Proton-induced membrane fusion role of phospholipid composition and protein-mediated intermembrane contact

Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectinmediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occured (pH ? 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.     

Synthesis and Characterization of Binding of 5-[76Br] Bromo-3-[[2(S)-Azetidinyl]methoxy]pyridine, a Novel Nicotinic Acetylcholine Receptor Ligand, in Rat Brain

5-[76Br]Bromo-3-[[2(S)-azetidinyl]methoxy]pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half-life of [76Br]BAP was 51 +/- 6 min in cortical membranes and 56 +/- 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (K(D)) values of 36 +/- 9 and 30 +/- 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(-)-nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by approximately 60% with cytisine and by 50% with (S)(-)-nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.     

Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

Clear cell renal cell carcinoma (ccRCC) is a primary kidney cancer with high aggressive phenotype and extremely poor prognosis. Accumulating evidence suggests that circular RNAs (circRNAs) play pivotal roles in the occurrence and development of various human cancers. However, the expression, clinical significance and regulatory role of circRNAs in ccRCC remain largely unclear. Here we report that circDVL1 to be reduced in the serums and tissues from ccRCC patients, and to negatively correlate with ccRCC malignant features. Overexpression of circDVL1 inhibits proliferation, induces G1/S arrest, triggers apoptosis, and reduces migration and invasion in different ccRCC cells in vitro. Correspondingly, circDVL1 overexpression suppresses ccRCC tumorigenicity in a mouse xenograft model. Mechanistically, circDVL1 serves as a sponge for oncogenic miR-412-3p, thereby preventing miR-412-3p-mediated repression of its target protocadherin 7 (PCDH7) in ccRCC cells. Collectively, our results demonstrate that circDVL1 exerts tumor-suppressive function during ccRCC progression through circDVL1/miR-412-3p/PCDH7 axis, and suggest that circDVL1 could be a novel diagnostic and prognositc marker and therapeutic target for ccRCC.     

Structure-activity studies of a macrocyclic peptide inhibitor of histone lysine demethylase 4A

The combination of genetic code reprogramming and mRNA display is a powerful approach for the identification of macrocyclic peptides with high affinities to a target of interest. We have previously used such an approach to identify a potent inhibitor (CP2) of the human KDM4A and KDM4C lysine demethylases; important regulators of gene expression. In the present study, we have used genetic code reprogramming to synthesise very high diversity focused libraries (>10¹² compounds) based on CP2 and, through affinity screening, used these to delineate the structure activity relationship of CP2 binding to KDM4A. In the course of these experiments we identified a CP2 analogue (CP2f-7) with ～4-fold greater activity than CP2 in in vitro inhibition assays. This work will facilitate the development of more potent, selective inhibitors of lysine demethylases.     

Mitogen-activated protein kinase signaling is involved in suramin-induced neurite outgrowth in a neuronal cell line

Suramin is a well-known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers. Previous study showed that suramin is an activator of extracellular signal-regulated kinase (ERK1/2) signaling in several cell lines including Chinese hamster ovary cells, although the physiological relevance of this activation remains uncertain. Here, it was shown that suramin enhances neurite outgrowth concomitant with activation of ERK1/2 in Neuro-2a cells, a neuronal cell line. These neurite outgrowth and ERK1/2 activation were significantly inhibited by PD98059, an inhibitor of mitogen-activated protein kinase kinase, as well as by activation of endogenous adenosine A2A receptors. The suramin-induced phosphorylation of ERK1/2 was also inhibited by inhibitors of Src family kinases. This attenuation of ERK1/2 activity was accompanied by a significant decrease in suramin-induced neurite outgrowth. These results suggest that suramin activates the Src/ERK1/2 signaling pathway that induces neurite outgrowth, both of which are negatively regulated by cAMP produced in response to activation of endogenous adenosine A2A receptors.     

Mitochondria-targeted penetrating cations as carriers of hydrophobic anions through lipid membranes

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain (n) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10-12 and substantially decreased at n = 16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n = 16 or n = 18 as compared to that with n = 12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n = 18 and n = 12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.