Structure-Based Design of Hepatitis C Virus E2 Glycoprotein Improves Serum Binding and Cross-Neutralization

Copyright © 2020 American Society for Microbiology. An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target

Unusual Case of Coexisting Renal Malignancies: Mucinous Tubular and Spindle Cell Carcinoma Kidney with Sarcomatoid Change

Mucinous tubular and spindle cell carcinoma (MTSCC) is a recent entity introduced in the World Health Organization 2004 Classification. It is a tumour of low malignant potential. MTSCC is a subtype of renal cell carcinoma (RCC), which is characterized by a polymorphous histology, wherein the spindled epithelial cell is an inherent carcinomatous component. We report the case of a 57-year-old man presenting with loin pain and dragging sensation. Imaging revealed a large mass arising from the left kidney. Radical nephrectomy was performed, and histopathology revealed spindle cell elements of MTSCC with low-grade cytology, which occasionally blended with tubular structures in variable mucinous stroma admixed with spindle sarcomatoid cells with marked nuclear pleomorphism, associated with significant necrosis and mitoses of up to 5/10 high-power field. A final diagnosis of MTSCC along with high-grade areas consistent with sarcomatoid dedifferentiation was made. Sarcomatoid dedifferentiation has been well documented in various subtypes of RCC, and its presence signifies a worse prognosis in RCC.</p

Synthesis, crystal structures, cation-binding properties and the influence of intramolecular C-H...O interactions on the complexation behaviour of a family of cone p-tert-Butylcalix[4]arene-crown-5 compounds

A family of cone p-tert-butylcalix[4]arene-crown-5 compounds with various substituents (H, COCH<SUB>3</SUB>, CH<SUB>2</SUB>CO<SUB>2</SUB>C<SUB>2</SUB>H<SUB>5</SUB> and CH<SUB>2</SUB>CO<SUB>2</SUB>H) appended at the opposite phenolic oxygen atoms have been synthesised to evaluate their efficiency and selectivity towards different alkali and alkaline-earth metal ions, and also to ascertain the role of the appended side-arms in the complexation process. The selectivity of these ionophores towards Na<SUP>+</SUP>, K<SUP>+</SUP>, Mg<SUP>2+</SUP>, and Ca<SUP>2+</SUP> has been evaluated with an aqueous solution containing an equimolar mixture of these ions. The concentration of metal ion in the extract (organic phase) has been estimated by ion chromatographic assay. Among these ions, K<SUP>+</SUP> shows the highest selectivity in all cases except one, where the two phenolic oxygen atoms contain COCH<SUB>3</SUB> substituents. All the ionophores show poor selectivity towards Mg<SUP>2+</SUP> and Ca<SUP>2+</SUP>. Association constants (Ka) for the binding of Na<SUP>+</SUP> and K<SUP>+</SUP> to these ionophores have been determined spectrophotometrically. Ka (7.2 × 107) is highest for the binding of K<SUP>+</SUP> to the ionophore with CH<SUB>2</SUB>CO<SUB>2</SUB>C<SUB>2</SUB>H<SUB>5</SUB> substituents. The molecular structures of four of the ionophores and four of the metal complexes have been established by single-crystal X-ray crystallography. Analysis of the structures revealed that in case of the ionophore with two COCH<SUB>3</SUB> substituents, the C-H...O interactions form an eight-membered zigzag ring almost perpendicular to the crown ring, which prevents entry of the metal ions into the calix-crown cavity. The ionophore with CH<SUB>2</SUB>CO<SUB>2</SUB>C<SUB>2</SUB>H<SUB>5</SUB> substituents, where no such interaction is observed, forms metal complexes easily and exhibits the highest association constant. <SUP>1</SUP>H and <SUP>13</SUP>C NMR studies have also been carried out to investigate the conformational behaviour of these ionophores and their metal complexes in solution

Characterization of a Novel Putative S-Adenosylmethionine Decarboxylase-Like Protein from <i>Leishmania donovani</i>

<div><p>In addition to the S-adenosylmethionine decarboxylase (AD) present in all organisms, trypanosomatids including <i>Leishmania spp.</i> possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL). Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In <i>Trypanosoma</i> ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from <i>Leishmania spp</i>. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of <i>Leishmania donovani</i> (<i>L. donovani</i>) ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, <i>L. donovani</i> ADL binds S-adenosylmethionine (SAM) and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. <i>In silico</i> protein-protein interaction study reveals that <i>L. donovani</i> ADL can interact with AD. These results indicate that <i>L. donovani</i> ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.</p></div

Selective extraction of alkali metal ions from bittern using picrate anion and crown ethers as ligand: An experimental and theoretical study

2439-2446Dibenzo-21-crown-7 (DB21C7) and dibenzo-24-crown-8 (DB24C8) have been used to extract the metal ions (Na+, K+,Mg2+ and Ca2+) present in bittern, schoenite and in an artificial solution containing equimolar amount of the above mentioned metal ions. Ion chromatographic study of the organic extract shows that DB21C7 extracts potassium with high selectivity, whereas DB24C8 extracts comparable amount of potassium and sodium. The extraction equilibrium constants (Ke) for potassium and sodium with both the ligands have been evaluated spectrophotometrically by two-phase (waterdichloromethane) extraction process. For DB21C7, Ke value for potassium is 4.5×104 M-2, which is 6.3×102 time higher than that of sodium, whereas DB24C8 shows Ke values 3.3×104 and 2.4×104 M-2 for potassium and sodium, respectively. Molecular mechanics calculations have been performed to examine the selectivity of the cations. In the gas phase calculations, DB21C7 and DB24C8 are found to be selective towards the sodium ion. However, K+ is preferred by DB21C7 in the aqueous solution, which is in qualitative agreement with our experimental results

Conformation profiles of ADL as analysed by fluorescence and far-UV CD spectroscopy.

<p>(A) Intrinsic tryptophan fluorescence profile of <i>L. donovani</i> ADL shows emission maxima at 341 nm, indicating tryptophans are partially exposed. (B) Far-UV CD spectra (260 nm-200 nm) of <i>L. donovani</i> ADL protein shows ADL is comprised of mixture of α-helix and β-sheet. (C) Thermal denaturation curve (θ₂₂₂) of <i>L. donovani</i> ADL showing co-operative unfolding with midpoint at ∼70°C. (D) Far-UV CD spectra of <i>L. donovani</i> ADL at pH range (4–9) showing maximum stability near to biological pH (7.0).</p

Study of the competitive binding of mixed alkali and alkaline earth metal ions with dibenzo-30-crown-10

Dibenzo-30-crown-10 (DB30C10) has been used for the extraction of potassium ion into organic solution (CH2Cl2) from aqueous potassium picrate generated from the in situ reaction between picric acid and KOH. The extraction equilibrium constant (Ke) was found to be 2.9 × 106. The high value of Ke is ascribed to the facile transport of the K+-complex from the aqueous to the organic phase as a result of ion-pair interaction between [K-DB30C10]+ and the picrate counter ion. The crystal structure of [K-DB30C10][picrate] shows evidence of such interactions (C-H...π, π...π and C-H...O) between [K-DB30C10]+ and the counter ion. MMFF94 molecular mechanics/Monte Carlo conformational search yielded five low energy conformations (5 kcal/mol energy window) of which one of the conformations was similar to the conformation of the complex in the above crystal, indicating that the crystal formation is thermodynamically controlled. The combination of DB30C10 and the picrate anion was employed to determine the selectivity of K+ extraction from an equimolar mixture of Na+, K+, Mg2+ and Ca2+; the molar percentage of the metal ions in the extract was found to be 7.8%, 82.9%, 7.9% and 1.5%, respectively. When the same experiment was conducted with highly concentrated salt bitterns, the corresponding values were 7.5%, 75.6%, 16.3% and 0.5%, respectively. The hierarchy of selectivity was qualitatively reproduced by molecular mechanics calculations when experimental cation hydration data was taken into account

Selective precipitation of alkaline earth metal cations with dipicrylamine anion: Structure-selectivity correlation

Salts of dipicrylamine anion and alkaline earth metal ions are highly soluble in water, unlike the salts formed with K<SUP>+</SUP>, Rb<SUP>+</SUP>, and Cs<SUP>+</SUP>. We report here that when DPA- is added to a solution containing Mg<SUP>2+</SUP>, Ca<SUP>2+</SUP>, Sr<SUP>2+</SUP>, and Ba<SUP>2+</SUP>, Ba<SUP>2+</SUP> can be precipitated out with high yield and selectivity by optimizing the concentrations. When [DPA<SUP>-</SUP>] = 0.17 M and the molar ratio of each metal ion to DPA<SUP>-</SUP> is 1:1, Sr<SUP>2+</SUP> and Ba<SUP>2+</SUP> are precipitated out in about 90 % overall yield (with respect to DPA<SUP>-</SUP>) and 85 % selectivity of Ba<SUP>2+</SUP>. Salts of Mg<SUP>2+</SUP> and Ca<SUP>2+</SUP> are virtually absent. Single-crystal XRD of the pure Sr<SUP>2+</SUP> and Ba<SUP>2+</SUP> compounds has been performed. Layered structures are observed with clear evidence that the salts exist in the form of complexes with the composition [M(DPA)<SUB>2</SUB>(H<SUB>2</SUB>O)<SUB>4</SUB>]. This is different to the case of alkali metal ions, where the complexes have no coordinated water molecules. The Ba<SUP>2+</SUP> complex has a more compact layered structure than the Sr<SUP>2+</SUP> complex, with reduced exposure of the polar entities. As a result it would be expected to have relatively lower solubility in water, which would account for its selective precipitation over the Sr<SUP>2+</SUP> complex. That it is legitimate to rationalize the precipitation behavior from the structure deduced by single crystal XRD is verified due to the close similarity between the experimental powder XRD data of the precipitate and the simulated powder XRD pattern obtained from the single-crystal data

Putrescine binding analysis by fluorescence and far-UV CD spectroscopy.

<p>(A) Effect of increasing concentration of putrescine on tryptophan fluorescence shows putrescine quench <i>L. donovani</i> ADL in non-interpretable manner. (B) Far-UV CD spectra with increasing concentration of putrescine show change in secondary structure with increasing putrescine concentration. (C) Change in secondary structure, with increasing concentration of putrescine (0–200 µM) was monitored by molar ellipticity value at θ₂₂₂, curve showing change in secondary structure with increasing concentration of putrescine upto 50 µM. (D) Limited proteolysis with increasing concentration of putrescine (10–90 µM) also does not show effect on folding pattern of <i>L. donovani</i> ADL. (E) Thermal denaturation profiles of native ADL (black), <i>L. donovani</i> ADL in complex with putrescine (blue), <i>L. donovani</i> ADL in complex with SAM (green) and <i>L. donovani</i> ADL in complex with both SAM and putrescine (red) reveals substrates binding causes decrease in thermal stability of <i>L. donovani</i> ADL.</p

Multiple sequence alignment of the amino acid sequences of AD and ADLs of <i>L.</i> donovani, L. infantum, L. major, L. brazilensis, T. brucei and T. cruzi and human AD.

<p>Based on this alignment, the residues of human AD involved in autocatalysis are shown by a green asterisk; SAM positioning by red asterisk; SAM binding by brown asterisk and putrescine binding shown in cyan asterisk. Molecular docking studies of SAM and putrescine with homology model of L. <i>donovani</i> ADL suggest that SAM binding residues are not conserved while putrescine binding residues are found partially conserved. The residue comprising SAM binding pocket and involved in H-bond interaction with SAM in <i>L. donovani</i> ADL are enclosed in blue boxes. Putrescine binding residues of <i>L. donovani</i> ADL are represented by black boxes. Alignment is made with the help of Espript 2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065912#pone.0065912-Gouet1" target="_blank">[54]</a>.</p