Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at lambda >495 nm. Subsequent irradiation of the complex at lambda310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with T(alpha). For identification of the sites of crosslinks in T(alpha), the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the T(alpha) species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T(alpha) derivatives and isolation of T(alpha) peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T(alpha) containing the amino acid sequences 310-313 and 342-345.     

A folding-dependent mechanism of antimicrobial peptide resistance to degradation unveiled by solution structure of distinctin

Many bioactive peptides, presenting an unstructured conformation in aqueous solution, are made resistant to degradation by posttranslational modifications. Here, we describe how molecular oligomerization in aqueous solution can generate a still unknown transport form for amphipathic peptides, which is more compact and resistant to proteases than forms related to any possible monomer. This phenomenon emerged from 3D structure, function, and degradation properties of distinctin, a heterodimeric antimicrobial compound consisting of two peptide chains linked by a disulfide bond. After homodimerization in water, this peptide exhibited a fold consisting of a symmetrical full-parallel four-helix bundle, with a well secluded hydrophobic core and exposed basic residues. This fold significantly stabilizes distinctin against proteases compared with other linear amphipathic peptides, without affecting its antimicrobial, hemolytic, and ion-channel formation properties after membrane interaction. This full-parallel helical orientation represents a perfect compromise between formation of a stable structure in water and requirement of a drastic structural rearrangement in membranes to elicit antimicrobial potential. Thus, distinctin can be claimed as a prototype of a previously unrecognized class of antimicrobial derivatives. These results suggest a critical revision of the role of peptide oligomerization whenever solubility or resistance to proteases is known to affect biological properties.     

Locations of the beta1 transmembrane helices in the BK potassium channel

BK channels are composed of α-subunits, which form a voltage- and Ca²⁺-gated potassium channel, and of modulatory β-subunits. The β1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca²⁺]_i in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltage-dependent Ca²⁺ channels and of smooth muscle tone. We explored the contacts between α and β1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two β1 transmembrane (TM) helices, TM1 and TM2, and to the seven α TM helices, consisting of S1–S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1–S4. We now find that the extracellular ends of β1 TM2 and α S0 are in contact and that β1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3–S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance–voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N- and C-terminal tails of β1, to the modulation of BK channel function.     

蛋白质二硫键异构酶在幽门螺杆菌感染者胃黏膜中的表达及意义

目的：了解蛋白质二硫键异构酶(protein disulfide isomerase,PDI)在幽门螺杆菌(H pylon)感染人胃黏膜组织中的表达情况．方法：分别应用半定量RT-PCR方法及Westem blot方法检测感染(n=32)与未感染(n=28)H pylori的人胃黏膜组织中PDI mRNA及蛋白的表达情况．结果：未感染组PDI mRNA及蛋白表达量分别为0．5704±0．0794,0．5198±0．0379,感染组PDI mRNA及蛋白表达量分别为1．0642±0．1533,0．8252±0．0321：两组相比差异显著(P＜0．01)．结论：正常情况下胃黏膜组织有PDI mRNA的表达及蛋白的合成,H pylori感染可使胃黏膜增加PDI mRNA的表达及蛋白的合成．     

NADPH oxidase activity controls phagosomal proteolysis in macrophages through modulation of the lumenal redox environment of phagosomes

The phagosomal lumen in macrophages is the site of numerous interacting chemistries that mediate microbial killing, macromolecular degradation, and antigen processing. Using a non-hypothesis-based screen to explore the interconnectivity of phagosomal functions, we found that NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the inhibition of phagosomal proteolysis in macrophages through reversible oxidative inactivation of local cysteine cathepsins. We also show that NOX2 activity significantly compromises the phagosome''s ability to reduce disulfides. These findings indicate that NOX2 oxidatively inactivates cysteine cathepsins through sustained ablation of the reductive capacity of the phagosomal lumen. This constitutes a unique mechanism of spatiotemporal control of phagosomal chemistries through the modulation of the local redox environment. In addition, this work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal physiologies, particularly of those pertinent to antigen processing.     

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 Å. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.     

Cysteine-Induced Hybridization of 2D Molybdenum Disulfide Films for Efficient and Stable Hydrogen Evolution Reaction

The noble, metal-free materials capable of efficiently catalyzing water splitting reactions currently hold a great deal of promise. In this study, we reported the structure and electrochemical performance of new MoS₂-based material synthesized with L-cysteine. For this, a facile one-pot hydrothermal process was developed and an array of densely packed nanoplatelet-shaped hybrid species directly on a conductive substrate were obtained. The crucial role of L-cysteine was determined by numerous methods on the structure and composition of the synthesized material and its activity and stability for hydrogen evolution reaction (HER) from the acidic water. A low Tafel slope of 32.6 mV dec⁻¹, close to a Pt cathode, was registered for the first time. The unique HER performance at the surface of this hybrid material in comparison with recently reported MoS₂-based electrocatalysts was attributed to the formation of more defective 1T, 2H-MoS₂/MoO_x, C nanostructures with the dominant 1T-MoS₂ phase and thermally degraded cysteine residues entrapped. Numerous stacks of metallic (1T-MoS₂ and MoO₂) and semiconducting (2H-MoS₂ and MoO₃) fragments relayed the formation of highly active layered nanosheets possessing a low hydrogen adsorption free energy and much greater durability, whereas intercalated cysteine fragments had a low Tafel slope of the HER reaction. X-ray photoelectron spectroscopy, scanning electron microscopy, thermography with mass spectrometry, high-resolution transmission electron microscopy, Raman spectroscopy techniques, and linear sweep voltammetry were applied to verify our findings.     

Strandwise translocation of a DNA glycosylase on undamaged DNA

Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.