Helical Regions Research Articles

Sequence and functional features of a novel scavenger receptor homolog, SCARA5 from Yellow drum (Nibea albiflora)

As an important member in SR-As, member 5 (SCARA5) can swallow apoptotic cells and foreign bodies, and participate multiple signaling pathways to inhibit tumor occurrence, development growth and metastasis. To explore its immune function, SCARA5 was identified from the yellow drum (Nibea albiflora) according to its transcriptome data, and its full-length cDNA was 6968 bp (named as NaSCARA5, GenBank accession no: MW070211) encoding 497 amino acids with a calculated molecular weight of 55.12 kDa, which had the typical motifs of SR family, such as transmembrane helix region, coil region, Pfam collagens region and SR region. BLASTp and the phylogenetic relationship analysis illustrated that the sequences shared high similarity with known SCARA5 of teleosts. Quantitative real time RT-PCR analysis showed that NaSCARA5 was expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the spleen (24.42-fold compared with that in heart). After being infected with Polyinosinic:polycytidylic acid (PolyI:C), Vibrio alginolyticus and Vibrio parahaemolyticus, NaSCARA5 mRNA were up-regulated with time dependent mode in spleen, which suggested that NaSCARA5 might play an important role in the immune process of fish. The extracellular domain of NaSCARA5 was successfully expressed in BL21 (DE3), and yielded the target protein of the expected size with many active sites for their conferring protein-protein interaction functions. After being purified by Ni-NAT Superflow resin and renatured, it was found to bind all the tested bacteria (V.parahaemolyticus,V.alginolyticus and Vibrio harveyi). The eukaryotic expression vector of the NaSCARA5-EGFP fusion protein was constructed and transferred into epithelioma papulosum cyprini (EPC) cells, and it was mainly expressed on the cell membrane indicating that NaSCARA5 was a typical transmembrane protein. The aforementioned results indicated that NaSCARA5 played a significant role in the defense against pathogenic bacteria infection as PRRs, which may provide some further understandings of the regulatory mechanisms in the fish innate immune system for SR family.

Structured globules with twisted arrangement of helical blocks: Computer simulation

This report reveals the influence of local helical structure on the globular morphology. For this purpose we performed molecular dynamics simulations of coil-globule transition in alternating multi-block copolymer of helical and flexible blocks. The blocks were composed of identical beads and were equal in length. It was found that in poor solvent the macromolecules form structured globules, with regions mostly composed of either helical or flexible blocks. The morphology of the globules depends on the block length and the stiffness of the helical blocks. In the helix-rich regions the blocks have cholesteric-like arrangement, with the neighboring blocks aligned locally at a non-zero preferential skew angle, similar to the wide-spread packing motifs of biological macromolecules. • Block copolymers with alternating helical and flexible blocks are considered. • In poor solvent structured globules are formed with helical and flexible regions. • Morphology of the globules depends on block length and stiffness of helical blocks. • At moderate stiffness, blocks aligned locally at a non-zero preferential skew angle.

Interpretable Deep Learning Model Reveals Subsequences of Various Functions for Long Non-Coding RNA Identification.

Long non-coding RNAs (lncRNAs) play crucial roles in many biological processes and are implicated in several diseases. With the next-generation sequencing technologies, substantial unannotated transcripts have been discovered. Classifying unannotated transcripts using biological experiments are more time-consuming and expensive than computational approaches. Several tools are available for identifying long non-coding RNAs. These tools, however, did not explain the features in their tools that contributed to the prediction results. Here, we present Xlnc1DCNN, a tool for distinguishing long non-coding RNAs (lncRNAs) from protein-coding transcripts (PCTs) using a one-dimensional convolutional neural network with prediction explanations. The evaluation results of the human test set showed that Xlnc1DCNN outperformed other state-of-the-art tools in terms of accuracy and F1-score. The explanation results revealed that lncRNA transcripts were mainly identified as sequences with no conserved regions, short patterns with unknown functions, or only regions of transmembrane helices while protein-coding transcripts were mostly classified by conserved protein domains or families. The explanation results also conveyed the probably inconsistent annotations among the public databases, lncRNA transcripts which contain protein domains, protein families, or intrinsically disordered regions (IDRs). Xlnc1DCNN is freely available at https://github.com/cucpbioinfo/Xlnc1DCNN.

Functional and structural segregation of overlapping helices in HIV-1.

Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays, and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1.

A novel small-molecule selective activator of homomeric GIRK4 channels

G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.

FC015: High Salt Environment Disrupts the Regulatory Effect of Podocin on the Nephrin-Nephrin Distance

Abstract BACKGROUND AND AIMS The podocin encoding NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. Podocin homooligomerizes through C-terminal helical regions [1] and binds nephrin in the glomerular slit diaphragm [2]. We formerly showed that podocin regulates the distance between two neighbouring nephrin molecules in cis, i.e. the shortest dimension of the glomerular pore (3.5-4nm) [3, 4]. High sodium diet has been shown to promote glomerular injury and proteinuria [5]. We aimed to assess the effect of high salt environment on the nephrin-nephrin distance in the presence and absence of podocin. METHODS To generate extracellularly tagged nephrin constructs, cDNA sequences encoding YPet or mCherry were inserted in the human wild type nephrin cDNA (YPet-N1 plasmid) replacing the c.3136-3162 nucleotides. Thus, the p.Glu1046-Pro1054 residues of the fibronectin domain, located in the juxtamembraneous extracellular part of nephrin was replaced with either YPet or mCherry. HEK-293 cells were co-transfected with both nephrin constructs with or without wild type HA-tagged podocin construct (pLEX-MCS plasmid). To measure the effect of salt overload, DMEM was supplemented with either 25 mM NaCl or 50 mM mannitol as osmotic control 24-h prior to the FRET measurements. The fluorescence decay curves measured by time-correlated single photon counting (TCSPC) (Chronos BH, ISS Inc.) were decomposed into lifetime components and the longest lifetime population—most suitable for long-range FRET—was used for further calculations. Nonparametric tests were used for statistical analysis. RESULTS High salt environment did not influence the nephrin-nephrin distance in the absence of podocin. In accordance with our previous results, podocin decreased the nephrin-nephrin distance in physiological salt environment, as reflected by an increased FRET efficiency between the extracellular YPet and mCherry tags. High salt environment (+25 mM) completely disrupted this effect. Mannitol of similar osmotic concentration did not modify the effect of podocin (Figure 1). CONCLUSION High salt environment increases the distance between the nephrin molecules in cis by disrupting the organizing effect of podocin. The deleterious effect of high salt is not explained by an increased osmotic pressure.

Mechanism of Mixed-Valence Fe2.5+···Fe2.5+ Formation in Fe4S4 Clusters in the Ferredoxin Binding Motif.

Most low-potential Fe4S4 clusters exist in the conserved binding sequence CxxCxxC (CnCn+3Cn+6). Fe(II) and Fe(III) at the first (Cn) and third (Cn+6) cysteine ligand sites form a mixed-valence Fe2.5+···Fe2.5+ pair in the reduced Fe(II)3Fe(III) cluster. Here, we investigate the mechanism of how the conserved protein environment induces mixed-valence pair formation in the Fe4S4 clusters, FX, FA, and FB in photosystem I, using a quantum mechanical/molecular mechanical approach. Exchange coupling between Fe sites is predominantly determined by the shape of the Fe4S4 cluster, which is stabilized by the preorganized protein electrostatic environment. The backbone NH and CO groups in the conserved CxxCxxC and adjacent helix regions orient along the FeCn···FeC(n+6) axis, generating an electric field and stabilizing the FeCn(II)FeC(n+6)(III) state in FA and FB. The overlap of the d orbitals via -S- (superexchange) is observed for the single FeCn(II)···FeC(n+6)(III) pair, leading to the formation of the mixed-valence Fe2.5+···Fe2.5+ pair. In contrast, several superexchange Fe(II)···Fe(III) pairs are observed in FX due to the highly symmetric pair of the CDGPGRGGTC sequences. This is likely the origin of FX serving as an electron acceptor in the two electron transfer branches.

The PentUnFOLD algorithm as a tool to distinguish the dark and the light sides of the structural instability of proteins.

Intrinsically disordered proteins are frequently involved in important regulatory processes in the cell thanks to their ability to bind several different targets performing sometimes even opposite functions. The PentUnFOLD algorithm is a physicochemical method that is based on new propensity scales for disordered, nonstable and stable elements of secondary structure and on the counting of stabilizing and destabilizing intraprotein contacts. Unlike other methods, it works with a PDB file, and it can determine not only those fragments of alpha helices, beta strands, and random coils that can turn into disordered state (the “dark” side of the disorder), but also nonstable regions of alpha helices and beta strands which are able to turn into random coils (the “light” side), and vice versa (H ↔ C, E ↔ C). The scales have been obtained from structural data on disordered regions from the middle parts of amino acid sequences only, and not on their expectedly disordered N- and C-termini. Among other tendencies we have found that regions of both alpha helices and beta strands that can turn into the disordered state are relatively enriched in residues of Ala, Met, Asp, and Lys, while regions of both alpha helices and beta strands that can turn into random coil are relatively enriched in hydrophilic residues, and Cys, Pro, and Gly. Moreover, PentUnFOLD has the option to determine the effect of secondary structure transitions on the stability of a given region of a protein. The PentUnFOLD algorithm is freely available at http://3.17.12.213/pent-un-fold and http://chemres.bsmu.by/PentUnFOLD.htm.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00726-022-03153-5.

Abstract P1-10-05: Evidence that body mass index modifies breast tissue collagen peptide response pattern to treatment with the non-steroidal anti-inflammatory drug sulindac

Abstract Background. In observational studies, regular use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced breast cancer risk and disease recurrence with evidence that the benefit is greater in overweight/obese women. This parallels findings that obesity is associated with an increase in proinflammatory processes with possible effects on the breast stroma and extracellular matrix. Methods. To assess if body mass index (BMI kg/m2) alters breast tissue response to NSAIDs, we conducted exploratory analyses of change in breast tissue collagen-associated peptides following 6 months of treatment with the NSAID sulindac (150 mg bid) by BMI category: normal (18.5-24.9 n=6), overweight (25-29.9 n=14) and obese (≥30 n=11). Samples for this study were non-cancer core needle breast biopsies from postmenopausal women with a history of hormone receptor positive breast cancer enrolled to study sulindac effect on breast tissue biomarkers. At completion, 36 of 50 patients underwent baseline biopsy and 31 had sufficient tissue for paired analyses. Tissue collagen-associated peptides were studied using whole slide tissue imaging mass spectrometry proteomics. Individual peptide signals were normalized to total ion current and mean peak intensity per area across the entire biological specimen were used to generate a score per patient. Change in stroma peptides was evaluated by BMI status using the Wilcoxon matched pairs signed rank test. Unsupervised hierarchical clustering and heatmap visualization were used to assess differential expression of the peaks. Results. Approximately, 550 peptide peaks were found by targeted collagen tissue imaging proteomics. Striking differences in response to sulindac were observed by BMI. In overweight patients, 6 peptides related to COL1A1, COL6A1, COL6A3 and VIM on database matching differed significantly before and after sulindac treatment. Three were also altered in obese women. Two of the peptides overlapped 10 peptide changes identified in unstratified analyses reported in a separate submitted abstract on the main effect of sulindac on tissue collagen. Interestingly, these peptides were not altered in patients with normal BMI. In addition, on study 15 patients experienced a decrease in BMI and 12 experienced an increase. Two collagen peptides showed inverse relationships dependent on change in BMI status during the study. Conclusion. Six-month treatment with the non-selective NSAID sulindac was associated with changes in collagen-associated peptide differently by BMI status and by weight change. Our findings are most consistent with changes in post-translational hydroxylated proline modifications of collagen variation in the triple helical region. Ongoing work may provide insights on inflammation/adiposity-associated inflammation and effects on breast tissue collagen. Citation Format: Denys Rujchanarong, Peggi M. Angel, Alison Stopeck, Christina Preece, Pavani Chalasani, Patricia A. Thompson. Evidence that body mass index modifies breast tissue collagen peptide response pattern to treatment with the non-steroidal anti-inflammatory drug sulindac [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-10-05.

Structural basis of neuropeptide Y signaling through Y1 receptor

Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y1R) in complex with Gi1 protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y1R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y1R extracellular loops, contributing to the high affinity of NPY for Y1R. The structural analysis of NPY-bound Y1R and mutagenesis studies provide molecular insights into the activation mechanism of Y1R upon NPY binding.

A human antibody reveals a conserved site on beta-coronavirus spike proteins and confers protection against SARS-CoV-2 infection.

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses (β-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 Å resolution and found that the peptide adopted a mainly helical structure. Conserved residues in β-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on β-CoV spike proteins for protective antibodies that may facilitate the development of pan-β-CoV vaccines.

Molecular cloning, sequence characterization, and expression analysis of C-type lectin (CTL) and ER-Golgi intermediate compartment 53-kDa protein (ERGIC-53) homologs from the freshwater prawn, Macrobrachium rosenbergii.

Lectin protein families are diverse and multi-functional in crustaceans. The carbohydrate-binding domains (CRDs) of lectins recognize the molecular patterns associated with pathogens and orchestrate important roles in crustacean defense. In this study, two lectin homologs, a single CRD containing C-type lectin (CTL) and an L-type lectin (LTL) domain containing endoplasmic reticulum Golgi intermediate compartment 53 kDa protein (ERGIC-53) were identified from the freshwater prawn, Macrobrachium rosenbergii. The open reading frames of MrCTL and MrERGIC-53 were 654 and 1,515 bp, encoding polypeptides of 217 and 504 amino acids, respectively. Further, MrCTL showed a 20-amino acid transmembrane helix region and 10 carbohydrate-binding residues within the CRD. MrERGIC-53 showed a signal peptide region, a type-I transmembrane region, and a coiled-coil region at the C-terminus. Phylogenetic analysis revealed a close relationship between MrCTL and MrLectin and M. nipponense CTL (MnCTL), whereas MrERGIC-53 shared high sequence identity with Eriocheir sinensis ERGIC-53 and Penaeus vannamei MBL-1. A homology-based model predicted small carbohydrate-combining sites with a metal-binding site for ligand binding (Ca2+ binding site) in MrCTL and beta-sheets connected by short loops and beta-bends forming a dome-shaped beta-barrel structure representing the LTL domain of MrERGIC-53. Quantitative real-time polymerase chain reaction detected MrCTL and MrERGIC-53 transcripts in all examined tissues, with particularly high levels observed in hemocytes, hepatopancreas, and mucosal-associated tissues, such as the stomach and intestine. Further, the expression levels of MrCTL and MrERGIC-53 transcripts were remarkably altered after V. harveyi challenge, suggesting putative function in host innate immunity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10499-022-00845-3.

Helical region near poly-Q tract in prion-like domain of Arabidopsis ELF3 plays role in temperature-sensing mechanism

Many plants, such as Arabidopsis thaliana, have adapted to sense increasing temperature and respond by up-regulating genes responsible for growth. Recent studies have identified a three-protein circadian clock component, the Evening Complex (EC), a transcription repressor thought to be responsible for integrating temporal information with thermal signals from the environment to quickly enable this genetic response. One of these proteins, ELF3, contains a C-terminal prion-like domain (PrD) responsible for aggregation of the protein into large condensates, removing the complex from DNA and freeing up growth-related genes for transcription.

Global nature of magnetic reconnection during sawtooth crash in ASDEX Upgrade

This paper discusses the toroidal localisation of magnetic reconnection during sawtooth crashes. Numerical analysis with realistic heat diffusion coefficients shows that heat distributes itself helically along the torus faster than the temporal resolution of any existing ECE diagnostics. It makes local and global (helically axisymmetric) magnetic reconnection indistinguishable for an observer, while a local crash where the heat stays confined within a finite helical region could be distinguished. Statistical analysis of sawtooth crashes with the ECEI diagnostic is conducted in ASDEX Upgrade. The displacement of the heat within a finite helical region has not been observed. The statistical data supports global magnetic reconnection.

Is Cys(MTSL) the Best α-Amino Acid Residue to Electron Spin Labeling of Synthetically Accessible Peptide Molecules with Nitroxides?

Electron paramagnetic resonance spectroscopy, particularly its pulse technique double electron–electron resonance (DEER) (also termed PELDOR), is rapidly becoming an extremely useful tool for the experimental determination of side chain-to-side chain distances between free radicals in molecules fundamental for life, such as polypeptides. Among appropriate probes, the most popular are undoubtedly nitroxide electron spin labels. In this context, suitable biosynthetically derived, helical regions of proteins, along with synthetic peptides with amphiphilic properties and antibacterial activities, are the most extensively investigated compounds. A strict requirement for a precise distance measurement has been identified in a minimal dynamic flexibility of the two nitroxide-bearing α-amino acid side chains. To this end, in this study, we have experimentally compared in detail the side-chain mobility properties of the two currently most widely utilized residues, namely, Cys(MTSL) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, two double-labeled, chemically synthesized 20-mer peptide molecules have been adopted as appropriate templates for our investigation on the determination of the model intramolecular separations. These double-Cys(MTSL) and double-TOAC compounds are both analogues of the almost completely rigid backbone peptide ruler which we have envisaged and 3D structurally analyzed as our original, unlabeled compound. Here, we have clearly found that the TOAC side-chain labels are largely more 3D structurally restricted than the MTSL labels. From this result, we conclude that the TOAC residue offers more precise information than the Cys(MTSL) residue on the side chain-to-side chain distance distribution in synthetically accessible peptide molecules.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals a Novel Binding Region of a Neutralizing Fully Human Monoclonal Antibody to Anthrax Protective Antigen.

Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.g., 5–10 min) for the characterization of protein interactions. We recently developed a long-gradient (e.g., 90 min), sub-zero temperature, ultra-high performance liquid chromatography HDX-MS (UPLC-HDX-MS) platform that has significantly increased separation power and limited back-exchange for the analysis of protein samples with high complexity. In this study, we demonstrated the utility of this platform for mapping antibody–antigen epitopes by examining four fully human monoclonal antibodies to anthrax PA. Antibody p1C03, with limited neutralizing activity in vivo, bound to a region on domain 1A of PA. p6C04 and p1A06, with no neutralizing activities, bound to the same helix on domain 3 to prevent oligomerization of PA. We found p6C01 strongly bound to domain 3 on a different helix region. We also identified a secondary epitope for p6C01, which likely leads to the blocking of furin cleavage of PA after p6C01 binding. This novel binding of p6C01 results in highly neutralizing activity. This is the first report of this distinct binding mechanism for a highly neutralizing fully human antibody to anthrax protective antigen. Studying such epitopes can facilitate the development of novel therapeutics against anthrax.

Clinical and Genetic Characteristics of COL2A1-Associated Skeletal Dysplasias in 60 Russian Patients: Part I.

The significant variability in the clinical manifestations of COL2A1-associated skeletal dysplasias makes it necessary to conduct a clinical and genetic analysis of individual nosological variants, which will contribute to improving our understanding of the pathogenetic mechanisms and prognosis. We presented the clinical and genetic characteristics of 60 Russian pediatric patients with type II collagenopathies caused by previously described and newly identified variants in the COL2A1 gene. Diagnosis confirmation was carried out by new generation sequencing of the target panel with subsequent validation of the identified variants using automated Sanger sequencing. It has been shown that clinical forms of spondyloepiphyseal dysplasias predominate in childhood, both with more severe clinical manifestations (58%) and with unusual phenotypes of mild forms with normal growth (25%). However, Stickler syndrome, type I was less common (17%). In the COL2A1 gene, 28 novel variants were identified, and a total of 63% of the variants were found in the triple helix region resulted in glycine substitution in Gly-XY repeats, which were identified in patients with clinical manifestations of congenital spondyloepiphyseal dysplasia with varying severity, and were not found in Stickler syndrome, type I and Kniest dysplasia. In the C-propeptide region, five novel variants leading to the development of unusual phenotypes of spondyloepiphyseal dysplasia have been identified.

Lipoprotein-associated phospholipase A2: A paradigm for allosteric regulation by membranes

Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2's specificity toward oxidized phospholipids.

IN SILICO CHARACTERIZATION OF HUMAN INTERFERON ALPHA/BETA RECEPTOR 2 (ISOFORM A, B AND C) PROTEIN

Objective: To predict the tertiary structure of human interferon alpha/beta receptor 2 protein.
 Study Design: Structure prediction by using bio informatics tools.
 Place and Duration of Study: Department of Biochemistry, Swat Medical College (STMC), Saidu Shareef, Swat, Pakistan, from Aug 2019 to Dec 2019.
 Methodology: All protein sequences of human interferon alpha/beta receptor 2 (isoforma, b and c) (IFNAR-2) were retrieved through the BLAST search (The Basic Local Alignment Search Tool) from available databases ‘NCBI’ (National Centre for Biotechnology Information) and ‘Uni Prot KB’ (The Universal Protein Resource). Sequence alignment was conducted by using Clustal Omega, to get the consensus sequence for IFNAR-2 protein. Consensus protein sequence of human IFNAR-2 was used for the prediction of the three-dimensional structure by employing Swiss-Model Server. Moreover, subcellular localization analysis was also performed by using CELLO2GO program.
 Results: Structural model of human IFNAR-2 protein was predicted and evaluated by Ramachandran dimension. Cellular localization of tertiary topological domains of the predicted models were revealed probability of localization of IFNAR-2 protein (isoform a, b & c) is highest in the plasma membrane due to the presence of the transmembrane alpha helical regions.
 Conclusion: This study predicted the tertiary structural dimensions of human IFNAR-2 protein, including the specific topological domains that contribute towards the subcellular compartmentalization and functional characteristics.

Conformational Analysis of Fluoro-, Chloro-, and Proteo-Alkene Gly-Pro and Pro-Pro Isosteres to Mimic Collagen.

Collagen is the most abundant human protein, with the canonical sequence (Gly-Pro-Hyp)n in its triple helix region. Cis-trans isomerization of the Xaa-Pro amide has made two of these amide bonds the target of alkene replacement: the Gly-Pro and the Pro-Hyp positions. The conformations of Gly-Pro and Pro-Pro (as a Pro-Hyp model) fluoro-, chloro-, and proteo-alkene mimic models were investigated computationally to determine whether these alkenes can stabilize the polyproline type II (PPII) conformation of collagen. Second-order Møller-Plesset (MP2) calculations with various basis sets were used to perform the conformational analyses and locate stationary points. The calculation results predict that fluoro- and chloro-alkene mimics of Gly-Pro and Pro-Pro can participate in n→π* donation to stabilize PPII conformations, yet they are poor n→π* acceptors, shifting the global minima away from PPII conformations. For the proteo-alkene mimics, the lack of significant n→π* interactions and unstable PPII-like geometries explains their known destabilization of the triple helix in collagen-like peptides.