Protein-protein interaction in transport

The transport of histidine in the gram negative bacterium S. typhimurium has been studied over a number of years and found to occur through five transport systems (Ames, 1972). Of these, the one with the highest affinity has been studied in detail from the genetic, physiological and biochemical point of view. This system, known as the high-affinity histidine permease, is composed of two subsystems, the J-P and K-P systems, which have a component in common, the P protein, presumed to be membrane-bound. The J-P system, moreover, is known to require the presence of a periplasmic histidine-binding protein, the J protein. The J protein is coded for by the hisJ gene and the P protein is coded for by the hisP gene. Both of these genes have been mapped at 75 min on the Salmonella chromosomal map. Adjacent to them is a regulatory gene, the dhuA gene. The periplasmic histidine-binding protein J has been shown to interact directly with the second component of transport, the P protein (Ames and Spudich, 1976). In accordance with this, histidine-binding protein J has been shown to contain, besides the histidine-binding site, a second site, essential for function, the interaction site (Kustu and Ames, 1974). We have recently shown that a mutant J protein with a defective interaction site but an intact histidine-binding site cannot function in histidine transport, unless an appropriate compensating mutation is introduced in the P protein. The interaction between the J and P proteins is an obligatory step in transport. The mutation in the interaction site of the J protein has been shown to map in the hisJ gene, and the compensating supressor mutation in the P protein has been shown to map in the hisP gene. Our contention that the J and P proteins engage in a functional interaction assumes further strength from other studies on protein-protein interaction in bacteriophage development and in ribosomal structure. Among the possible functions of the J-P interaction in histidine transport, a likely one is the transmission of information to the P protein, concerning whether or not the histidine-binding site on the J protein is occupied. Appropriate conformational changes then can occur in either the J or the P protein, or both, such that the histidine is released in the correct location and direction on the inside of the cell. This could occur either by a pore-formation mechanism or by binding-site translocation. Another alternative is that the P protein is part of an energy transducing mechanism in which energy is transmitted to the J protein, through the interaction site, as a prerequisite for the J protein participation in translocation. Among the interesting findings coming out of this work, is also the fact that the P protein performs a central function in transport being involved in the permeation of other substrates besides histidine. It is likely that other binding proteins besides the J protein require the P protein. Thus an interesting question which we are trying to answer at present is whether the P protein has separate interaction sites for each of these other binding proteins requiring its function, or whether they all interact at one common site.     

Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K_m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca²⁺ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.     

Constitutive heterochromatin: a surprising variety of expressed sequences

The organization of chromosomes into euchromatin and heterochromatin is amongst the most important and enigmatic aspects of genome evolution. Constitutive heterochromatin is a basic yet still poorly understood component of eukaryotic chromosomes, and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Although recent evidence indicates that the presence of transcribed genes in constitutive heterochromatin is a conserved trait that accompanies the evolution of eukaryotic genomes, the term heterochromatin is still considered by many as synonymous of gene silencing. In this paper, we comprehensively review data that provide a clearer picture of transcribed sequences within constitutive heterochromatin, with a special emphasis on Drosophila and humans.     

Exogenous HIV-1 Nef upsets the IFN-γ-induced impairment of human intestinal epithelial integrity

Background

The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.

Methodology/Principal Findings

We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepitelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.

Conclusion/Significance

Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

Land-use intensification systematically alters the size structure of aquatic communities in the Neotropics

Land-use and land-cover transitions can affect biodiversity and ecosystem functioning in a myriad of ways, including how energy is transferred within food-webs. Size spectra (i.e. relationships between body size and biomass or abundance) provide a means to assess how food-webs respond to environmental stressors by depicting how energy is transferred from small to larger organisms. Here, we investigated changes in the size spectrum of aquatic macroinvertebrates along a broad land-use intensification gradient (from Atlantic Forest to mechanized agriculture) in 30 Brazilian streams. We expected to find a steeper size spectrum slope and lower total biomass in more disturbed streams due to higher energetic expenditure in physiologically stressful conditions, which has a disproportionate impact on large individuals. As expected, we found that more disturbed streams had fewer small organisms than pristine forest streams, but, surprisingly, they had shallower size spectrum slopes, which indicates that energy might be transferred more efficiently in disturbed streams. Disturbed streams were also less taxonomically diverse, suggesting that the potentially higher energy transfer in these webs might be channelled via a few efficient trophic links. However, because total biomass was higher in pristine streams, these sites still supported a greater number of larger organisms and longer food chains (i.e. larger size range). Our results indicate that land-use intensification decreases ecosystem stability and enhances vulnerability to population extinctions by reducing the possible energetic pathways while enhancing efficiency between the remaining food-web linkages. Our study represents a step forward in understanding how land-use intensification affects trophic interactions and ecosystem functioning in aquatic systems.     

Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from<Emphasis Type="Italic"> Alicyclobacillus acidocaldarius</Emphasis> and<Emphasis Type="Italic"> Archaeoglobus fulgidus</Emphasis> by random mutagenesis and screening

The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively. The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop. In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction. The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations. Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.     

Dynamic of VE-cadherin-mediated spermatid–Sertoli cell contacts in the mouse seminiferous epithelium

Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid–Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity.