Single-molecule fluorescence measurements reveal the reaction mechanisms of the core-RISC,composed of human Argonaute 2 and a guide RNA

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection. [BMB Reports 2015; 48(12): 643-644]     

Epitope Analysis of Soybean Major Allergen Gly m Bd 30K Recognized by the Mouse Monoclonal Antibody Using Overlapping Peptides

The epitopes of the major soybean allergen, Gly m Bd 30K, recognized by mouse monoclonal antibodies H6 and F5 were investigated by using synthetic peptides bound to pins. The epitopes are shown to be localized in peptide ³¹QGGCGRGWAFSATGAI-EA⁴⁸ for H6, and in ¹¹⁵ DKVTIDGYETLIMSDEST¹³² for F5.     

Immunochemical specificity of antisera raised against the synthetic encephalitogenic peptide SH624, residues 59–74 of the myelin basic protein

Synthetic peptide SH624 (SHHPARTAHYGSLPQK), residues 59–74 of human myelin basic protein (MBP) was found to be encephalitogenic in the rabbit. Four antisera raised, against the peptide were employed in a liquid-phase equilibrium competitive radioimmunoassay with a series of synthetic peptide analogs of the region to probe the structural requirements of the B-cell determinant subsumed within SH624. The cross-reactivities of the four antisera with intact MBP were also examined. Immunochemical analyses of the four antisera suggested specificities directed against a conformational determinant dependent upon residues from the more phylogenetically conserved carboxyl C-terminal region, residues 65–74 (TAHYGSLPQK) of the synthetic immunogen. Peptide analogs shorter than SH624 from the C-terminal end showed no cross-reactivity with any of the reagent antisera while analogs shorter from the N-terminal end and including the encephalitogenic sequence TTHYGSLPQK, as well as, HYGSLPQK were reactive under equilibrium competitive conditions. SH624-reactive antibodies, cross-reactive with purified heterologous MBPs from 10 different species were also identified in all four reagent antisera. The results of these experiments support previous investigations demonstrating the accessibility of the encephalitogenic 65–74 region in intact MBP. They also underscore the importance of B-cell recognition of organ specific antigenic determinants with respect to MBP immunology and, in particular, the recognition of autoreactive determinants in the neighborhood of encephalitogenic centers.     

An immunodominant site of γ-zein1 is in the region of tandem hexapeptide repeats

The immunochemical data from studies with polyclonal antisera to -zein₁, the 27 kD component of the maize prolamin, indicated that the region containing 8 tandem repeats of the sequence PPPVHL is an immunodominant site. In one case, the entire antibody repertoire of an antiserum recognized epitope(s) within this region. Three 17-mer oligopeptides corresponding to the predicted antigenic epitopes of -zein₁ were synthesized and reacted with three different anti--zein₁ sera in order to map antigenic sites in the intact protein. These antisera yielded positive reactions with a 17-mer peptide (peptide 37), which was not in a hydrophilic maximum but derived from the repeat region. The same antisera gave little or no reaction with other peptides (peptides 38 and 39), both of which were in a hydrophilic maximum. In addition, an antiserum to peptide 37 reacted strongly with both the homologous antigen and the intact -zein₁. Peptide 37 also blocked the binding of antisera to -zein₁ in competition assays. Subsequently, the shorter 6-mer (peptide 82) and 12-mer (peptide 80) versions of peptide 37 were synthesized, and both reacted with anti-peptide 37 serum and also with each of the three anti--zein₁ sera. In these reactions and in competition assays, the reactivity and the blocking ability increased in proportion to the length of the peptide. Based on these data, it was concluded that the repeat region of -zein₁ is the site of one or more continuous immunodominant epitopes. The data also suggest that the repeat region is exposed on the surface of the folded protein and probably occur as a mobile, random coil.     

The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays

A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of (1–4)galactosyltransferase, (1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

X-ray and model-building studies on the specificity of the active site of proteinase K

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca²⁺ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.