scholarly journals Structural characterization of the PCV2d genotype at 3.3 Å resolution reveals differences to PCV2a and PCV2b genotypes, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes

2019 ◽  
Author(s):  
Reza Khayat ◽  
Ke Wen ◽  
Aleksandra Alimova ◽  
Boris Gavrilov ◽  
Al Katz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Structural Changes ◽  
Expression System ◽  
Structural Difference ◽  
Underlying Mechanism ◽  
Porcine Circovirus ◽  
Swine Industry ◽  
Vaccination Programs ◽  
N Terminus ◽  
Axes Of Symmetry

AbstractPorcine circovirus 2 (PCV2) is a T=1 non-enveloped icosahedral virus that has a major impact on the swine industry as an agent of porcine circovirus associate disease. PCV2 capsid protein sequences have been employed by others to provide a temporal description of the emerging genotypes. PCV2a is believed to be the earliest genotype and responsible for giving rise to PCV2b, which gives rise to PCV2d. The underlying mechanism responsible for the emerging genotypes is not understood. To determine if a change in the PCV2d capsid accompanies the emergence of this genotype, we determined the cryo-electron microscopy image reconstruction of PCV2d VLP at 3.3 Å resolution and compared it to the previously reported PCV2a and PCV2b structures. Differences between the CD and GH loops identify structural changes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. We also model additional amino acids for the N-terminus near the icosahedral 3-fold axes of symmetry and a tetranucleotide between the 5- and 2-fold axes of symmetry. To interpret the sequence diversity that defines the PCV2 genotypes on a structural platform we have performed structure-based sequence comparison. Our analysis demonstrates that each genotype possesses a unique set of amino acids located on the surface of the capsid that experience a high degree of substitution. These substitutions may be a response to the PCV2 vaccination program. The structural difference between PCV2a, b and d genotypes indicate that it is important to determine the PCV2 capsid structure as the virus evolves into different genotypes.ImportancePCV2 is a significant epidemic agricultural pathogen that is the causative agent of a variety of swine illnesses. PCV2 infections have significant economic impact in the swine industry and must be controlled by vaccination. Outbreaks in farms vaccinated for PCV2 suggest that improvements to the current vaccination programs are needed. Better understanding of the assembly, structure, replication and evolution of these viruses is necessary for production of improved vaccines. The ability of PCV2 to rapidly shift genotypes suggests that expression systems capable of rapidly producing large quantities of virus-like particles should be pursued. To these ends we have established a mammalian cell-based virus-like particle expression system and performed high resolution structural studies of a new PCV2 genotype. Differences between the structure of this genotype and earlier genotypes demonstrate that it is important to study the PCV2 structure as it shifts genotypes.

scholarly journals Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity

2007 ◽  
Vol 88 (8) ◽  
pp. 2320-2328 ◽  
Author(s):  
Christian Klewitz ◽  
Hans-Dieter Klenk ◽  
Jan ter Meulen
Keyword(s):  
Amino Acids ◽  
Structural Changes ◽  
Point Mutations ◽  
Lassa Virus ◽  
Virus Envelope ◽  
Glycoprotein Precursor ◽  
Alanine Scan ◽  
N Terminus ◽  
Hydrophobic Amino Acids ◽  
Ph Dependent

Lassa virus glycoprotein 2 (LASV GP-2) belongs to the class I fusion protein family. Its N terminus contains two stretches of highly conserved hydrophobic amino acids (residues 260–266 and 276–298) that have been proposed as N-terminal or internal fusion peptide segments (N-FPS, I-FPS) by analogy with similar sequences of other viral glycoproteins or based on experimental data obtained with synthetic peptides, respectively. By using a pH-dependent, recombinant LASV glycoprotein mediated cell–cell fusion assay and a retroviral pseudotype infectivity assay, an alanine scan of all hydrophobic amino acids within both proposed FPSs was performed. Fusogenicity and infectivity were correlated, both requiring correct processing of the glycoprotein precursor. Most point mutations in either FPS accounted for reduced or abolished fusion or infection, respectively. Some mutations also had an effect on pre-fusion steps of virus entry, possibly by inducing structural changes in the glycoprotein. The data demonstrate that several amino acids from both hydrophobic regions of the N terminus, some of which (W264, G277, Y278 and L280) are 100 % conserved in all arenaviruses, are involved in fusogenicity and infectivity of LASV GP-2.

A Novel Synthetic Chemokine Containing D-Amino Acids That Binds to the CXCR4 Receptor and Inhibits HIV-1 Infection.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 603-603
Author(s):  
Ziwei Huang ◽  
Dongxiang Liu ◽  
Navid Madani ◽  
Santosh Kumar ◽  
Won-Tak Choi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Binding Affinity ◽  
Chemokine Receptors ◽  
Structural Changes ◽  
Virus Entry ◽  
Structural Features ◽  
Loop Region ◽  
N Terminus ◽  
Hiv 1

Abstract The viral inflammatory protein II (vMIP-II) is a mammalian chemokine homologue encoded by Kaposi’s Sarcoma-associated herpesvirus. vMIP-II shows a broad spectrum binding activity to CXC, CC and CX3C chemokine receptors. Particularly, vMIP-II binds to CXCR4 and CCR5 which are two major coreceptors for HIV-1 virus entry and infection. In our previous study, we reported an all D-amino acids peptide (DV1) derived from the N-terminus of vMIP-II showing high binding affinity to CXCR4. To study the binding mechanism of vMIP-II with CXCR4, we attached the first 10 D-amino acid residues of this peptide to the N-terminus of vMIP-II, before the first cysteine and synthesized a new chemokine analogue of vMIP-II which we named D10-vMIP-II. Despite of the chiral inverse of the N-terminal residues, D10-vMIP-II shows high CXCR4 binding affinity and inhibits the HIV-1 virus entry as vMIP-II. To explore the structural features of D10-vMIP-II, we determined its crystal structure in high resolution. The crystal structure shows that D10-vMIP-II adopts the same structural folding pattern as vMIP-II except for structural changes at the N-terminus, N-loop and the 30’s loop region, suggesting that the local structures at the N-terminus, N-Loop and the 30’s loop region can be adjusted coordinately. This novel synthetic SMM-Chemokine strongly inhibits the entry and replication of HIV-1 via CXCR4. Our studies demonstrate a new promising lead for the development of therapeutic agents targeted to the HIV-1 entry mechanism.

Post-proline endopeptidase, further characterization of the enzyme from pig kidneys

1984 ◽  
Vol 49 (8) ◽  
pp. 1846-1853 ◽  
Author(s):  
Karel Hauzer ◽  
Tomislav Barth ◽  
Linda Servítová ◽  
Karel Jošt
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Relative Molecular Mass ◽  
Enzyme Molecule ◽  
Thiol Compounds ◽  
Modified Method ◽  
N Terminus

A post-proline endopeptidase (EC 3.4.21.26) was isolated from pig kidneys using a modified method described earlier. The enzyme was further purified by ion exchange chromatography on DEAE-Sephacel. The final product contained about 95% of post-proline endopeptidase. The enzyme molecule consisted of one peptide chain with a relative molecular mass of 65 600 to 70 000, containing a large proportion of acidic and alifatic amino acids (glutamic acid, aspartic acid and leucine) and the N-terminus was formed by aspartic acid or asparagine. In order to prevent losses of enzyme activity, thiol compounds has to be added.

scholarly journals Porcine Gammaherpesviruses in Italian Commercial Swine Population: Frequent but Harmless

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 47
Author(s):  
Giovanni Franzo ◽  
Michele Drigo ◽  
Matteo Legnardi ◽  
Laura Grassi ◽  
Maria Luisa Menandro ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Signs ◽  
Northern Italy ◽  
Porcine Circovirus ◽  
Swine Industry ◽  
Positive Interaction ◽  
High Prevalence ◽  
Swine Population ◽  
Clinical Conditions

Differently from alpha- and betaherpesviruses affecting swine, interest in the recently discovered Suid gammaherpesvirus 3, Suid gammaherpesvirus 4, and Suid gammaherpesvirus 5, also known as porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, and PLHV-3), has largely focused on their role as potential zoonotic agents in cases of xenotransplantation. However, their role as primary pathogens of swine or as co-factors for other lymphotropic infections has essentially been neglected. The present study aims at filling this gap, evaluating the association between PLHVs infection and different clinical conditions and/or porcine circovirus (PCV) co-infection. One hundred seventy-six samples were obtained from different animals located in a high-density pig area of Northern Italy in the period 2017–2020. The presence of PLHVs and PCVs was tested and quantified by specific real-time PCR: PLHVs were widespread among pigs (PLHV-1, PLHV-2, and PLHV-3 prevalence was 28.97%, 10.79%, and 4.54%, respectively) and detected in all considered tissues and clinical conditions. Frequent co-infections were also observed among PLHVs and with PCVs, although a significant association was not detected with the exception of a positive interaction between PLHV-1 and PLHV-3, and a negative one between PLHV-2 and PCV-2. Significantly, no association between PLHVs, alone or in co-infection, emerged with any of the considered clinical signs, their frequency being comparable between healthy and diseased animals. Based on these pieces of evidence and despite their high prevalence, PLHVs’ relevance for the swine industry appears negligible, either as primary pathogens or as predisposing factors for circovirus-induced diseases.

scholarly journals Generation of Antibodies against Foot-and-Mouth-Disease Virus Capsid Protein VP4 Using Hepatitis B Core VLPs as a Scaffold

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 338
Author(s):  
Jessica Swanson ◽  
Rennos Fragkoudis ◽  
Philippa C. Hawes ◽  
Joseph Newman ◽  
Alison Burman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis B ◽  
Capsid Protein ◽  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Specific Antibodies ◽  
Mouth Disease Virus ◽  
N Terminus ◽  
Foot And Mouth

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the economically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neutralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

2004 ◽  
Vol 70 (4) ◽  
pp. 2529-2534 ◽  
Author(s):  
Hyungdon Yun ◽  
Seongyop Lim ◽  
Byung-Kwan Cho ◽  
Byung-Gee Kim
Keyword(s):  
Amino Acids ◽  
Kinetic Resolution ◽  
Specific Activity ◽  
Expression System ◽  
Enantiomeric Excess ◽  
Selective Enrichment ◽  
Alcaligenes Denitrificans ◽  
Keto Acids ◽  
Optically Pure ◽  
High Stereoselectivity

ABSTRACT Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and V max for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and V max for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion.

Tyrosine-protein kinase Src regulates Kv1.5 channel activity and membrane expression through interaction with the N-terminus of the channel

2020 ◽  
Author(s):  
Taylore Dodd ◽  
Tingzhong Wang ◽  
Shetuan Zhang
Keyword(s):  
Protein Kinase ◽  
Cell Voltage ◽  
Underlying Mechanism ◽  
Delayed Rectifier ◽  
Binding Motif ◽  
Membrane Expression ◽  
Binding Motifs ◽  
Delayed Rectifier Potassium Current ◽  
N Terminus ◽  
Tyrosine Protein Kinase

Kv1.5 is a voltage-gated potassium channel that generates the ultra-rapid delayed rectifier potassium current (IKur) important in the repolarization of the atrial action potential. Malfunction of the Kv1.5 channel often results in atrial fibrillation (AFib). A reduction in Kv1.5 current (IKv1.5) occurs upon activation of the endogenous tyrosine-protein kinase Src. The Src SH3 domain binds to proline-rich motifs located within the N-terminus of Kv1.5. Disruption of these binding motifs has been involved in the development of familial AFib. The mechanism underlying the reduction of IKv1.5 upon Src activation has not yet been established and the relationship between Kv1.5 and Src is poorly understood. Therefore, the present study aims to further elucidate the mechanism behind IKv1.5  reduction. The hypothesis that Src regulates Kv1.5 activity by altering the density of mature membrane-localized channels was tested using whole-cell voltage clamp and Western blot analysis. We demonstrate that Src tonically inhibits Kv1.5 activity and decreases the density of mature membrane-localized channels. Kv1.5 channels possessing mutations within the Src binding motifs were also investigated and it was determined that each binding motif contributes to the Kv1.5-Src relationship, however, the binding of Src to an individual motif is sufficiently effective. Our findings indicate that Src regulates Kv1.5 through an interaction with the N-terminal binding motifs and suggests that the inhibition of forward trafficking may be involved in the underlying mechanism. (Supported by the Heart and Stroke foundation of Canada and The Canadian Institutes of Health Research).

Glycoprotein Ibα clustering induces macrophage-mediated platelet clearance in the liver

2015 ◽  
Vol 113 (01) ◽  
pp. 107-117 ◽  
Author(s):  
Na Ma ◽  
Lili Zhao ◽  
Cao Lijuan ◽  
Yiwen Zhang ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Intravenous Injection ◽  
Guinea Pigs ◽  
Immune Thrombocytopenia ◽  
Underlying Mechanism ◽  
Therapeutic Strategies ◽  
Integrin Αiibβ3 ◽  
N Terminus ◽  
Cynomolgus Macaques ◽  
Independent Mechanism

SummaryMany immune thrombocytopenia (ITP) patients, particularly patients with anti-glycoprotein (GP) Ib-IX autoantibodies, do not respond to the conventional treatments such as splenectomy. However, the underlying mechanism remains unclear. Here we found that anti-GPIbα N-terminus antibody AN51, but not other anti-GPIbα antibodies (AK2, HIP1, VM16d, or WM23), induced GPIbα clustering that led to integrin αIIbβ3-dependent platelet aggregation. After intravenous injection, AN51 dose-dependently induced thrombocytopenia in guinea pigs, and the platelets were mainly removed by macrophages in the liver. N-acetyl-D-glucosamine, previously shown to inhibit integrin αMβ2-mediated phagocytosis of refrigerated platelets, dose-dependently inhibited AN51-induced platelet clearance. Furthermore, AN51 but not VM16d, induced rapid platelet clearance in the liver of cynomolgus macaques. Five of 22 chronic ITP patients had anti-GPIbα autoantibodies, and the autoantibodies from four of the five patients competed with AN51 for binding to platelets. These data indicate that GPIbα clustering induced by anti-GPIbα N-terminus antibody causes integrin αIIbβ3-dependent platelet aggregation, phagocytosis, and rapid platelet clearance in the liver. Our findings reveal a novel Fc-independent mechanism underlying the pathogenesis of ITP, and suggest new therapeutic strategies for ITP patients with anti-GPIbα autoantibodies.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

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