Macromolecular tool box to elucidate CLAVATA3/EMBRYO SURROUNDING REGION-RELATED-RLK binding, signaling, and downstream effects.

Plant peptides communicate by binding to a large family of receptor-like kinases (RLKs), and they share a conserved binding mechanism, which may account for their promiscuous interaction with several RLKs. In order to understand the in vivo binding specificity of the CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptide family in Arabidopsis, we have developed a novel set of CLAVATA3 (CLV3)-based peptide tools. After carefully evaluating the CLE peptide binding characteristics, using solid phase synthesis process, we modified the CLV3 peptide and attached a fluorophore and a photoactivable side group. We observed that the labeled CLV3 shows binding specificity within the CLAVATA1 clade of RLKs while avoiding the distantly related PEP RECEPTOR clade, thus resolving the contradictory results obtained previously by many in vitro methods. Furthermore, we observed that the RLK-bound CLV3 undergoes clathrin-mediated endocytosis and is trafficked to the vacuole via ARA7 (a Rab GTPase)-labeled endosomes. Additionally, modifying CLV3 for light-controlled activation enabled spatial and temporal control over CLE signaling. Hence, our CLV3 macromolecular toolbox can be used to study rapid cell specific down-stream effects. Given the conserved binding properties, in the future our toolbox can also be used as a template to modify other CLE peptides.

Long-term evolution of human seasonal influenza virus A(H3N2) is associated with an increase in polymerase complex activity.

Since the influenza pandemic in 1968, influenza A(H3N2) viruses have become endemic. In this state, H3N2 viruses continuously evolve to overcome immune pressure as a result of prior infection or vaccination, as is evident from the accumulation of mutations in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, phylogenetic studies have also demonstrated ongoing evolution in the influenza A(H3N2) virus RNA polymerase complex genes. The RNA polymerase complex of seasonal influenza A(H3N2) viruses produces mRNA for viral protein synthesis and replicates the negative sense viral RNA genome (vRNA) through a positive sense complementary RNA intermediate (cRNA). Presently, the consequences and selection pressures driving the evolution of the polymerase complex remain largely unknown. Here, we characterize the RNA polymerase complex of seasonal influenza A(H3N2) viruses representative of nearly 50 years of influenza A(H3N2) virus evolution. The H3N2 polymerase complex is a reassortment of human and avian influenza virus genes. We show that since 1968, influenza A(H3N2) viruses have increased the transcriptional activity of the polymerase complex while retaining a close balance between mRNA, vRNA, and cRNA levels. Interestingly, the increased polymerase complex activity did not result in increased replicative ability on differentiated human airway epithelial (HAE) cells. We hypothesize that the evolutionary increase in polymerase complex activity of influenza A(H3N2) viruses may compensate for the reduced HA receptor binding and avidity that is the result of the antigenic evolution of influenza A(H3N2) viruses.

Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.

Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.

Continued adaptation of A/H2N2 viruses during pandemic circulation in humans.

Influenza A viruses of the H2N2 subtype sparked a pandemic in 1957 and circulated in humans until 1968. Because A/H2N2 viruses still circulate in wild birds worldwide and human population immunity is low, the transmissibility of six avian A/H2N2 viruses was investigated in the ferret model. None of the avian A/H2N2 viruses was transmitted between ferrets, suggesting that their pandemic risk may be low. The transmissibility, receptor binding preference and haemagglutinin (HA) stability of human A/H2N2 viruses were also investigated. Human A/H2N2 viruses from 1957 and 1958 bound to human-type α2,6-linked sialic acid receptors, but the 1958 virus had a more stable HA, indicating adaptation to replication and spread in the new host. This increased stability was caused by a previously unknown stability substitution G205S in the 1958 H2N2 HA, which became fixed in A/H2N2 viruses after 1958. Although individual substitutions were identified that affected the HA receptor binding and stability properties, they were not found to have a substantial effect on transmissibility of A/H2N2 viruses via the air in the ferret model. Our data demonstrate that A/H2N2 viruses continued to adapt during the first year of pandemic circulation in humans, similar to what was previously shown for the A/H1N1pdm09 virus.

Longitudinal single-cell analysis of a patient receiving adoptive cell therapy reveals potential mechanisms of treatment failure.

Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) is being studied in multiple tumor types. However, little is known about clonal cell expansion in vitro and persistence of the ACT product in vivo. We performed single-cell RNA and T-Cell Receptor (TCR) sequencing on serial blood and tumor samples from a patient undergoing ACT, who did not respond. We found that clonal expansion varied during preparation of the ACT product, and only one expanded clone was preserved in the ACT product. The TCR of the preserved clone which persisted and remained activated for five months was previously reported as specific for cytomegalovirus and had upregulation of granzyme family genes and genes associated with effector functions (HLA-DQB1, LAT, HLA-DQA1, and KLRD1). Clones that contracted during TIL preparation had features of exhaustion and apoptosis. At disease progression, all previously detected clonotypes were detected. New clonotypes appearing in blood or tumor at disease progression were enriched for genes associated with cytotoxicity or stemness (FGFBP2, GNLY, GZMH, GZMK, IL7R, SELL and KLF2), and these might be harnessed for alternative cellular therapy or cytokine therapy. In-depth single-cell analyses of serial samples from additional ACT-treated patients is warranted, and viral- versus tumor-specificity should be carefully analyzed.

Re-Emergence of Circulation of Seasonal Influenza during COVID-19 Pandemic in Russia and Receptor Specificity of New and Dominant Clade 3C.2a1b.2a.2 A(H3N2) Viruses in 2021-2022.

The circulation of seasonal influenza in 2020-2021 around the world was drastically reduced after the start of the COVID-19 pandemic and the implementation of mitigation strategies. The influenza virus circulation reemerged in 2021-2022 with the global spread of the new genetic clade 3C.2a1b.2a.2 of A(H3N2) viruses. The purpose of this study was to characterize influenza viruses in the 2021-2022 season in Russia and to analyze the receptor specificity properties of the 3C.2a1b.2a.2 A(H3N2) viruses. Clinical influenza samples were collected at the local Sanitary-and-Epidemiological Centers of Rospotrebnadzor. Whole genome sequencing was performed using NGS. The receptor specificity of hemagglutinin was evaluated using molecular modeling and bio-layer interferometry. Clinical samples from 854 cases of influenza A and B were studied; A(H3N2) viruses were in the majority of the samples. All genetically studied A(H3N2) viruses belonged to the new genetic clade 3C.2a1b.2a.2. Molecular modeling analysis suggested a higher affinity of hemagglutinin of 3C.2a1b.2a.2. A(H3N2) viruses to the α2,6 human receptor. In vitro analysis using a trisaccharide 6'-Sialyl-N-acetyllactosamine receptor analog did not resolve the differences in the receptor specificity of 3C.2a1b.2a.2 clade viruses from viruses belonging to the 3C.2a1b.2a.1 clade. Further investigation of the A(H3N2) viruses is required for the evaluation of their possible adaptive advantages. Constant monitoring and characterization of influenza are critical for epidemiological analysis.

In Planta, In Vitro and In Silico Studies of Chiral N6-Benzyladenine Derivatives: Discovery of Receptor-Specific S-Enantiomers with Cytokinin or Anticytokinin Activities.

Cytokinins, classical phytohormones, affect all stages of plant ontogenesis, but their application in agriculture is limited because of the lack of appropriate ligands, including those specific for individual cytokinin receptors. In this work, a series of chiral N6-benzyladenine derivatives were studied as potential cytokinins or anticytokinins. All compounds contained a methyl group at the α-carbon atom of the benzyl moiety, making them R- or S-enantiomers. Four pairs of chiral nucleobases and corresponding ribonucleosides containing various substituents at the C2 position of adenine heterocycle were synthesized. A nucleophilic substitution reaction by secondary optically active amines was used. A strong influence of the chirality of studied compounds on their interaction with individual cytokinin receptors of Arabidopsis thaliana was uncovered in in vivo and in vitro assays. The AHK2 and CRE1/AHK4 receptors were shown to have low affinity for the studied S-nucleobases while the AHK3 receptor exhibited significant affinity for most of them. Thereby, three synthetic AHK3-specific cytokinins were discovered: N6-((S)-α-methylbenzyl)adenine (S-MBA), 2-fluoro,N6-((S)-α-methylbenzyl)adenine (S-FMBA) and 2-chloro,N6-((S)-α-methylbenzyl)adenine (S-CMBA). Interaction patterns between individual receptors and specific enantiomers were rationalized by structure analysis and molecular docking. Two other S-enantiomers (N6-((S)-α-methylbenzyl)adenosine, 2-amino,N6-((S)-α-methylbenzyl)adenosine) were found to exhibit receptor-specific and chirality-dependent anticytokinin properties.

Receptor-Binding Assay for Avian Influenza Viruses.

It is well known that influenza viruses utilize host cell glycans for virus attachment factors via their major glycoprotein, hemagglutinin (HA), to initiate their invasion to host cells. Unlike well-known theories in human and avian influenza viruses, barriers laying between interspecies transmission of influenza viruses among bird species are not well understood. Recently, it was speculated that glycan binding of the HA to fucosylated Siaα2-3Gal is related to the expansion in the host range of the virus in avian species. Accordingly, the binding specificity of avian influenza viruses to fucosylated Siaα2-3Gal glycans should be monitored for the better control of avian influenza in both poultry and wild birds. Here, general methods and points for the glycan-binding assay that are specifically modified to target fucosylated Siaα2-3Gal glycans are provided.

Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs.

Humans can be infected by zoonotic avian, pandemic and seasonal influenza A viruses (IAVs), which differ by receptor specificity and conformational stability of their envelope glycoprotein hemagglutinin (HA). It was shown that receptor specificity of the HA determines the tropism of IAVs to human airway epithelial cells, the primary target of IAVs in humans. Less is known about potential effects of the HA properties on viral attachment, infection and activation of human immune cells. To address this question, we studied the infection of total human peripheral blood mononuclear cells (PBMCs) and subpopulations of human PBMCs with well characterized recombinant IAVs differing by the HA and the neuraminidase (NA) but sharing all other viral proteins. Monocytes and all subpopulations of lymphocytes were significantly less susceptible to infection by IAVs with avian-like receptor specificity as compared to human-like IAVs, whereas plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells were equally susceptible to IAVs with avian-like and human-like receptor specificity. This tropism correlated with the surface expression of 2-3-linked sialic acids (avian-type receptors) and 2-6-linked sialic acids (human-type receptors). Despite a reduced infectivity of avian-like IAVs for PBMCs, these viruses were not less efficient than human-like IAVs in terms of cell activation as judged by the induction of cellular mRNA of IFN-α, CCL5, RIG-I, and IL-6. Elevated levels of IFN-α mRNA were accompanied by elevated IFN-α protein secretion in primary human pDC. We found that high basal expression in monocytes of antiviral interferon-induced transmembrane protein 3 (IFITM3) limited viral infection in these cells. siRNA-mediated knockdown of IFITM3 in monocytes demonstrated that viral sensitivity to inhibition by IFITM3 correlated with the conformational stability of the HA. Our study provides new insights into the role of host- and strain-specific differences of HA in the interaction of IAVs with human immune cells and advances current understanding of the mechanisms of viral cell tropism, pathogenesis and markers of virulence.

Receptor Specificity Engineering of TNF Superfamily Ligands.

The tumor necrosis factor (TNF) ligand family has nine ligands that show promiscuity in binding multiple receptors. As different receptors transduce into diverse pathways, the study on the functional role of natural ligands is very complex. In this review, we discuss the TNF ligands engineering for receptor specificity and summarize the performance of the ligand variants in vivo and in vitro. Those variants have an increased binding affinity to specific receptors to enhance the cell signal conduction and have reduced side effects due to a lowered binding to untargeted receptors. Refining receptor specificity is a promising research strategy for improving the application of multi-receptor ligands. Further, the settled variants also provide experimental guidance for engineering receptor specificity on other proteins with multiple receptors.

Peptide-Enabled Targeted Delivery Systems for Therapeutic Applications.

Receptor-targeting peptides have been extensively pursued for improving binding specificity and effective accumulation of drugs at the site of interest, and have remained challenging for extensive research efforts relating to chemotherapy in cancer treatments. By chemically linking a ligand of interest to drug-loaded nanocarriers, active targeting systems could be constructed. Peptide-functionalized nanostructures have been extensively pursued for biomedical applications, including drug delivery, biological imaging, liquid biopsy, and targeted therapies, and widely recognized as candidates of novel therapeutics due to their high specificity, well biocompatibility, and easy availability. We will endeavor to review a variety of strategies that have been demonstrated for improving receptor-specificity of the drug-loaded nanoscale structures using peptide ligands targeting tumor-related receptors. The effort could illustrate that the synergism of nano-sized structures with receptor-targeting peptides could lead to enrichment of biofunctions of nanostructures.

Adaptation of influenza B virus by serial passage in human airway epithelial cells.

Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. To understand their adaptation capability, we examined the genetic changes that occurred following 15 serial passages of two influenza B viruses, B/Brisbane/60/2008 and B/Victoria/504/2000, in human epithelial cells. Thirteen distinct amino acid mutations were found in the PB1, PA, hemagglutinin (HA), neuraminidase (NA), and M proteins after serial passage in the human lung epithelial cell line, Calu-3, and normal human bronchial epithelial (NHBE) cells. These changes were associated with significantly decreased viral replication levels. Our results demonstrate that adaptation of influenza B viruses for growth in human airway epithelial cells is partially conferred by selection of HA1, NA, and polymerase mutations that regulate receptor specificity, functional compatibility with the HA protein, and polymerase activity, respectively.

Highly Pathogenic Avian Influenza H5N1 A/Chicken/France/150169a/2015 Presents In Vitro Characteristics Consistent with Its Predicted Tropism for Avian Species.

Avian influenza A viruses are a major threat to animal and public health. Since 1997, several highly pathogenic H5N1 avian viruses have been directly transmitted from poultry to humans, caused numerous human deaths, and had considerable economic impact on poultry markets. During 2015-2016, a highly pathogenic avian influenza outbreak occurred in southwestern France. Different subtypes circulated, including the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus, which did not possess the full set of genomic determinants known to promote transmission to humans. In order to evaluate the predicted absence of zoonotic potential, a quick method based on in vitro tests was developed to analyze some genetic and phenotypic host restriction determinants. A receptor-binding assay showed that the virus preferentially recognizes avian cell receptors. Temperature sensitivity revealed a cold-sensitive phenotype of the virus at 33 C as virus replication was reduced in contrast with what is expected for human influenza viruses, according to their primary infection sites. Altogether, our quick evaluation method suggests that the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus has an avian phenotype in vitro, in accordance with in silico predictions based on genomic markers.

Nota de Investigación - El virus de la influenza aviar altamente patógeno H5N1 A/Pollo/Francia/150169a/2015 presenta características in vitro consistentes con el tropismo que ha sido predicho para especies aviares. Los virus de la influenza aviar A son una amenaza importante para la salud animal y pública. Desde el año 1997, varios virus aviares H5N1 altamente patógenos que se han transmitido directamente de la avicultura comercial a los humanos, han causado numerosas muertes humanas y han tenido un impacto económico considerable en los mercados avícolas. Durante los años 2015-2016, se produjo un brote de influenza aviar altamente patógena en el suroeste de Francia. Diferentes subtipos circularon, incluido el virus H5N1 A/pollo/Francia/150169a/2015, altamente patógeno, que no poseía completamente el conjunto de determinantes genómicos conocidos por promover la transmisión a los humanos. Para evaluar la ausencia prevista de potencial zoonótico, se desarrolló un método rápido basado en pruebas in vitro para analizar algunos determinantes genéticos y fenotípicos de restricción para el hospedero. Un ensayo de unión al receptor mostró que el virus reconoce preferentemente los receptores de células aviares. La sensibilidad a la temperatura reveló un fenotipo del virus sensible al frío a 33 C ya que la replicación del virus se redujo en contraste con lo esperado para los virus de la influenza humana, según sus sitios de infección primaria. En conjunto, el presente método de evaluación rápida sugiere que el virus altamente patógeno A/pollo/Francia/150169a/2015 H5N1 tiene un fenotipo aviar in vitro, que está de acuerdo con las predicciones in silico basadas en marcadores genómicos.

Phenotypic Effects of Substitutions within the Receptor Binding Site of Highly Pathogenic Avian Influenza H5N1 Virus Observed during Human Infection.

Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans.IMPORTANCE H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

Targeting arrestin interactions with its partners for therapeutic purposes.

Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds of G protein-coupled receptors, certain receptors of other types, and numerous non-receptor partners. Arrestins have no enzymatic activity and work by interacting with other proteins, often assembling multi-protein signaling complexes. Arrestin binding to every partner affects cell signaling, including pathways regulating cell survival, proliferation, and death. Thus, targeting individual arrestin interactions has therapeutic potential. This requires precise identification of protein-protein interaction sites of both participants and the choice of the side of each interaction which would be most advantageous to target. The interfaces involved in each interaction can be disrupted by small molecule therapeutics, as well as by carefully selected peptides of the other partner that do not participate in the interactions that should not be targeted.

Plethora of functions packed into 45 kDa arrestins: biological implications and possible therapeutic strategies.

Mammalian arrestins are a family of four highly homologous relatively small ~ 45 kDa proteins with surprisingly diverse functions. The most striking feature is that each of the two non-visual subtypes can bind hundreds of diverse G protein-coupled receptors (GPCRs) and dozens of non-receptor partners. Through these interactions, arrestins regulate the G protein-dependent signaling by the desensitization mechanisms as well as control numerous signaling pathways in the G protein-dependent or independent manner via scaffolding. Some partners prefer receptor-bound arrestins, some bind better to the free arrestins in the cytoplasm, whereas several show no apparent preference for either conformation. Thus, arrestins are a perfect example of a multi-functional signaling regulator. The result of this multi-functionality is that reduction (by knockdown) or elimination (by knockout) of any of these two non-visual arrestins can affect so many pathways that the results are hard to interpret. The other difficulty is that the non-visual subtypes can in many cases compensate for each other, which explains relatively mild phenotypes of single knockouts, whereas double knockout is lethal in vivo, although cultured cells lacking both arrestins are viable. Thus, deciphering the role of arrestins in cell biology requires the identification of specific signaling function(s) of arrestins involved in a particular phenotype. This endeavor should be greatly assisted by identification of structural elements of the arrestin molecule critical for individual functions and by the creation of mutants where only one function is affected. Reintroduction of these biased mutants, or introduction of monofunctional stand-alone arrestin elements, which have been identified in some cases, into double arrestin-2/3 knockout cultured cells, is the most straightforward way to study arrestin functions. This is a laborious and technically challenging task, but the upside is that specific function of arrestins, their timing, subcellular specificity, and relations to one another could be investigated with precision.

[Evolution of pandemic influenza virus A(H1N1)pdm09 in 2009-2016: dynamics of receptor specificity of the first hemagglutinin subunit (HA1).]

INTRODUCTION: The new reassortant of the swine flu virus A(H1N1)pdm09, which emerged in 2009, overcame the species barrier and caused the 2009-2010 pandemic. One of the key points required for the influenza virus to overcome the species barrier and adapt it to humans is its specific binding to the receptors on the epithelium of the human respiratory tract. PURPOSE: Studying the dynamics of changes in receptor specificity (RS) of the HA1 subunit of the hemagglutinin of the influenza A(H1N1)pdm09 virus strains isolated during the period 2009-2016 on the territory of the Russian Federation, and an analysis of the possible impact of these changes on the incidence rates of the population of the Russian Federation of pandemic influenza in certain epidemic seasons. MATERIAL AND METHODS: Standard methods of collecting clinical materials, isolation of influenza viruses, their typing and genome sequencing were used. For the study of RS of influenza A virus (H1N1)pdm09, the method of solid phase sialosidenzyme analysis was used. RESULTS: It is shown that the change in the parameter W3/6 , which characterizes the degree of a2-3 receptor specificity (a2-3-RS) of the influenza virus A(H1N1) pdm09 over a2-6-RS, coincides with the change in the incidence rates of the Russian Federation's pandemic flu in separate epidemic seasons. There is a tendency to increase the affinity of the virus A(H1N1)pdm09 to α2-3 analogs of the sialyl-glycan receptors of the human respiratory tract epithelium - α2-3-sialoglycopolymers (α2-3-SGP), and falls to α2-6-SGP, with the virus showing the greatest affinity for sulfated sialoglycopolymers. DISCUSSION: Screening for RS strains of influenza A (H1N1)pdm09 virus isolated on the territory of the Russian Federation in 2009-2016 revealed a decrease in the affinity of viruses for a2-6-sialosides, especially for 6'SL-SGP, which is probably due to the presence of amino acid substitutions in the 222 and 223 positions of RBS HA1 viruses. Previous studies have shown that the presence of such substitutions correlates with an increase in the virulence of the influenza A virus (H1N1)pdm09 [16, 23]. Probably, the pandemic virus has evolved towards the selection of more virulent pneumotropic variants. CONCLUSION: Monitoring of the receptor specificity of a pandemic influenza virus makes it possible to identify strains with altered RS to the epithelium of the human respiratory tract and an increased ability to transfer from person to person. Change in the period 2009-2016 the W3/6 parameter characterizing the degree of α2-3-RS excess of the influenza A(H1N1)pdm09 virus over α2-6-RS, coincides with the change in the incidence rates of the pandemic influenza population of the Russian Federation in certain epidemic seasons.

Arrestin mutations: Some cause diseases, others promise cure.

Arrestins play a key role in homologous desensitization of G protein-coupled receptors (GPCRs) and regulate several other vital signaling pathways in cells. Considering the critical roles of these proteins in cellular signaling, surprisingly few disease-causing mutations in human arrestins were described. Most of these are loss-of-function mutations of visual arrestin-1 that cause excessive rhodopsin signaling and hence night blindness. Only one dominant arrestin-1 mutation was discovered so far. It reduces the thermal stability of the protein, which likely results in photoreceptor death via unfolded protein response. In case of the two nonvisual arrestins, only polymorphisms were described, some of which appear to be associated with neurological disorders and altered response to certain treatments. Structure-function studies revealed several ways of enhancing arrestins' ability to quench GPCR signaling. These enhanced arrestins have potential as tools for gene therapy of disorders associated with excessive signaling of mutant GPCRs.

The structural basis of the arrestin binding to GPCRs.

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins targeted by more clinically used drugs than any other protein family. GPCR signaling via G proteins is quenched (desensitized) by the phosphorylation of the active receptor by specific GPCR kinases (GRKs) followed by tight binding of arrestins to active phosphorylated receptors. Thus, arrestins engage two types of receptor elements: those that contain GRK-added phosphates and those that change conformation upon activation. GRKs attach phosphates to serines and threonines in the GPCR C-terminus or any one of the cytoplasmic loops. In addition to these phosphates, arrestins engage the cavity that appears between trans-membrane helices upon receptor activation and several other non-phosphorylated elements. The residues that bind GPCRs are localized on the concave side of both arrestin domains. Arrestins undergo a global conformational change upon receptor binding (become activated). Arrestins serve as important hubs of cellular signaling, emanating from activated GPCRs and receptor-independent.

Arrestins: Introducing Signaling Bias Into Multifunctional Proteins.

Arrestins were discovered as proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) and block their interactions with G proteins, i.e., for their role in homologous desensitization of GPCRs. Mammals express only four arrestin subtypes, two of which are largely restricted to the retina. Two nonvisual arrestins are ubiquitous and interact with hundreds of different GPCRs and dozens of other binding partners. Changes of just a few residues on the receptor-binding surface were shown to dramatically affect GPCR preference of inherently promiscuous nonvisual arrestins. Mutations on the cytosol-facing side of arrestins modulate their interactions with individual downstream signaling molecules. Thus, it appears feasible to construct arrestin mutants specifically linking particular GPCRs with signaling pathways of choice or mutants that sever the links between selected GPCRs and unwanted pathways. Signaling-biased "designer arrestins" have the potential to become valuable molecular tools for research and therapy.