1A (human adenovirus 1), NS5A (HCV), PB2 (influenza), HN (human parainfluenza virus 3), L83L (African swine fever), MC155R (molluscum contagiosum virus), other Poxviridae proteins, and some bacteriophages of human associated bacteria. If confirmed experimentally, these findings may aid in dissection of molecular functions of uncharacterized viral proteins and provide a novel rationale for statin and FT/GGT1-based inhibition of viral infections. Prenylation of bacteriophage proteins may aid in moderation of microbial infections.

Туб и В1 лимфоциты являются важнейшими компонентами мукозальной иммунной системы, отличающиеся способностью усвоения бактериальных и вирусных лигандов, без костимулирующих сигналов и предварительного процессинга другими эффекторами иммунитета. Это обстоятельство обеспечивает быструю защиту от различных патогенов и вносит свой вклад в расшифровку механизма меньшей сенсибилизирующей активности антигенов при мукозальном введении, поскольку при взаимодействии этих клеток экспрессируются IgM и IgA антитела, но не IgE. В исследованиях подтверждено положение о том, что происходит интенсивный обмен лимфоцитами не только между лимфоидными образованиями, ассоциированными со слизистыми оболочками респираторного и желудочно-кишечного трактов, но и с селезенкой, что обеспечивает при мукозальных методах иммунизации развитие не только местного, но и системного иммунитета.

AIM:Study the production of cytokines in mice during vaccination with polycomponent Immunovac-VP-4 vaccine containing TLR ligands with various administration methods.MATERIALS AND METHODS:Immunovac-VP-4 was administered to mice by subcutaneous, intranasal or per oral methods. The preparation was administered nasally at a single dose of 500 microg in the volume of 30 microl. Per oral single dose was 2000 microg in the volume of 0.5 ml. 200 microg of the preparation was administered subcutaneously. Cytokines in blood sera were determined by EIA 8 hours after the administration of the vaccine.RESULTS:In mice 8 hours afterthe single administration of Immunovac-VP-4 the levels of IL-1beta, IL-6, IL- 12, IL-5 increased significantly. However their concentration differed depending on the method of administration. The most active expression of cytokines was observed during subcutaneous administration. The indexes of cytokine expression were significantly higher (p < 0.05) than during non-parenteral administration methods.CONCLUSION:Mucosal application methods along with parenteral were established to be able to activate effector mechanisms of immune repose with its consequent polarization by Th1/Th2 pathways. These mechanisms lay the groundwork for development of antigen-specific immune responses against antigens/pathogens.

Biofilms, multicellular communities of bacteria, may be an environmental survival and transmission mechanism of Francisella tularensis. Chitinases of F. tularensis ssp. novicida (Fn) have been suggested to regulate biofilm formation on chitin surfaces. However, the underlying mechanisms of how chitinases may regulate biofilm formation are not fully determined. We hypothesized that Fn chitinase modulates bacterial surface properties resulting in the alteration of biofilm formation. We analyzed biofilm formation under diverse conditions using chitinase mutants and their counterpart parental strain. Substratum surface charges affected biofilm formation and initial attachments. Biophysical analysis of bacterial surfaces confirmed that the chi mutants had a net negative-charge. Lectin binding assays suggest that chitinase cleavage of its substrates could have exposed the concanavalin A-binding epitope. Fn biofilm was sensitive to chitinase, proteinase and DNase, suggesting that Fn biofilm contains exopolysaccharides, proteins and extracellular DNA. Exogenous chitinase increased the drug susceptibility of Fn biofilms to gentamicin while decreasing the amount of biofilm. In addition, chitinase modulated bacterial adhesion and invasion of A549 and J774A.1 cells as well as intracellular bacterial replication. Our results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties. Our findings position chitinase as a potential anti-biofilm enzyme in Francisella species.

For their protection from host cell immune defense, intracellular pathogens of eukaryotic cells developed a variety of mechanisms, including secretion systems III and IV which can inject bacterial effectors directly into eukaryotic cells. These effectors may function inside the host cell and may be posttranslationally modified by host cell machinery. Recently, prenylation was added to the list of possible posttranslational modifications of bacterial proteins. In this work we describe the current state of the knowledge about the prenylation of eukaryotic and prokaryotic proteins and prenylation inhibitors. The bioinformatics analyses suggest the possibility of prenylation for a number of Francisella genus proteins.

Существующие методы исследования гликозилирования белков остаются громоздкими. В своем развитии они отстают от высокопроизводительных протеомных технологий. Существует реальная потребность в разработке простого, недорогого и специфичного метода выявления сайтов гликозилирования в белках, совместимого с общепринятыми процедурами подготовки биологического материала к протеомному и фосфопротеомному анализу. В статье описан новый метод дегликозилирования гликопротеинов с помощью бета-элиминирования с последующим введением ковалентной метки и визуализацией паттерна первично негликозилированных и дегликозилированных белков в предобработанном клеточном гомогенате с использованием J-агрегатов, образующихся в геле in situ. Новый метод подготовки материала к протеомному и фосфопротеомному анализу может быть использован при проведении фосфопротеомного анализа в формате RPMA для регистрации изменения фосфорилирования белкового состава клеток, в том числе в условиях развития вирусной инфекции.

The KCTD family includes tetramerization (T1) domain containing proteins with diverse biological effects. We identified a novel member of the KCTD family, BTBD10. A comprehensive analysis of protein-protein interactions (PPIs) allowed us to put forth a number of testable hypotheses concerning the biological functions for individual KCTD proteins. In particular, we predict that KCTD20 participates in the AKT-mTOR-p70 S6k signaling cascade, KCTD5 plays a role in cytokinesis in a NEK6 and ch-TOG-dependent manner, KCTD10 regulates the RhoA/RhoB pathway. Developmental regulator KCTD15 represses AP-2α and contributes to energy homeostasis by suppressing early adipogenesis. TNFAIP1-like KCTD proteins may participate in post-replication DNA repair through PCNA ubiquitination. KCTD12 may suppress the proliferation of gastrointestinal cells through interference with GABAb signaling. KCTD9 deserves experimental attention as the only eukaryotic protein with a DNA-like pentapeptide repeat domain. The value of manual curation of PPIs and analysis of existing high-throughput data should not be underestimated.

High fuel costs have encouraged producers of greenhouse tomato (Solanum lycopersicum L.) in the mid-Atlantic region to reduce air temperatures during the day. However, effects on fruit ripening and yield are not known, especially under the low light conditions found in off-season production. This 2-yr study compared fruit ripening and yield of tomato under two temperature regimes during the fall season. Two sets of 18 tomato plants, three rows of six, were grown in soilless culture under either a warm or cool temperature regime. Temperatures were similar during night hours but allowed to rise to at least 2124 oC in the cool greenhouse section and 23-26 oC in the warm section, depending on daily solar heating. Mean 24 hour temperature difference between zones was less than 2 oC. Ripe tomato fruit were harvested and weighed 3 times per week for 8 weeks and the remaining un-ripened green tomatoes were weighed at the termination of the experiment to obtain total fruit biomass. The warm zone produced significantly greater weight of ripe tomatoes (23%) than the cool zone. However, total fruit weight (ripe and green), was not significantly different. Thus, a relatively small increase in temperature (2 oC) during the mid-day was associated with a significant increase in fruit ripening but not in total fruit weight. This study showed that greenhouse temperature could be used to better manage fruit production to match weekly market demand without affecting total fruit weight and that consistently maintaining a cool greenhouse would delay tomato ripening and likely increase the potential for plant stress due to high fruit loads remaining on the vines.

Внутриклеточные паразиты эукариот выработали множество механизмов для защиты от активной среды и внутриклеточных компонентов клетки хозяина, ограничивающих микробные инфекции. В частности, это компоненты так называемых систем секреции прокариотических белков III и IV, способные обеспечивать проникновение эффекторных молекул, производимых микроорганизмами, непосредственно в эукариотическую клетку. Многие из этих белков проходят посттрансляционную модификацию с использованием ферментов клетки хозяина и имитируют функции белков эукариот. Предполагают, что одной из таких посттрансляционных модификаций является пренилирование, которое заключается в присоединении липофильной изопренильной группировки к синтезирующемуся полипептиду. В обзоре представлены современные данные о механизмах пренилирования на примере эукариотических и прокариотических белков, описаны их функции, рассмотрены ингибиторы пренилирования и механизм их действия, а также белки бактерий рода Francisella как вероятные мишени пренилирования.

This review is dedicated to structural and functional properties of IgA1-proteases in pathogenic microorganisms. The interest to structural and functional studies of IgA-proteases is caused essentially by their putatively great impact on set up and progression of infectious diseases. IgA degradation at mucosal surfaces is commonly suggested to be the main function of IgA1-proteases. In turn, this effect may lead to abatement of the immune response efficiency at the epithelial barrier. In our review, structural comparison of “classic” IgA1-proteases with their functional analogues and structural homologues allows to hypothesize IgA-protease dependent effector mechanisms mediated by serum IgA.

В статье приведены результаты по созданию и испытанию биологической активности модельных штаммов E. coli, экспрессирующих полусинтетические гены соматолиберина курицы и свиньи. Отличием испытанных штаммов от ранее описанных аналогов является то, что ген предшественника соматолиберина разделен на фрагменты, кодирующие истинные пептидные гормоны GHRH и PACAP. Таким образом, впервые получены данные о физиологическом действии этих гормонов на животных моделях при пероральной доставке. Впервые показана возможность тестирования GHRH курицы на модели мышат.