Consensus Amino Acid Sequence Research Articles

PCR-based detection and mutation dynamics of fusion protein gene of orthoaviula viruses sequestered during 2023 field outbreaks in Pakistan

: To isolate and detect a Newcastle disease virus in commercial poultry, Molecular characterization and phylogenetic analysis of the confirmed isolate and Multiple sequence alignment and achievement of accession numbers against our submissions in NCBI bankit.: Genetic and antigenic diversity in the fusion protein gene of New Castle disease virus strains has been recognized and the progressive changes over sequential years indicate that it is a continuously evolving virus. The current vaccines containing conventional vaccinal strains can protect birds to a certain level but do not prevent infection and virus shedding. : The partial fusion protein gene of the 14 NDV isolates during the 2023 outbreaks from different areas of Pakistan was determined and analyzed. The antigenic protein translational segment of the fusion gene nucleotide fragment was targeted with a specifically designed primer executed 202 bp size of predictable amplicon during PCR amplification. The nucleotide sequence analysis of studied isolates showed closed similarity to the NCBI bankit numbers. Phylogenetic analysis revealed that 3 isolates belong to genotype II while, 2 isolates positions near genotype VIII of class II. The 6 isolates were located near genotype XVII and only 1 was presented on genotype V branch in calss II. Mutation analysis results revealed various mutations at nucleotide intervals and even found altered amino acids during translation. The results revealed that nucleotide mutation at various positions attributes amino acid substitution that enables wild prevailing strains to evade artificial active immunity. In such a scenario Chimeric and genotype match vaccines prepared from indigenous isolates may be useful in developing candidate vaccines to prevent virus shedding and infection. Further studies are suggested at molecular level to determine the consensus amino acid sequence for virulent, mesogenic, and avirulent prevailing NDV strains.

N-Myristoyltransferase: A Novel Therapeutic Target for Cancer, Inflammation and Radiation Response Modifications

N-Myristoyltransferase (NMT) is a well conserved monomeric protein that catalyzes myristoyl group transfer to various target proteins carrying consensus amino acid sequence motif GXXXS/T (where ‘X’ is any amino acid). NMT mediated myristoylation promotes protein-protein and protein-membrane interactions, leading to diverse signaling including cell proliferation, carcinogenesis, inflammation and immune responses. However, inter-networking linkage between NMT over-expression and protein myristoylation under oxidative stress coupled with inflammation and carcinogenesis is completely missing and needs further investigation. Various types of tumor and cancer tissue showed significant increase in NMT expression. Therefore, inhibition of NMT expression either by using chemical NMT inhibitors or gene silencing could be a promising approach in controlling carcinogenesis. Various scientific evidence suggests that NMT mediated myristoylation promotes membrane association of some cell cycle and cell proliferation regulatory proteins and thus may contribute to cell cycle regulation and cell survival homeostasis. Several NMT inhibitors have been evaluated and tested for their probable anticancer potential. Molecular linkage between oxidative stress and carcinogenesis has been established in several studies. In this review, the authors cover the role and therapeutic potential of NMT in cancer, inflammatory disease and radiation response modulation.

Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections

Neuraminidase is suggested as an important component for developing a universal influenza vaccine. Targeted induction of neuraminidase-specific broadly protective antibodies by vaccinations is challenging. To overcome this, we rationally select the highly conserved peptides from the consensus amino acid sequence of the globular head domains of neuraminidase. Inspired by the B cell receptor evolution process, a reliable sequential immunization regimen is designed to result in immuno-focusing by steering bulk immune responses to a selected region where broadly protective B lymphocyte epitopes reside. After priming neuraminidase protein-specific antibody responses in C57BL/6 or BALB/c inbred mice strains by immunization or pre-infection, boost immunizations with certain neuraminidase-derived peptide-keyhole limpet hemocyanin conjugates significantly strengthened serum neuraminidase inhibition activities and cross-protections. Overall, this study provides proof of concept for a peptide-based sequential immunization strategy for achieving targeted induction of cross-protective antibody response, which provides references for designing universal vaccines against other highly variable pathogens.

Cholesterol affects voltage-gated proton channels indirectly.

A number of membrane proteins, including ion channels, interact directly with cholesterol within the plasma membrane. This interaction may affect physiologically relevant properties of the protein. Often, protein-sterol interaction is facilitated by a cholesterol recognition amino acid consensus sequence (CRAC) domain with (L/V)-X1-5-(Y)-X1-5-(K/R) or the inverse sequence CARC (K/R)-X1-5-(Y/F)-X1-5-(L/V) (Fantini & Barrantes, 2013, Front. Physiol. 4:31). The human proton channel hHV1 contains a putative CARC sequence KNNYAAMV (amino-acid residues 131-138) within the extrafacial part of its S2 transmembrane domain. We explored effects of cholesterol depletion from or addition to the membrane. Modulation of cholesterol levels using methyl-β-cyclodextrin (MBCD) dramatically altered current amplitude in inside-out membrane patches. Mutations to the CARC domain did not alter the effects of cholesterol, indicating that cholesterol binding to the CARC domain is not responsible. We propose that hHV1 molecules preferentially associate with cholesterol-dependent domains within the membrane. Cholesterol might affect proton currents if varying cholesterol changes the distribution of hHV1 molecules in the membrane between the cholesterol-dependent lipid domains and the rest of the membrane. A previous study reported that suction increased proton currents in excised patches and this was interpreted as evidence for mechanosensitivity of the HV1 protein (Pathak et al, 2016, J. Gen. Physiol. 148:405). We have confirmed that suction applied to the pipette increases proton currents. This observation is consistent with our hypothesis since membrane tension will undoubtedly affect the association of individual Hv1 molecules with lipid domains without the need for bona fide channel mechanosensitivity. Increase in membrane tension would result in the increased proton currents due to the dispersal of condensed lipid domains in a similar way to the cholesterol depletion. This study provides the first indication that HV1 associates with membrane lipid domains, sometimes called lipid rafts.

A novel role of catalase in cholesterol uptake of Helicobacter pylori

Helicobacter pylori infection is known to be a significant risk factor for the development of gastric cancers in humans. This pathogen exhibits unique biological characteristics in membrane lipid composition. Specifically, H. pylori incorporates exogenous cholesterol into biomembranes and uses cholesterol as the membrane lipid constituents. A previous study by our group demonstrated that phosphatidylethanolamine of H. pylori functions as the cholesterol-binding lipid. It is, however, unclear whether H. pylori is equipped with protein molecules involved in the cholesterol uptake. We, therefore, examined H. pylori proteins that tightly bind to cholesterol. As a consequence, H. pylori catalase (KatA) turned out to be a candidate of the cholesterol uptake-associated protein. In addition, an H. pylori mutant strain that expresses KatA protein lacking catalase activity was significantly lower in total cholesterol contents than the wild-type H. pylori strain. The putative amino acid sequence of KatA found out to contain a number of the cholesterol recognition/interaction amino acid consensus sequence domains (CRAC and CARC domains). These results suggest that H. pylori KatA with normal folding conformation acts as the cholesterol-binding or -storage protein.

Role of cholesterol-recognition motifs in the infectivity of SARS-CoV-2 variants

The presence of linear amino acid motifs with the capacity to recognize the neutral lipid cholesterol, known as Cholesterol Recognition/interaction Amino acid Consensus sequence (CRAC), and its inverse or mirror image, CARC, has recently been reported in the primary sequence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike S homotrimeric glycoprotein. These motifs also occur in the two other pathogenic coronaviruses, SARS-CoV, and Middle-East respiratory syndrome CoV (MERS-CoV), most conspicuously in the transmembrane domain, the fusion peptide, the amino-terminal domain, and the receptor binding domain of SARS-CoV-2 S protein. Here we analyze the presence of cholesterol-recognition motifs in these key regions of the spike glycoprotein in the pathogenic CoVs. We disclose the inherent pathophysiological implications of the cholesterol motifs in the virus-host cell interactions and variant infectivity.

The First EGF Domain of Coagulation Factor IX Increases PAR1 Distributionin Lipid Rafts and Modulates the Response to Thrombin in Endothelial Cells

The first epidermal growth factor 1 (EGF-1) domain of coagulation factor IX (FIX) contains a CXDXXXXYXCXC consensus amino acid sequence, which reportedly induces endocytosis and clustering of lipid rafts in adhesive cells. Endocytosis and lipid rafts regulate signaling via receptors on the plasma membrane. In this study, weinvestigated the effect of FIX EGF-1 domains on intracellular signaling by thrombin (FIIa) in endothelial cells.Recombinant EGF-1 protein of FIX (EGF-FIX) alone did not induce ROCK-dependent phosphorylation ofmyosin light chain (MLC) in human umbilical vein endothelial cells (HUVECs). On the other hand, a high concentration (100 ng/ml) of FIIa increased phosphorylated MLC, and EGF-FIX enhanced it by 1.3-fold. Interestingly, low concentrations (0.3 ng/ml) of FIIa suppressed the phosphorylation of MLC, and the combination oflow-concentration FIIa and EGF-FIX promoted MLC phosphorylation. Methyl-beta-cyclodextrin (MβCD), whichinhibits the formation of lipid rafts, suppressed the phosphorylation of MLC regardless of the FIIa concentration,whereas EGF-FIX attenuated the effects of MβCD. EGF-FIX increased the distribution of the FIIa receptor(PAR1) in the lipid raft fraction. Active FIX could modulate intracellular signaling by other molecules in endothelial cells via its EGF domain.

MTR4 adaptor PICT1 functions in two distinct steps during pre-rRNA processing

Ribosome biogenesis proceeds with the successive cleavage and trimming of the large 47S rRNA precursor, where the RNA exosome plays major roles in concert with the Ski2-like RNA helicase, MTR4. The recent finding of a consensus amino acid sequence, the arch-interacting motif (AIM), for binding to the arch domain in MTR4 suggests that recruitment of the RNA processing machinery to the maturing pre-rRNA at appropriate places and timings is mediated by several adaptor proteins possessing the AIM sequence. In yeast Saccharomyces cerevisiae, Nop53 plays such a role in the maturation of the 3′-end of 5.8S rRNA. Here, we investigated the functions of PICT1 (also known as GLTSCR2 or NOP53), a mammalian ortholog of Nop53, during ribosome biogenesis in human cells. PICT1 interacted with MTR4 and exosome in an AIM-dependent manner. Overexpression of PICT1 mutants defecting AIM sequence and siRNA-mediated depletion of PICT1 showed that PICT1 is involved in two distinct pre-rRNA processing steps during the generation of 60S ribosomes; first step is the early cleavage of 32S intermediate RNA, while the second step is the late maturation of 12S precursor into 5.8S rRNA. The recruitment of MTR4 and RNA exosome via the AIM sequence was required only during the late processing step. Although, the depletion of MTR4 and PICT1 induced stabilization of the tumor suppressor p53 protein in cancer cell lines, the depletion of the exosome catalytic subunits, RRP6 and DIS3, did not exert such an effect. These results suggest that recruitment of the RNA processing machinery to the 3′-end of pre-5.8S rRNA may be involved in the induction of the nucleolar stress response, but the pre-rRNA processing capabilities themselves were not involved in this process.

Characterization of chicken p53 transcriptional function via parallel genome-wide chromatin occupancy and gene expression analysis

The tumor suppressor p53, which acts primarily as a transcription factor, can regulate infections from various viruses in chickens. However, the underlying mechanisms of the antiviral functions of chicken p53 (chp53) remain unclear due to the lack of detailed information on its transcriptional regulation. Here, to gain comprehensive insights into chp53 transcriptional regulatory function in a global and unbiased manner, we determined the genome-wide chromatin occupancy of chp53 by chromatin immunoprecipitation, which was followed by sequencing and chp53-mediated gene expression profile by RNA sequencing using chemically immortalized leghorn male hepatoma (LMH) cells with ectopic expression of chp53 as the model. The integrated parallel genome-wide chromatin occupancy and gene expression analysis characterized chp53 chromatin occupancy and identified 754 direct target genes of chp53. Furthermore, functional annotation and cross-species comparative biological analyses revealed the conserved key biological functions and DNA binding motifs of p53 between chickens and humans, which may be due to the consensus amino acid sequence and structure of p53 DNA-binding domains. The present study, to our knowledge, provides the first comprehensive characterization of the chp53 transcriptional regulatory network, and can possibly help to improve our understanding of p53 transcriptional regulatory mechanisms and their antiviral functions in chickens.

Membrane Cholesterol Prediction from Human Receptor using Rough Set based Mean-Shift Approach

In human physiology, cholesterol plays an imperative part in membrane cells which regulates the function of G-protein-coupled receptors (GPCR) family. Cholesterol is an individual type of lipid structure and about 90 percent of cellular cholesterol is present at plasma membrane region. Cholesterol Recognition/interaction Amino acid Consensus (CRAC) sequence, generally referred as the CRAC (L/V)-X1−5-(Y)-X1−5-(K/R) and the new cholesterol-binding domain is similar to the CRAC sequence, but exhibits the inverse orientation along the polypeptide chain i.e. CARC (K/R)-X1−5-(Y/F)-X1−5-(L/V). GPCR is treated as a biggest super family in human physiology and probably more than 900 protein genes included in this family. Among all membrane proteins GPCR is responsible for novel drug discovery in all pharmaceuticals industry. In earlier researches the researchers did not find the required number of valid motifs in terms of helices and motif types so they were lacking clinical relevance. The research gap here is that they were not able to predict the motifs effectively which are belonging to multiple motif types. To find out better motif sequences from human GPCR, we explored a hybrid computational model consisting of hybridization of Rough Set with Mean-Shift algorithm. In this paper we made comparison among our resulted output with other techniques such as fuzzy C-means (FCM), FCM with spectral clustering and we concluded that our proposed method targeted well on CRAC region in comparison to CARC region which have higher biological relevance in medicine industry and drug discovery.

Epitope identification of SARS-CoV-2 structural proteins using in silico approaches to obtain a conserved rational immunogenic peptide

The short time between the first cases of COVID-19 and the declaration of a pandemic initiated the search for ways to stop the spread of SARS-CoV-2. There are great expectations regarding the development of effective vaccines that protect against all variants, and in the search for it, we hypothesized the obtention of a predicted rational immunogenic peptide from structural components of SARS-CoV-2 might help the vaccine research direction. In the search for a candidate of an immunogenic peptide of the SARS-CoV-2 envelope (E), membrane (M), nucleocapsid (N), or spike (S) proteins, we access the predicted sequences of each protein after the genome sequenced worldwide. We obtained the consensus amino acid sequences of about 14,441 sequences of each protein of each continent and the worldwide consensus sequence. For epitope identification and characterization from each consensus structural protein related to MHC-I or MHC-II interaction and B-cell receptor recognition, we used the IEDB reaching 68 epitopes to E, 174 to M, 245 to N, and 833 to S proteins. To select an epitope with the highest probability of binding to the MHC or BCR, all epitopes of each consensus sequence were aligned. The curation indicated 1, 4, 8, and 21 selected epitopes for E, M, N, and S proteins, respectively. Those epitopes were tested in silico for antigenicity obtaining 16 antigenic epitopes. Physicochemical properties and allergenicity evaluation of the obtained epitopes were done. Ranking the results, we obtained one epitope of each protein except for the S protein that presented two epitopes after the selection. To check the 3D position of each selected epitope in the protein structure, we used molecular homology modeling. Afterward, each selected epitope was evaluated by molecular docking to reference MHC-I or MHC-II allelic protein sequences. Taken together, the results obtained in this study showed a rational search for a putative immunogenic peptide of SARS-CoV-2 structural proteins that can improve vaccine development using in silico approaches. The epitopes selected represent the most conserved sequence of new coronavirus and may be used in a variety of vaccine development strategies since they are also presented in the described variants of SARS-CoV-2.

Investigating the Role of CRAC in FSH Receptor Function and Structure

Human infertility is a complex disorder that can often be attributed to a dysfunction of the endocrine system. Follicle‐stimulating hormone (FSH) is one of many hormones that participate in a complex process in both women and men to regulate normal reproduction. The dysfunction of this hormone and its receptor are some of the many causes of infertility. FSH is secreted by the anterior pituitary and, in women, initiates a cascade of biological events that enable ovulation. FSH carries out its function by binding and activating specific receptors. The FSH receptor (FSHR) is a G protein‐coupled receptor (GPCR) that is located in the cell membrane of target cells in the ovaries and testes. GPCRs often interact with a variety of molecules beyond their ligands including, in some cases, cholesterol in the plasma membrane. The Cholesterol Recognition/Interaction Amino Acid Consensus Sequence (CRAC) is one such binding domain and is represented by the sequence LV‐XXXX‐Y‐XXXXX‐K/R. The human FSHR contains a sequence consistent with the CRAC motif in the first intracellular loop L‐TTLQ‐Y‐KLTVP‐R. We hypothesized that mutating this domain will negatively affect the signaling of the receptor upon ligand activation. To test this hypothesis tyrosine 375 was mutated to phenylalanine (Y375F) which should render the CRAC inactive. After establishing a cell line stably expressing the mutant receptor, activation of the p44/42 (ERK1/2) pathway was measured. Preliminary data suggest that signaling of the hFSHR‐Y375F mutant is decreased relative to the wild‐type receptor. As we learn more about this receptor, we continue to gain knowledge about conditions such as infertility. This information can facilitate the development of innovative treatments which can help many families who struggle to conceive.

A novel chimeric dengue vaccine candidate composed of consensus envelope protein domain III fused to C-terminal-modified NS1 protein

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate.

The role of cholesterol recognition (CARC/CRAC) mirror codes in the allosterism of the human organic cation transporter 2 (OCT2, SLC22A2)

The human organic cation transporter 2 (OCT2) is a multispecific transporter with cholesterol-dependent allosteric features. The present work elucidates the role of evolutionarily conserved cholesterol recognition/interaction amino acid consensus sequences (CRAC and CARC) in the allosteric binding to 1-methyl-4-phenylpyridinium (MPP+) in human embryonic kidney 293 cells stably or transiently expressing OCT2. Molecular blind simulations docked two mirroring cholesterol molecules in the 5th putative transmembrane domain, where a CARC and a CRAC sequence lie. The impact of the conserved amino acids that may constitute the CARC/CRAC mirror code was studied by alanine-scanning mutagenesis. At a saturating extracellular concentration of substrate, at which the impact of cholesterol depletion is maximal, five mutants transported MPP+ at a significantly lower rate than the wild-type OCT2 (WT), resembling the behavior of the WT upon cholesterol depletion. MPP+ influx rate as a function of the extracellular concentration of substrate was measured for the mutants R234A, R235A, L252A and R263A. R234A kinetic behavior was similar to that of the WT, whereas R235A, L252A and R263A activity shifted from allosteric to one-binding site kinetics, very much like the WT upon cholesterol depletion. The impact of cholesterol on protein thermal stability was assessed for WT, R234A and R263A. While the thermal stability of WT and R234A was improved by the supplementation with cholesterol, R263A was not sensitive to the presence of cholesterol. To conclude, the disruption of the CARC/CRAC mirror code in the 5th putative transmembrane domain is sufficient to abolish the allosteric interaction between OCT2 and MPP+.

In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon.

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca2+. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the “off signal” afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5′NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca2+ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.

Phylogenetic Structure and Sequential Dominance of Sub-Lineages of PRRSV Type-2 Lineage 1 in the United States.

The genetic diversity and frequent emergence of novel genetic variants of porcine reproductive and respiratory syndrome virus type-2 (PRRSV) hinders control efforts, yet drivers of macro-evolutionary patterns of PRRSV remain poorly documented. Utilizing a comprehensive database of >20,000 orf5 sequences, our objective was to classify variants according to the phylogenetic structure of PRRSV co-circulating in the U.S., quantify evolutionary dynamics of sub-lineage emergence, and describe potential antigenic differences among sub-lineages. We subdivided the most prevalent lineage (Lineage 1, accounting for approximately 60% of available sequences) into eight sub-lineages. Bayesian coalescent SkyGrid models were used to estimate each sub-lineage’s effective population size over time. We show that a new sub-lineage emerged every 1 to 4 years and that the time between emergence and peak population size was 4.5 years on average (range: 2–8 years). A pattern of sequential dominance of different sub-lineages was identified, with a new dominant sub-lineage replacing its predecessor approximately every 3 years. Consensus amino acid sequences for each sub-lineage differed in key GP5 sites related to host immunity, suggesting that sub-lineage turnover may be linked to immune-mediated competition. This has important implications for understanding drivers of genetic diversity and emergence of new PRRSV variants in the U.S.

In Vitro Glycosylation of Membrane Proteins Using N-Glycosyltransferase.

Glycoproteins are post-translationally modified proteins that take part in nearly every biological process and make up a large percent of the proteome. N-Linked glycosylation can be performed by N-glycosyltransferase (NGT), which recognizes the consensus amino acid sequence, -Asn-X-Ser/Thr- (NXT), within the protein. The enzyme catalyzes glycosidic bond formation between the oligosaccharide donor, containing nucleoside phosphatase, and the amide nitrogen of the asparagine residue. The attachment of the sugar moiety can influence physiological and biological properties of the protein by affecting their folding, modulating interactions with other biomolecules, and modifying their functions at the cellular level. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. Therefore, the use of in vitro protein glycosylation can help further evaluate the effects of the properties for these important macromolecules. In vitro studies of N-linked glycosylation were done in a stepwise fashion in a membrane-mimetic environment to confirm that the methods for glycosylating soluble proteins could be applicable to membrane proteins. Detergent and lipid systems were used since hydrophobic peptides and membrane proteins are insoluble in aqueous solvents. The stepwise method consisted of the glycosylation of a soluble 7-residue peptide, a hydrophobic WALP-NVT peptide, and a γ-sarcoglycan membrane protein, all of which contained the glycosylation site Asn-Val-Thr (NVT). Glycosylation of the samples was performed using Escherichia coli-expressed NGT from the Actinobacillus pleuropneumoniae genome, and a single sugar moiety of glucose, provided from a nucleotide-linked donor, was added to the glycosylation site. Gel electrophoresis, mass spectrometry, and NMR studies were used for the detection of glycosyltransferase activity and to show the attachment of a single glucose molecule. Our experiments demonstrated that small or large membrane proteins that contain an N-glycosylation consensus sequence can be glycosylated by NGT in membrane-mimetic environments.

Biochemical Investigation of the Interaction of pICln, RioK1 and COPR5 with the PRMT5–MEP50 Complex

The PRMT5–MEP50 methyltransferase complex plays a key role in various cancers and is regulated by different protein–protein interactions. Several proteins have been reported to act as adaptor proteins that recruit substrate proteins to the active site of PRMT5 for the methylation of arginine residues. To define the interaction between these adaptor proteins and PRMT5, we employed peptide truncation and mutation studies and prepared truncated protein constructs. We report the characterisation of the interface between the TIM barrel of PRMT5 and the adaptor proteins pICln, RioK1 and COPR5, and identify the consensus amino acid sequence GQF[D/E]DA[E/D] involved in binding. Protein crystallography revealed that the RioK1 derived peptide interacts with a novel PPI site.

Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β2-adrenergic receptor (β2AR) and the cholecystokinin (CCK) receptor subfamily. The β2AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous β2AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK1R shows functional sensitivity to membrane cholesterol while CCK2R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-Tm) shows that mutation of a conserved CRAC sequence on TM7 of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.

1191. Evolution of Hepatitis C Virus Points to Postpartum Recovery of CD8+ T-Cell Selection Pressure

BackgroundIn acute persisting hepatitis C virus (HCV) infection, brief control of viral replication can occur with onset of a cellular immune response but is eventually lost due to selection of viral variants with escape mutations in CD8 T cell epitopes or development of T cell exhaustion. In chronic infection, the CD8s targeting remaining intact epitopes are exhausted, viral levels are stable, and selection of new escape mutations is rare. Postpartum (PP), some chronically infected women experience a drop in viremia which we hypothesize is due to recovery of the CD8 response and will be associated with selection of de novo escape mutations as seen in acute persisting infection.MethodsHCV genomic evolution was assessed in 2 women with different patterns of PP viral control through consecutive pregnancies. Subject M016 experienced > 2.5 log10 drops in viral loads after each pregnancy while M062 had stable viral levels (Table 1). Longitudinal viral genomes were assessed by Illumina sequencing of near full length PCR products of viral cDNA. Reads were initially aligned to a genotype reference and then iterative consensus sequences until deep coverage was achieved across the entire PCR amplicon. To identify potential CD8 escape mutations, amino acid substitutions were counted if their frequency increased from < 10% of reads peripartum to > 90% at follow up. We focused on the nonstructural region to avoid substitutions related to antibody selection pressure.Viral Load Levels ResultsM016 developed 3 nonsynonomous (NS) mutations after her 1st pregnancy and 1 more after her 2nd. Of these, 3 were directed away from the consensus amino acid sequence, and one differed from consensus at baseline and changed tangentially. M062 developed 1 NS mutation within a known class I epitope following her second pregnancy which was directed toward consensus for her genotype (Fig 1). All mutations occurred within predicted class I epitopes.Nonsynonomous Substitutions in Nonstructural Region ConclusionWhile these pilot data require verification in additional subjects, the observed emergence of non-synonymous mutations directed away from genotype consensus in viral class I epitopes of a woman with postpartum viral control but not in a women with stable viral levels support our hypothesis enhanced CD8 T cell selection pressure contribute to enhanced control of HCV replication after childbirth.Disclosures All Authors: No reported disclosures