Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

Synthesis and alkylation of aza‐glycinyl dipeptide building blocks

Aza‐glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the aza‐glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of oxadiazalone, hydantoin and symmetric urea by‐products. Employing N,N′‐disuccinimidyl carbonate to activate benzophenone hydrazone, we have developed a more efficient approach for the synthesis of aza‐glycinyl dipeptides. Alkylation of the semicarbazone of the resulting protected aza‐glycinyl dipeptides using tetraethylammonium hydroxide and propargyl bromide provided an efficient entry into the aza‐propargylglycinyl peptide building blocks, which have served previously in various reactions including Sonogashira cross‐couplings, dipolar cycloadditions and intramolecular exo‐dig cycloadditions to furnish a variety of azapeptide building blocks. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

NADPH oxidase 5 (NOX5) interacts with and is regulated by calmodulin

Superoxide generation by NADPH oxidase 5 (NOX5) is regulated by Ca(2+) through intramolecular activation of the C-terminal catalytic domain by the EF-hand-containing N-terminal regulatory domain. The C terminus contains a consensus calmodulin-binding domain (CaMBD), which, however, is not the binding site of the N-terminal regulatory domain. Here we show by pull down, cross-linking, fluorimetry and by enzymatic assays, that calmodulin binds to this CaMBD in a Ca(2+)-dependent manner, changes its conformation and increases the Ca(2+) sensitivity of the N terminus-regulated enzymatic activity. This mechanism represents an additional sophistication in the regulation of superoxide production by NOX5.     

Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute

Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the twoα subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pI of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.     

A novel pocket in 14-3-3? is required to mediate specific complex formation with cdc25C and to inhibit cell cycle progression upon activation of checkpoint pathways

Mitotic progression requires the activity of the dual specificity phosphatase, cdc25C. Cdc25C function is inhibited by complex formation with two 14-3-3 isoforms, 14-3-3? and 14-3-3γ. To understand the molecular basis of specific complex formation between 14-3-3 proteins and their ligands, chimeric 14-3-3 proteins were tested for their ability to form a complex with cdc25C in vivo. Specific complex formation between cdc25C and 14-3-3? in vivo requires a phenylalanine residue at position 135 (F135) in 14-3-3?. Mutation of this residue to the corresponding residue present in other 14-3-3 isoforms (F135V) leads to reduced binding to cdc25C and a decrease in the ability to inhibit cdc25C function in vivo. Similarly, F135V failed to rescue the incomplete S phase and the G2 DNA damage checkpoint defects observed in cells lacking 14-3-3?. A comparative analysis of the 14-3-3 structures present in the database suggested that the F135 in 14-3-3? was required to maintain the integrity of a pocket that might be involved in secondary interactions with cdc25C. These results suggest that the specificity of the 14-3-3 ligand interaction may be dependent on structural motifs present in the individual 14-3-3 isoforms.     

Specificity of covalently stabilized complexes of 125I-labeled human somatotropin and components of the lactogenic binding sites of rat liver

¹²⁵I-labeled human somatotropin specifically bound to the lactogenic sites of microsomal membranes from pregnant rat liver, originated a radioactive covalent complex of M_r 63,000 upon reaction with dimethyl suberimidate, disuccinimidyl suberate (DSS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The amoun of this species was closely parallel with the preexisting amount of the ligand-receptor complex. Photoactivation of a hormone derivative bound to the receptor also gave rise to the 63 K species. A ternary complex of receptor, hormone and Triton X-100 cross-linked with DSS yielded the 63 K species and a new one of 96 K. The data indicate that the 63 K complex involves the radioactive hormone and a constituent of the binding site. The 96 K species could comprise a second component of the receptor.     

Solution structure of the TatB component of the twin-arginine translocation system

The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC–substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended ‘L-shape’ conformation comprising four helices: a transmembrane helix (TMH) α1, an amphipathic helix (APH) α2, and two solvent exposed helices α3 and α4. The packing of TMH and APH is relatively rigid, whereas helices α3 and α4 display notably higher mobility. The observed floppiness of helices α3 and α4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.     

Mim1 functions in an oligomeric form to facilitate the integration of Tom20 into the mitochondrial outer membrane

The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Despite its central role in the biogenesis of mitochondria, the assembly process of this complex is not completely understood. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein with an undefined role in the assembly of the TOM complex. The protein is composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space. Here we show that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. We further investigated what the structural characteristics allowing Mim1 to fulfil its function are. The N- and C-terminal domains of Mim1 are crucial neither for the function of the protein nor for its biogenesis. Thus, the TMS of Mim1 is the minimal functional domain of the protein. We show that Mim1 forms homo-oligomeric structures via its TMS, which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs did not form oligomeric structures and was inactive. With all these data taken together, we propose that the homo-oligomerization of Mim1 allows it to fulfil its function in promoting the integration of Tom20 into the mitochondrial outer membrane.     

Affinity labelling of the tetrodotoxin-binding component of the Na+ channel

Tetrodotoxin-binding sites were covalently labelled with a highly tritiated derivative of tetrodotoxin. Cross-linking experiments, using dissucinimidyl suberate, on partially purified tetrodotoxin-binding component from electroplax of Electrophorus electricus, revealed covalent labelling of a single polypeptide chain of MW 270,000.