NS1-mediated upregulation of ZDHHC22 acyltransferase in influenza a virus infected cells.

Influenza A viruses contain two S-acylated proteins, the ion channel M2 and the glycoprotein hemagglutinin (HA). Acylation of the latter is essential for virus replication. Here we analysed the expression of each of the 23 members of the family of ZDHHC acyltransferases in human airway cells, the site of virus replication. RT-PCR revealed that every ZDHHC acyltransferase (except ZDHHC19) is expressed in A549 and Calu cells. Interestingly, expression of one ZDHHC, ZDHHC22, is upregulated in virus-infected cells; this effect is more pronounced after infection with an avian compared to a human virus strain. The viral protein NS1 triggers ZDHHC22 expression in transfected cells, whereas recombinant viruses lacking a functional NS1 gene did not cause ZDHHC22 upregulation. CRISPR/Cas9 technology was then used to knock-out the ZDHHC22 gene in A549 cells. However, acylation of M2 and HA was not reduced, as analysed for intracellular HA and M2 and the stoichiometry of S-acylation of HA incorporated into virus particles did not change according to MALDI-TOF mass spectrometry analysis. Comparative mass spectrometry of palmitoylated proteins in wt and ΔZDHHC22 cells identified 25 potential substrates of ZDHHC22 which might be involved in virus replication.

The Mechanism of Selective Recognition of Lipid Substrate by hDHHC20 Enzyme.

S-acylation is a post-translational linkage of long chain fatty acids to cysteines, playing a key role in normal physiology and disease. In human cells, the reaction is catalyzed by a family of 23 membrane DHHC-acyltransferases (carrying an Asp-His-His-Cys catalytic motif) in two stages: (1) acyl-CoA-mediated autoacylation of the enzyme; and (2) further transfer of the acyl chain to a protein substrate. Despite the availability of a 3D-structure of human acyltransferase (hDHHC20), the molecular aspects of lipid selectivity of DHHC-acyltransferases remain unclear. In this paper, using molecular dynamics (MD) simulations, we studied membrane-bound hDHHC20 right before the acylation by C12-, C14-, C16-, C18-, and C20-CoA substrates. We found that: (1) regardless of the chain length, its terminal methyl group always reaches the "ceiling" of the enzyme's cavity; (2) only for C16, an optimal "reactivity" (assessed by a simple geometric criterion) permits the autoacylation; (3) in MD, some key interactions between an acyl-CoA and a protein differ from those in the reference crystal structure of the C16-CoA-hDHHS20 mutant complex (probably, because this structure corresponds to a non-native dimer). These features of specific recognition of full-size acyl-CoA substrates support our previous hypothesis of "geometric and physicochemical selectivity" derived for simplified acyl-CoA analogues.

Molecular Dynamics of DHHC20 Acyltransferase Suggests Principles of Lipid and Protein Substrate Selectivity.

Lipid modification of viral proteins with fatty acids of different lengths (S-acylation) is crucial for virus pathogenesis. The reaction is catalyzed by members of the DHHC family and proceeds in two steps: the autoacylation is followed by the acyl chain transfer onto protein substrates. The crystal structure of human DHHC20 (hDHHC20), an enzyme involved in the acylation of S-protein of SARS-CoV-2, revealed that the acyl chain may be inserted into a hydrophobic cavity formed by four transmembrane (TM) α-helices. To test this model, we used molecular dynamics of membrane-embedded hDHHC20 and its mutants either in the absence or presence of various acyl-CoAs. We found that among a range of acyl chain lengths probed only C16 adopts a conformation suitable for hDHHC20 autoacylation. This specificity is altered if the small or bulky residues at the cavity's ceiling are exchanged, e.g., the V185G mutant obtains strong preferences for binding C18. Surprisingly, an unusual hydrophilic ridge was found in TM helix 4 of hDHHC20, and the responsive hydrophilic patch supposedly involved in association was found in the 3D model of the S-protein TM-domain trimer. Finally, the exchange of critical Thr and Ser residues in the spike led to a significant decrease in its S-acylation. Our data allow further development of peptide/lipid-based inhibitors of hDHHC20 that might impede replication of Corona- and other enveloped viruses.

COVID-19: Myths and Reality.

COVID-19, a new human respiratory disease that has killed nearly 3 million people in a year since the start of the pandemic, is a global public health challenge. Its infectious agent, SARS-CoV-2, differs from other coronaviruses in a number of structural features that make this virus more pathogenic and transmissible. In this review, we discuss some important characteristics of the main SARS-CoV-2 surface antigen, the spike (S) protein, such as (i) ability of the receptor-binding domain (RBD) to switch between the "standing-up" position (open pre-fusion conformation) for receptor binding and the "lying-down" position (closed pre-fusion conformation) for immune system evasion; (ii) advantage of a high binding affinity of the RBD open conformation to the human angiotensin-converting enzyme 2 (ACE2) receptor for efficient cell entry; and (iii) S protein preliminary activation by the intracellular furin-like proteases for facilitation of the virus spreading across different cell types. We describe interactions between the S protein and cellular receptors, co-receptors, and antagonists, as well as a hypothetical mechanism of the homotrimeric spike structure destabilization that triggers the fusion of the viral envelope with the cell membrane at physiological pH and mediates the viral nucleocapsid entry into the cytoplasm. The transition of the S protein pre-fusion conformation to the post-fusion one on the surface of virions after their treatment with some reagents, such as ß-propiolactone, is essential, especially in relation to the vaccine production. We also compare the COVID-19 pathogenesis with that of severe outbreaks of "avian" influenza caused by the A/H5 and A/H7 highly pathogenic viruses and discuss the structural similarities between the SARS-CoV-2 S protein and hemagglutinins of those highly pathogenic strains. Finally, we touch on the prospective and currently used COVID-19 antiviral and anti-pathogenetic therapeutics, as well as recently approved conventional and innovative COVID-19 vaccines and their molecular and immunological features.

The Structure of the Potato Virus A Particles Elucidated by Small Angle X-Ray Scattering and Complementary Techniques.

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen.

Filamentous versus Spherical Morphology: A Case Study of the Recombinant A/WSN/33 (H1N1) Virus.

Influenza A virus is a serious human pathogen that assembles enveloped virions on the plasma membrane of the host cell. The pleiomorphic morphology of influenza A virus, represented by spherical, elongated, or filamentous particles, is important for the spread of the virus in nature. Using fixative protocols for sample preparation and negative staining electron microscopy, we found that the recombinant A/WSN/33 (H1N1) (rWSN) virus, a strain considered to be strictly spherical, may produce filamentous particles when amplified in the allantoic cavity of chicken embryos. In contrast, the laboratory WSN strain and the rWSN virus amplified in Madin-Darby canine kidney cells exhibited a spherical morphology. Next-generation sequencing (NGS) suggested a rare Ser126Cys substitution in the M1 protein of rWSN, which was confirmed by the mass spectrometric analysis. No structurally relevant substitutions were found by NGS in other proteins of rWSN. Bioinformatics algorithms predicted a neutral structural effect of the Ser126Cys mutation. The mrWSN_M1_126S virus generated after the introduction of the reverse Cys126Ser substitution exhibited a similar host-dependent partially filamentous phenotype. We hypothesize that a shortage of some as-yet-undefined cellular components involved in virion budding and membrane scission may result in the appearance of filamentous particles in the case of usually "nonfilamentous" virus strains.

Selection and structural analysis of the NY25 peptide - A vaccine candidate from hemagglutinin of swine-origin Influenza H1N1.

The aim of this study was to construct a vaccine peptide candidate against pandemic Influenza H1N1 hemagglutinin and to test its structure. With the help of bioinformatic algorithms we showed that the sequence encoding the second polypeptide of pandemic Influenza H1N1 hemagglutinin (HA2) is protected from nonsynonymous mutations better than the sequence encoding its first polypeptide (HA1). With the help of secondary and ternary structure predicting algorithms we found the fragment of HA2 with the most reproducible secondary structure and synthesized the NY25 peptide corresponding to the residues Asn117 - Tyr141 of HA2. According to the circular dichroism spectra analysis, the peptide has short helix and beta hairpin. According to the analysis of differential fluorescence quenching results, two tyrosine residues are situated on a long distance from each other. These facts taken together with the positive results of affine chromatography with the serum of a person immunized by full-length hemagglutinin confirm that the structure of the fragment of viral full-length protein has been reproduced in the synthetic NY25 peptide. Amino acid sequence of the NY25 peptide (NLYEKVRSQLKNNAKEIGNGCFEFY) is relatively conserved in 18 subtypes of Influenza A virus hemagglutinin.

The alpha helix 1 from the first conserved region of HIV1 gp120 is reconstructed in the short NQ21 peptide.

Investigations of short peptides that can be used in the next phase of synthetic HIV1 vaccine development are an urgent goal, as well as investigations of peptides that can be used in immunological tests with the aim to check the titer of antibodies against the alpha helix 1 from the first conserved region of HIV1 gp120 that are known to cause antibody-dependent cellular cytotoxicity (ADCC). The aim of this work was to study the structure of the NQ21 peptide corresponding to the less mutable part of the first conserved region of HIV1 gp120 (residues 94-114). The NQ21 peptide and its conjugate with biotin (biotin-NQ21) are absolutely alpha-helical in phosphate buffer solutions at pH = 6.8, 7.4 and 8.0, as well as in the dried form, according to the results of surface-enhanced Raman scattering (SERS) spectroscopy. Results of the native gel electrophoresis and thermal analysis under the control of spectrofluorometer and near UV circular dichroism (CD) showed that the peptide exists in form of octamers and tetramers at pH = 7.4, that is important information for further vaccine development. Strong signal of interacting Trp residues in oligomers in the far UV CD obscures the signal from secondary structure, but becomes less intensive during the heating.

Site-specific S-acylation of influenza virus hemagglutinin: the location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate.

S-Acylation of hemagglutinin (HA), the main glycoprotein of influenza viruses, is an essential modification required for virus replication. Using mass spectrometry, we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus also be suitable to analyze cellular proteins. We observed cell type-specific differences in the fatty acid pattern of HA: more stearate was attached if human viruses were grown in mammalian compared with avian cells. No underacylated peptides were detected in the mass spectra, and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with [(3)H]palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern.

Differential effects of angiotensin II and aldosterone on human neutrophil adhesion and concomitant secretion of proteins, free amino acids and reactive oxygen and nitrogen species.

Invasion and adhesion of neutrophils into tissues and their concomitant secretion play an important role in the development of vascular pathologies, including abdominal aortic aneurysm (AAA). Chronic administration of angiotensin II is used to initiate AAA formation in mice. The role of aldosterone in this process is being studied. We conducted for the first time a complex comparative study of the effects of angiotensin II and aldosterone on the adhesion of human neutrophils to fibronectin and the concomitant secretion of proteins, free amino acids as well as reactive oxygen (ROS) and nitrogen (NO) species. Neither angiotensin II nor aldosterone affected the attachment of neutrophils to fibronectin and the concomitant production of ROS. We showed for the first time that aldosterone stimulated the release of amino acid hydroxylysine, a product of lysyl hydroxylase, the activity of which is positively correlated with cell invasiveness. Aldosterone also initiates the secretion of matrix metalloproteinase 9 (MMP-9) and cathepsin G, which may reorganize the extracellular matrix and stimulate the recruitment and adhesion of neutrophils to the aortic walls. Angiotensin II did not affect protein secretion. It may contribute to neutrophil-induced vascular injury by inhibiting the production of NO or by increasing the secretion of isoleucine. Our results suggest that it is aldosterone-induced neutrophil secretion that may play a significant role in neutrophil-induced vascular wall destruction in angiotensin II-induced AAA or other vascular complications.

Structural Insights into Plant Viruses Revealed by Small-Angle X-ray Scattering and Atomic Force Microscopy.

The structural study of plant viruses is of great importance to reduce the damage caused by these agricultural pathogens and to support their biotechnological applications. Nowadays, X-ray crystallography, NMR spectroscopy and cryo-electron microscopy are well accepted methods to obtain the 3D protein structure with the best resolution. However, for large and complex supramolecular structures such as plant viruses, especially flexible filamentous ones, there are a number of technical limitations to resolving their native structure in solution. In addition, they do not allow us to obtain structural information about dynamics and interactions with physiological partners. For these purposes, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are well established. In this review, we have outlined the main principles of these two methods and demonstrated their advantages for structural studies of plant viruses of different shapes with relatively high spatial resolution. In addition, we have demonstrated the ability of AFM to obtain information on the mechanical properties of the virus particles that are inaccessible to other experimental techniques. We believe that these under-appreciated approaches, especially when used in combination, are valuable tools for studying a wide variety of helical plant viruses, many of which cannot be resolved by classical structural methods.

Palmitoylation of influenza virus proteins.

Influenza viruses contain two palmitoylated (S-acylated) proteins: the major spike protein HA (haemagglutinin) and the proton-channel M2. The present review describes the fundamental biochemistry of palmitoylation of HA: the location of palmitoylation sites and the fatty acid species bound to HA. Finally, the functional consequences of palmitoylation of HA and M2 are discussed regarding association with membrane rafts, entry of viruses into target cells by HA-mediated membrane fusion as well as the release of newly assembled virus particles from infected cells.

Mass spectrometry analysis of influenza virus reassortant clones does not reveal an influence of other viral proteins on S-acylation of hemagglutinin.

Hemagglutinin (HA) of influenza virus is S-acylated with stearate at a transmembrane cysteine and with palmitate at two cytoplasmic cysteines. The amount of stearate varies from 35 (in avian strains) to 12% (in human strains), although the acylation region exhibits only minor or even no amino acid differences between HAs. To address whether matrix proteins and neuraminidase affect stearoylation of HA, we used mass spectrometry to analyze laboratory reassortants containing avian virus HA and the internal proteins from a human virus. Only minor fluctuations in the amount of stearate were observed, implying that other viral proteins do not affect acylation of HA.

Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine.

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.

Peptide Models of the Cytoplasmic Tail of Influenza A/H1N1 Virus Hemagglutinin Expand Understanding its pH-Dependent Modes of Interaction with Matrix Protein M1.

Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14-15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific "amyloid-like" aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed.

Linker and/or transmembrane regions of influenza A/Group-1, A/Group-2, and type B virus hemagglutinins are packed differently within trimers.

Influenza virus hemagglutinin is a homotrimeric spike glycoprotein crucial for virions' attachment, membrane fusion, and assembly reactions. X-ray crystallography data are available for hemagglutinin ectodomains of various types/subtypes but not for anchoring segments. To get structural information for the linker and transmembrane regions of hemagglutinin, influenza A (H1-H16 subtypes except H8 and H15) and B viruses were digested with bromelain or subtilisin Carlsberg, either within virions or in non-ionic detergent micelles. Proteolytical fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Within virions, hemagglutinins of most influenza A/Group-1 and type B virus strains were more susceptible to digestion with bromelain and/or subtilisin compared to A/Group-2 hemagglutinins. The cleavage sites were always located in the hemagglutinin linker sequence. In detergent, 1) bromelain cleaved hemagglutinin of every influenza A subtype in the linker region; 2) subtilisin cleaved Group-2 hemagglutinins in the linker region; 3) subtilisin cleaved Group-1 hemagglutinins in the transmembrane region; 4) both enzymes cleaved influenza B virus hemagglutinin in the transmembrane region. We propose that the A/Group-2 hemagglutinin linker and/or transmembrane regions are more tightly associated within trimers than type A/Group-1 and particularly type B ones. This hypothesis is underpinned by spatial trimeric structure modeling performed for transmembrane regions of both Group-1 and Group-2 hemagglutinin representatives. Differential S-acylation of the hemagglutinin C-terminal anchoring segment with palmitate/stearate residues possibly contributes to fine tuning of transmembrane trimer packing and stabilization since decreased stearate amount correlated with deeper digestion of influenza B and some A/Group-1 hemagglutinins.

The cytoplasmic tail of influenza A/H1N1 virus hemagglutinin is ß-structural.

Influenza A/H1N1 virus hemagglutinin (HA) is an integral type I glycoprotein that contains a large glycosylated ectodomain, a transmembrane domain, and a cytoplasmic tail (CT) of 10-14 amino acid residues. There are absolutely no data on the secondary or tertiary structure of the HA CT, which is important for virus pathogenesis. Three highly conserved cysteines are post-translationally modified by the attachment of fatty acid residues that pin the CT to the lipid membrane inside the virion. We applied circular dichroism (CD) and fluorescence spectroscopy analysis to examine four synthetic peptides corresponding to 14-15 C-terminal residues of H1 subtype HA (NH2-WMCSNGSLQCRICI-COOH; NH2-FWMCSNGSLQCRICI-COOH), with free or acetaminomethylated cysteines, in the reduced or non-reduced state, at various pH values and temperatures. The CD analysis detected the formation of a ß-structure (30-65% according to the new BeStSel algorithm), in addition to an unstructured random coil, in every peptide in various conditions. It was completely or partially recognized as an antiparallel ß-structure that was also confirmed by the multi-bounce Horizontal Attenuated Total Reflectance Fourier Transformed Infrared (HATR-FTIR) spectroscopy analysis. According to the experimental data, as well as 3 D modeling, we assume that the amino acid sequence corresponding to the HA CT may form a short antiparallel ß-structure under the lipid membrane within a virion.Communicated by Ramaswamy H. Sarma.

Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution.

An interplay between monomeric and dimeric forms of human epidermal growth factor (EGF) affecting its interaction with EGF receptor (EGFR) is poorly understood. While EGF dimeric structure was resolved at pH 8.1, the possibility of EGF dimerization under physiological conditions is still unclear. This study aimed to describe the oligomeric state of EGF in a solution at physiological pH value. With centrifugal ultrafiltration followed by blue native gel electrophoresis, we showed that synthetic human EGF in a solution at a concentration of 0.1 mg/ml exists mainly in the dimeric form at pH 7.4 and temperature of 37 °C, although a small fraction of its monomers was also observed. Based on bioinformatics predictions, we introduced the D46G substitution to examine if EGF C-terminal part is directly involved in the intermolecular interface formation of the observed dimers. We found a reduced ability of the resulting EGF D46G dimers to dissociate at temperatures up to 50 °C. The D46G substitution also increased the intermolecular antiparallel ß-structure content within the EGF peptide in a solution according to the CD spectra analysis that was confirmed by HATR-FTIR results. Additionally, the energy transfer between Tyr and Trp residues was detected by fluorescence spectroscopy for the EGF D46G mutant, but not for the native EGF. This allowed us to suggest the elongation and rearrangement of the intermolecular ß-structure that leads to the observed stabilization of EGF D46G dimers. The results imply EGF dimerization under physiological pH value and temperature and the involvement of EGF C-terminal part in this process.

Stearic acid blunts growth-factor signaling via oleoylation of GNAI proteins.

Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

The Cytoplasmic Tail of Influenza A Virus Hemagglutinin and Membrane Lipid Composition Change the Mode of M1 Protein Association with the Lipid Bilayer.

Influenza A virus envelope contains lipid molecules of the host cell and three integral viral proteins: major hemagglutinin, neuraminidase, and minor M2 protein. Membrane-associated M1 matrix protein is thought to interact with the lipid bilayer and cytoplasmic domains of integral viral proteins to form infectious virus progeny. We used small-angle X-ray scattering (SAXS) and complementary techniques to analyze the interactions of different components of the viral envelope with M1 matrix protein. Small unilamellar liposomes composed of various mixtures of synthetic or "native" lipids extracted from Influenza A/Puerto Rico/8/34 (H1N1) virions as well as proteoliposomes built from the viral lipids and anchored peptides of integral viral proteins (mainly, hemagglutinin) were incubated with isolated M1 and measured using SAXS. The results imply that M1 interaction with phosphatidylserine leads to condensation of the lipid in the protein-contacting monolayer, thus resulting in formation of lipid tubules. This effect vanishes in the presence of the liquid-ordered (raft-forming) constituents (sphingomyelin and cholesterol) regardless of their proportion in the lipid bilayer. We also detected a specific role of the hemagglutinin anchoring peptides in ordering of viral lipid membrane into the raft-like one. These peptides stimulate the oligomerization of M1 on the membrane to form a viral scaffold for subsequent budding of the virion from the plasma membrane of the infected cell.