Intramolecular azo-bridge as a cystine disulfide bond surrogate: Somatostatin-14 and brain natriuretic peptide (BNP) analogs

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst? and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst? in the nM range with best results obtained with peptide 2, that is, IC?? = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC?? = 60 nM.     

Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.     

Ligand binding to cytochrome P450 3A4 in phospholipid bilayer nanodiscs: the effect of model membranes

The membrane-bound protein cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme. Most studies of ligand binding by CYP3A4 are currently carried out in solution, in the absence of a model membrane. Therefore, there is little information concerning the membrane effects on CYP3A4 ligand binding behavior. Phospholipid bilayer Nanodiscs are a novel model membrane system derived from high density lipoprotein particles, whose stability, monodispersity, and consistency are ensured by their self-assembly. We explore the energetics of four ligands (6-(p-toluidino)-2-naphthalenesulfonic acid (TNS), alpha-naphthoflavone (ANF), miconazole, and bromocriptine) binding to CYP3A4 incorporated into Nanodiscs. Ligand binding to Nanodiscs was monitored by a combination of environment-sensitive ligand fluorescence and ligand-induced shifts in the fluorescence of tryptophan residues present in the scaffold proteins of Nanodiscs; binding to the CYP3A4 active site was monitored by ligand-induced shifts in the heme Soret band absorbance. The dissociation constants for binding to the active site in CYP3A4-Nanodiscs were 4.0 microm for TNS, 5.8 microm for ANF, 0.45 microm for miconazole, and 0.45 microm for bromocriptine. These values are for CYP3A4 incorporated into a lipid bilayer and are therefore presumably more biologically relevant that those measured using CYP3A4 in solution. In some cases, affinity measurements using CYP3A4 in Nanodiscs differ significantly from solution values. We also studied the equilibrium between ligand binding to CYP3A4 and to the membrane. TNS showed no marked preference for either environment; ANF preferentially bound to the membrane, and miconazole and bromocriptine preferentially bound to the CYP3A4 active site.     

GABA protects pancreatic beta cells against apoptosis by increasing SIRT1 expression and activity

We have previously shown that GABA protects pancreatic islet cells against apoptosis and exerts anti-inflammatory effects. Notably, GABA inhibited the activation of NF-κB in both islet cells and lymphocytes. NF-κB activation is detrimental to beta cells by promoting apoptosis. However, the mechanisms by which GABA mediates these effects are unknown. Because the above-mentioned effects mimic the activity of sirtuin 1 (SIRT1) in beta cells, we investigated whether it is involved. SIRT1 is an NAD⁺-dependent deacetylase that enhances insulin secretion, and counteracts inflammatory signals in beta cells. We found that the incubation of a clonal beta-cell line (rat INS-1) with GABA increased the expression of SIRT1, as did GABA receptor agonists acting on either type A or B receptors. NAD⁺ (an essential cofactor of SIRT1) was also increased. GABA augmented SIRT1 enzymatic activity, which resulted in deacetylation of the p65 component of NF-κB, and this is known to interfere with the activation this pathway. GABA increased insulin production and reduced drug-induced apoptosis, and these actions were reversed by SIRT1 inhibitors. We examined whether SIRT1 is similarly induced in newly isolated human islet cells. Indeed, GABA increased both NAD⁺ and SIRT1 (but not sirtuins 2, 3 and 6). It protected human islet cells against spontaneous apoptosis in culture, and this was negated by a SIRT1 inhibitor. Thus, our findings suggest that major beneficial effects of GABA on beta cells are due to increased SIRT1 and NAD⁺, and point to a new pathway for diabetes therapy.     

A new subfamily LIP of the major intrinsic proteins

Background

Proteins of the major intrinsic protein (MIP) family, or aquaporins, have been detected in almost all organisms. These proteins are important in cells and organisms because they allow for passive transmembrane transport of water and other small, uncharged polar molecules.

Results

We compared the predicted amino acid sequences of 20 MIPs from several algae species of the phylum Heterokontophyta (Kingdom Chromista) with the sequences of MIPs from other organisms. Multiple sequence alignments revealed motifs that were homologous to functionally important NPA motifs and the so-called ar/R-selective filter of glyceroporins and aquaporins. The MIP sequences of the studied chromists fell into several clusters that belonged to different groups of MIPs from a wide variety of organisms from different Kingdoms. Two of these proteins belong to Plasma membrane intrinsic proteins (PIPs), four of them belong to GlpF-like intrinsic proteins (GIPs), and one of them belongs to a specific MIPE subfamily from green algae. Three proteins belong to the unclassified MIPs, two of which are of bacterial origin. Eight of the studied MIPs contain an NPM-motif in place of the second conserved NPA-motif typical of the majority of MIPs. The MIPs of heterokonts within all detected clusters can differ from other MIPs in the same cluster regarding the structure of the ar/R-selective filter and other generally conserved motifs.

Conclusions

We proposed placing nine MIPs from heterokonts into a new group, which we have named the LIPs (large intrinsic proteins). The possible substrate specificities of the studied MIPs are discussed.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-173) contains supplementary material, which is available to authorized users.     

Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam

Different classes of olfactory receptor neurons (ORNs) in Drosophila innervate distinct targets, or glomeruli, in the antennal lobe of the brain. Here we demonstrate that specific ORN classes require the cell surface protein Dscam (Down Syndrome Cell Adhesion Molecule) to synapse in the correct glomeruli. Dscam mutant ORNs frequently terminated in ectopic sites both within and outside the antennal lobe. The morphology of Dscam mutant axon terminals in either ectopic or cognate targets was abnormal. Target specificity for other ORNs was not altered in Dscam mutants, suggesting that different ORNs use different strategies to regulate wiring. Multiple forms of Dscam RNA were detected in the developing antenna, and Dscam protein was localized to developing ORN axons. We propose a role for Dscam protein diversity in regulating ORN target specificity.     

A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.     

Androgen receptor drives cellular senescence

The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence in vitro and in vivo. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor.     

Active MnOx Electrocatalysts Prepared by Atomic Layer Deposition for Oxygen Evolution and Oxygen Reduction Reactions

The ability to deposit conformal catalytic thin films enables opportunities to achieve complex nanostructured designs for catalysis. Atomic layer deposition (ALD) is capable of creating conformal thin films over complex substrates. Here, ALD‐MnO_x on glassy carbon is investigated as a catalyst for the oxygen evolution reaction (OER) and the oxygen reduction reaction (ORR), two reactions that are of growing interest due to their many applications in alternative energy technologies. The films are characterized by X‐ray photoelectron spectroscopy, X‐ray diffraction, scanning electron microscopy, ellipsometry, and cyclic voltammetry. The as‐deposited films consist of Mn(II)O, which is shown to be a poor catalyst for the ORR, but highly active for the OER. By controllably annealing the samples, Mn₂O₃ catalysts with good activity for both the ORR and OER are synthesized. Hypotheses are presented to explain the large difference in the activity between the MnO and Mn₂O₃ catalysts for the ORR, but similar activity for the OER, including the effects of surface oxidation under experimental conditions. These catalysts synthesized though ALD compare favorably to the best MnO_x catalysts in the literature, demonstrating a viable way to produce highly active, conformal thin films from earth‐abundant materials for the ORR and the OER.     

Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma-associated herpesvirus and functions as an RGD-dependent entry receptor

Kaposi's sarcoma-associated herpesvirus (KSHV) envelope-associated glycoprotein B (gB) is involved in the initial steps of binding to host cells during KSHV infection. gB contains an RGD motif reported to bind the integrin α₃β₁ during virus entry. Although the ligand specificity of α₃β₁ has been controversial, current literature indicates that α₃β₁ ligand recognition is independent of RGD. We compared α₃β₁ to the RGD-binding integrin, α_Vβ₃, for binding to envelope-associated gB and a gB(RGD) peptide. Adhesion assays demonstrated that β₃-CHO cells overexpressing α_Vβ₃ specifically bound gB(RGD), whereas α₃-CHO cells overexpressing α₃β₁ did not. Function-blocking antibodies to α_Vβ₃ inhibited the adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to α₃β₁ did not. Using affinity-purified integrins and confocal microscopy, α_Vβ₃ bound to gB(RGD) and KSHV virions, demonstrating direct receptor-ligand interactions. Specific α_Vβ₃ antagonists, including cyclic and dicyclic RGD peptides and α_Vβ₃ function-blocking antibodies, inhibited KSHV infection by 70 to 80%. Keratinocytes from α₃-null mice lacking α₃β₁ were fully competent for infection by KSHV, and reconstitution of α₃β₁ function by transfection with α₃ cDNA reduced KSHV infectivity from 74% to 55%. Additional inhibitory effects of α₃β₁ on the cell surface expression of α_Vβ₃ and on α_Vβ₃-mediated adhesion of α₃-CHO cells overexpressing α₃β₁ were detected, consistent with previous reports of transdominant inhibition of α_Vβ₃ function by α₃β₁. These observations may explain previous reports of an inhibition of KSHV infection by soluble α₃β₁. Our studies demonstrate that α_Vβ₃ is a cellular receptor mediating both the cell adhesion and entry of KSHV into target cells through binding the virion-associated gB(RGD).