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]     

Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors

Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM–TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.     

Preliminary X-ray study of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida N.C.I.B. 9869

Single crystals of p-cresol methylhydroxylase, a flavocytochrome c from Pseudomonas putida, have been prepared. The crystals are orthorhombic, space group P212121 with unit cell parameters; a = 140.3 A, b = 130.6 A and c = 74.1 A. They contain a single non-symmetric dimer per asymmetric unit and diffract to at least 2.5 A resolution.     

Anti-Inflammatory Effects of Acupuncture Stimulation via the Vagus Nerve

Although acupuncture therapy is widely used in traditional Asian medicine for the treatment of diverse internal organ disorders, its underlying biological mechanisms are largely unknown. Here, we investigated the functional involvement of acupuncture stimulation (AS) in the regulation of inflammatory responses. TNF-α production in mouse serum, which was induced by lipopolysaccharide (LPS) administration, was decreased by manual acupuncture (MAC) at the zusanli acupoint (stomach36, ST36). In the spleen, TNF-α mRNA and protein levels were also downregulated by MAC and were recovered by using a splenic neurectomy and a vagotomy. c-Fos, which was induced in the nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMV) by LPS and electroacupuncture (EAC), was further increased by focal administration of the AMPA receptor blocker CNQX and the purinergic receptor antagonist PPADS. TNF-α levels in the spleen were decreased by CNQX and PPADS treatments, implying the involvement of inhibitory neuronal activity in the DVC. In unanesthetized animals, both MAC and EAC generated c-Fos induction in the DVC neurons. However, MAC, but not EAC, was effective in decreasing splenic TNF-α production. These results suggest that the therapeutic effects of acupuncture may be mediated through vagal modulation of inflammatory responses in internal organs.     

Mutations affecting the catalytic activity of Bacillus cereus 5/B/6 beta-lactamase II

Random in vitro mutagenesis of a cloned Bacillus cereus 5/B/6 beta-lactamase II gene was used to select defective genes unable to confer ampicillin or cephalosporin C resistance to Escherichia coli. DNA sequencing of mutant genes identified histidine at position 28 as important to beta-lactamase II function. In addition, the isolation of six identical frameshift mutants established that the carboxyl-terminal end of beta-lactamase II is critical for enzyme function. Random mutagenesis also revealed that His88 (implicated previously as one of 4 residues acting as a zinc ligand) is crucial to enzymatic activity and that a glycine to glutamic acid substitution at position 148 produced a defective beta-lactamase. Oligonucleotide mutagenesis directed at Glu37 and Glu212 suggests that these residues are inconsequential to enzyme function but that histidine at position 28 may be involved in substrate binding or recognition.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.