Ribosomal structure: RNA-protein and protein-protein interactions

Vitamin A and its derivatives have been postulated to play an important role in renal tubulogenesis and compensatory hypertrophy. This study examined the effects of two carboxylic derivatives of vitamin A on Lewis lung carcinoma-porcine kidney-1 (LLC-PK1) renal tubular epithelial cell mito- and motogenesis and cell size. It was found that all-trans and 13-cis retinoic acids exerted modest, dose-dependent effects to stimulate incorporation of 3H-thymidine into acid-precipitable material of LLC-PK1 cells. The effects of all-trans retinoic acid to promote 3H-thymidine uptake in LLC-PK1 cells modestly enhanced that seen with acidic fibroblastic growth factor. Similar findings of these two retinoic acid derivatives to promote 3H-thymidine uptake and to enhance 3H-thymidine uptake stimulated by another growth factor (platelet-derived growth factor BB) were also observed in cultured bovine aortic smooth muscle cells. Both retinoic acids promoted healing of denuded areas made within confluent monolayers of serum-starved LLC-PK1 cells. All-trans retinoic acid also stimulated recovery of mechanically denuded areas within bovine aortic smooth muscle monolayers. Neither all-trans nor 13-cis retinoic acids s affected cell size as assessed by forward light scatter with flow cytometry, suggesting lack of effect to induce hypertrophy. These results demonstrate that two carboxylic acid derivatives of vitamin A are capable of stimulation of basal and growth factor-induced incorporation of 3H-thymidine uptake into acid-precipitable material and healing of denuded areas in disparate cell types. These findings are compatible with a role for vitamin A and its analogues in the tissue repair process.     

The CYP2 family: models, mutants and interactions

1. The construction of three-dimensional models of mammalian cytochromes P450 from the CYP2 family is reported based on protein sequence alignment with CYP102, a bacterial P450 of known crystal structure. 2. The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions. 3. The generation of these models from the recently reported crystal structure of substrate-bound CYP102 enables the exploration of likely active site contacts with specific substrates of CYP2 family isozymes.     

Peptide modulators of protein-protein interactions in intracellular signaling

Signal transduction cascades involve multiple enzymes and are orchestrated by selective protein-protein interactions that are essential for the progression of intracellular signaling events. Modulators of these protein-protein interactions have been used to dissect the role of individual components of each signaling cascade. We describe several methods that have been developed for the identification of peptides that inhibit the interaction between signaling proteins and hence selectively modulate their functions. Such peptide modulators provide important tools for basic research and have great potential as leads for the development of new classes of therapeutic drugs.     

The ras recruitment system, a novel approach to the study of protein-protein interactions

The yeast two-hybrid system represents one of the most efficient approaches currently available for identifying and characterizing protein-protein interactions [1-4]. Although very powerful, this procedure exhibits several problems and inherent limitations [5]. A new system, the Sos recruitment system (SRS), was developed recently [6] based on a different readout from that of the two-hybrid system [6-8]. SRS overcomes several of the limitations of the two-hybrid system and thus serves as an attractive alternative for studying protein-protein interactions between known and novel proteins. Nevertheless, we encountered a number of problems using SRS and so have developed an improved protein recruitment system, designated the Ras recruitment system (RRS), based on the absolute requirement that Ras be localized to the plasma membrane for its function [9-10]. Ras membrane localization and activation can be achieved through interaction between two hybrid proteins. We have demonstrated the effectiveness of the novel RRS system using five different known protein-protein interactions and have identified two previously unknown protein-protein interactions through a library screening protocol. The first interaction (detailed here) is between JDP2, a member of the basic leucine zipper (bZIP) family, and C/EBPgamma, a member of the CCAAT/enhancer-binding protein (C/EBP) family. The second interaction is between the p21-activated protein kinase Pak65 and a small G protein (described in the accompanying paper by Aronheim et al. [11]). The RRS system significantly extends the usefulness of the previously described SRS system and overcomes several of its limitations.     

A hybrid method for extraction of protein-protein interactions from literature

In this work, a hybrid method is proposed to eliminate the limitations of traditional protein-pro- tein interactions (PPIs) extraction methods, such as pattern learning and machine learning. Each sentence from the biomedical literature containing a protein pair describes a PPI which is predicted by first learning syntax patterns typical of PPIs from training corpus and then using their presence as features, along with bag-of-word features in a maximum entropy model. Tested on the BioCreAtIve corpus, the PPIs extraction method, which achieved a precision rate of 64%, recall rate of 60%, improved the performance in terms of F1 value by 11% compared with the component pure pattern- based and bag-of-word methods. The results on this test set were also compared with other three ex- traction methods and found to improve the performance remarkably.     

Structure-based engineering of environmentally sensitive fluorophores for monitoring protein-protein interactions

Single, extrinsic, environmentally sensitive fluorophores can be used to quantitate formation of protein-protein complexes. These can be prepared semi-synthetically by covalent coupling to single cysteine mutations introduced at positions where the fluorophore is predicted to respond to formation of the complex without adversely affecting the interaction. The three-dimensional structure of a protein-protein interface can be used to select such locations by identifying residues that are located at the edge of a buried interfacial region, and are in partial steric contact with both partners as indicated by a change in their static solvent-accessible surface area upon complex formation. Using this design approach, cysteine mutations were introduced into the B1 domain of protein G, which successfully monitor complex formation with minimal interference. Such constructs have great utility in the analysis of solution properties of interface mutants.     

The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing

The splicing factor U2AF (U2 snRNP auxiliary factor) is a heterodimer with subunits of 65 and 35 kD (U2AF65 and U2AF35). U2AF65 binds specifically to 3' splice sites, but previous studies failed to demonstrate a function for U2AF35. Here, we report that U2AF35 is required for constitutive splicing and also functions as a mediator of enhancer-dependent splicing. Nuclear extracts deficient in U2AF35 were inactive; however, both constitutive and enhancer-dependent splicing could be restored by the addition of purified recombinant U2AF35. In vitro protein-RNA interaction studies with pre-mRNAs containing either a constitutive or regulated splicing enhancer revealed that U2AF35 directly mediates interactions between U2AF65 and proteins bound to the enhancers. Thus, U2AF35 functions as a bridge between U2AF65 and the enhancer complex to recruit U2AF65 to the adjacent intron.     

Molecular mechanisms for focal adhesion assembly through regulation of protein-protein interactions

Focal adhesions provide a useful model for studying cell/extracellular matrix interactions and the subsequent cytoskeletal reorganization. Recent advances have suggested potential mechanisms by which cells may regulate focal adhesion assembly following integrin-mediated cell adhesion.     

Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein

The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.     

The G beta gamma complex of the yeast pheromone response pathway. Subcellular fractionation and protein-protein interactions

Genetic evidence suggests that the yeast STE4 and STE18 genes encode G beta and G gamma subunits, respectively, that the G betagamma complex plays a positive role in the pheromone response pathway, and that its activity is subject to negative regulation by the G alpha subunit (product of the GPA1 gene) and to positive regulation by cell-surface pheromone receptors. However, as yet there is no direct biochemical evidence for a G betagamma protein complex associated with the plasma membrane. We found that the products of the STE4 and STE18 genes are stably associated with plasma membrane as well as with internal membranes and that 30% of the protein pool is not tightly associated with either membrane fraction. A slower-migrating, presumably phosphorylated, form of Ste4p is enriched in the non-membrane fraction. The Ste4p and Ste18p proteins that had been extracted from plasma membranes with detergent were found to co-sediment as an 8 S particle under low salt conditions and as a 6 S particle in the presence of 0.25 M NaCl; the Ste18p in these fractions was precipitated with anti-Ste4p antiserum. Under the conditions of our assay, Gpa1p was not associated with either particle. The levels of Ste4p and Ste18p accumulation in mutant cells provided additional evidence for a G betagamma complex. Ste18p failed to accumulate in ste4 mutant cells, and Ste4p showed reduced levels of accumulation and an increased rate of turnover in ste18 mutant cells. The gpa1 mutant blocked stable association of Ste4p with the plasma membrane, and the ste18 mutant blocked stable association of Ste4p with both plasma membranes and internal membranes. The membrane distribution of Ste4p was unaffected by the ste2 mutation or by down-regulation of the cell-surface receptors. These results indicate that at least 40% of Ste4p and Ste18p are part of a G betagamma complex at the plasma membrane and that stable association of this complex with the plasma membrane requires the presence of G alpha.     

Structure and function of bovine growth hormone. Bovine growth hormone as an experimental model for studies of protein-protein interactions

Growth hormone (GH) is a polipeptide that controls the differentiation, growth and metabolism of many cell types, and is secreted from the hypophysis of all vertebrate species tested so far. Despite the overlapping evolutionary, structural, immunological and biological properties, it is well-known that GHs from distinct mammalian species have significant species-specific characteristics. The main purpose of this review is to highlight bovine GH (bGH) structural features related to its species-specific properties. Novel interest in bGH is also aroused by the advent of biotechnological methods for production of recombinant proteins. In fact recombinant bGH will have a great importance in veterinary medicine research and as a 'high tech' drug that needs to be monitored in zootechnical productions.     

Asparaginylendopeptidase: an enzyme probably responsible to post-translational proteolysis and transpeptidation of proconcanavalin A

Nineteen ewes were injected subcutaneously with the agent of enzootic ovine abortion, Chlamydia psittaci serovar 1, at 50 days gestation. Placental and fetal tissues were examined at 15 days postinfection and thereafter at ten day intervals. Placental infection was detected at 15 days postinfection. Only postinoculation sera collected from postinfected ewes contained antibodies reactive to C. psittaci. Five (26%) chlamydial infected ewes experienced inapparent fetal loss before day 105 of gestation. This finding is significant since C. psittaci infection in sheep is commonly associated with abortion and not infertility.     

Understanding parent-adolescent interactions: Perceptions of family members and outsiders.

To see whether adolescents view parent–child relationships more like their parents or more like outsiders, 41 mother–father–adolescent child triads were videotaped interacting over two tasks. Each family member then viewed both interactions and rated levels of anxiety, dominance, involvement, and friendliness, for each interactant. These same interactions were also watched and rated by another mother–father–adolescent triad (outsider family) who did not know the family. These interactions were then rated by a trained observer. For ratings of anxiety, dominance, involvement, and friendliness, analyses involved comparisons (a) between the insider and outsider families ratings, on correlations between insider family members and outsider family members with the trained observer, and (b) comparisons among the three types of observers. Ratings by members of the outsider family were generally more negative than those by members of the insider family. Ratings of self tended to be more objective than ratings of other family members. Ratings by the trained observer were more highly correlated with those of the outsider family, although differences between the means showed that trained observers used a similar metric to the insider family. The importance of looking at patterns of ratings as well as mean differences is emphasized. (PsycINFO Database Record (c) 2010 APA, all rights reserved)