MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

Phage display selection of ligand residues important for Src homology 3 domain binding specificity.

The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.

Analysis of homeodomain function by structure-based design of a transcription factor.

The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

Signal transduction in T lymphocytes using a conditional allele of Sos.

While Ras activation has been shown to play an important role in signal transduction by the T-lymphocyte antigen receptor, the mechanism of its activation in T cells is unclear. Membrane localization of the guanine nucleotide exchange factor Sos, but not Vav or Dbl, was sufficient for Ras-mediated signaling in T lymphocytes. Activation of Sos appears to involve membrane recruitment and not allosteric changes, because interaction of Sos with the linking molecule Grb-2 was not required for Ras activation. To extend this analysis, we constructed a modified Sos that could be localized to the membrane inducibly by using a rationally designed chemical inducer of dimerization, FK1012. The role of Grb-2 in signaling was mimicked with this technique, which induced the association of a modified Sos with the membrane, resulting in rapid activation of Ras-induced signaling. In contrast, inducible localization of Grb-2 to the membrane did not activate signaling and suggests that the interaction of Grb-2 with Sos in T cells is subject to regulation. This conditional allele of Sos demonstrates that membrane localization of Sos is sufficient for Ras activation in T cells and indicates that the role of Grb-2 is to realize the biologic advantages of linker-mediated dimerization: enhanced specificity and favorable kinetics for signaling. This method of generating conditional alleles may also be useful in dissecting other signal transduction pathways regulated by protein localization or protein-protein interactions.

Retinoid X receptor alpha forms tetramers in solution.

Protein-protein interactions allow the retinoid X receptor (RXR) to bind to cognate DNA as a homo- or a heterodimer and to participate in mediating the effects of a variety of hormones on gene transcription. Here we report a systematic study of the oligomeric state of RXR in the absence of a DNA template. We have used electrophoresis under nondenaturing conditions and chemical crosslinking to show that in solution, RXR alpha forms homodimers as well as homotetramers. The dissociation constants governing dimer and tetramer formation were estimated by fluorescence anisotropy studies. The results indicate that RXR tetramers are formed with a high affinity and that at protein concentrations higher than about 70 nM, tetramers will constitute the predominant species. Tetramer formation may provide an additional level of the regulation of gene transcription mediated by RXRs.

A single GAL4 dimer can maximally activate transcription under physiological conditions.

Most eukaryotic promoters contain multiple binding sites for one or more transcriptional activators that interact in a synergistic manner. A common view is that synergism is a manifestation of the need for many contacts between activators and the general transcription machinery that a single activator presumably cannot fulfill. In this model, various combinations of protein-protein interactions control the level of gene expression. However, we show here that under physiological conditions, a single binding site and presumably GAL4 can activate transcription to the maximum possible level in vivo. Synergistic effects in this natural system are shown to be consistent with cooperative DNA binding. These results point to DNA occupancy as the major element in fine tuning gene expression in the galactose regulon.

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs.

Mapeamento global de interações proteicas nas vias de sinalização mediadas por c-di-GMP de Pseudomonas aeruginosa

A persistência bacteriana correlacionada à formação de biofilmes bacterianos é, há algum tempo, fonte de grande preocupação médica em virtude de sua ampla associação com a dificuldade de tratamento de infecções crônicas. Por outro lado, as perspectivas de utilização de biofilmes bacterianos em novas aplicações biotecnológicas e até mesmo para fins terapêuticos são promissoras. Há, portanto, grande interesse em compreender os mecanismos que levam as células bacterianas a deixar o estado planctônico, de vida livre, e associarem-se nesses conglomerados celulares altamente complexos. Ao longo das últimas décadas, o segundo mensageiro c-di-GMP – em conjunto com as moléculas que catalisam sua síntese (diguanilato ciclases) e sua degradação (fosfodiesterases) e seus receptores – estabeleceu-se como um elemento central de regulação de uma série de respostas celulares que determinam a formação ou a dispersão de biofilmes. Curiosamente, as proteínas que participam do metabolismo deste segundo mensageiro estão, frequentemente, codificadas múltiplas vezes em um mesmo genoma bacteriano. Em vista dessa observação, estudos mais recentes apontam que, para reger paralelamente uma variedade tão ampla de fenótipos, este sistema opera em modo de alta especificidade de sinalização e que, portanto, o sinal metabolizado por determinados conjuntos de diguanilato ciclases e fosfodiesterases tem alvos celulares específicos. Evidências robustas, porém isoladas até o momento, apontaram que um dos meios pelo qual ocorre a segregação entre sinal produzido e alvo específico é a interação direta entre as proteínas componentes das vias de sinalização. Mais, demonstrou-se que, em algumas vias, a transmissão de sinal ocorre exclusivamente via interação proteica, dispensando a intermediação do sinalizador em si. Para avaliar a validade e relevância global deste mecanismo, propôs-se, neste estudo, a investigação da rede total de interações entre as proteínas tipicamente associadas às vias de sinalização por c-di-GMP em Pseudomonas aeruginosa, utilizando ensaios de duplo-hibrido bacteriano. Para tanto, foram construídas duas bibliotecas de DNA direcionadas e foram feitos testes de interação de forma estratégica para possibilitar o esgotamento e averiguação de todas as possíveis interações entre as proteínas alvo identificadas. O resultado obtido, um mapa inicial, porém abrangente, da rede de interações proteicas em P. aeruginosa, indica uma grande probabilidade de que os mecanismos previamente descritos sejam realmente recorrentes e relevantes para o intermédio da sinalização nesse organismo. Algumas das interações mais robustas encontradas são bastante interessantes e serão, em estudos futuros, mais extensivamente estudadas.

Componentes genéticos que afetam a via de direcionamento de proteínas organelares em Arabidopsis thaliana

Nos eucariotos, a evolução dos sistemas de transporte molecular foi essencial pois seu alto grau de compartimentalização requer mecanismos com maior especificidade para a localização de proteínas. Com o estabelecimento das mitocôndrias e plastídeos como organelas da célula eucariota, grande parte dos genes específicos para sua atividade e manutenção foram transferidos ao núcleo. Após a transferência gênica, a maioria das proteínas passaram a ser codificadas pelo núcleo, sintetizadas no citosol e direcionadas às organelas por uma maquinaria complexa que envolve receptores nas membranas das organelas, sequências de direcionamento nas proteínas e proteínas citossólicas que auxiliam o transporte. A importação depende em grande parte de uma sequência na região N-terminal das proteínas que contém sinais reconhecidos pelas membranas organelares. No entanto, muito ainda não é compreendido sobre o transporte de proteínas organelares e fatores ainda desconhecidos podem influenciar o direcionamento sub-celular. O objetivo deste trabalho foi a caracterização da General Regulatory Factor 9 (GRF9), uma proteína da família 14-3-3 de Arabidopsis thaliana potencialmente envolvida no direcionamento de proteínas organelares, e a geração de um genótipo para ser utilizado na obtenção de uma população mutante para genes que afetam o direcionamento da proteína Tiamina Monofosfato Sintetase (TH-1). Após experimentos in vivo e in planta, foi observado que GRF9 interage com as proteínas duplo-direcionadas Mercaptopyruvate Sulfurtransferase1 (MST1) e a Thiazole Biosynthetic Enzyme (THI1), e com a proteína direcionada aos cloroplastos TH-1. Experimentos de deleção e interação in vivo mostraram que a região Box1 de GRF9 é essencial para a interação com THI1 e MST1. Com a finalidade de dar continuidade a caracterização da GRF9 e para realização de testes com relação a sua função no direcionamento de proteínas organelares foi gerada uma linhagem homozigota que superexpressa GRF9. Plantas expressando o transgene TH-1 fusionado a Green Fluorescent Protein (GFP) em genótipo deficiente na TH-1 (CS3469/TH-1-GFP) foram obtidas para a geração de população mutante que possibilitará a descoberta de componentes genéticos ainda desconhecidos e responsáveis pelo direcionamento de proteínas aos cloroplastos.

Reductionism at the vertebrate kinetochore

The kinetochore forms the site of attachment for mitotic spindle microtubules driving chromosome segregation. The interdependent protein interactions in this large structure have made it difficult to dissect the function of its components. In this issue, Hori et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201210106) present a novel and powerful methodology to address the sufficiency of individual proteins for the creation of a functional de novo centromere.

Gene duplication and hierarchical modularity in intracellular interaction networks

Networks of interactions evolve in many different domains. They tend to have topological characteristics in common, possibly due to common factors in the way the networks grow and develop. It has been recently suggested that one such common characteristic is the presence of a hierarchically modular organization. In this paper, we describe a new algorithm for the detection and quantification of hierarchical modularity, and demonstrate that the yeast protein-protein interaction network does have a hierarchically modular organization. We further show that such organization is evident in artificial networks produced by computational evolution using a gene duplication operator, but not in those developing via preferential attachment of new nodes to highly connected existing nodes. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Interpretation of the temperature dependence of rate constants in biosensor studies

A comparison is made between Arrhenius and transition-state analyses of the temperature dependence of rate constants reported in four published biosensor studies. Although the Eyring transition-state theory seemingly affords a more definitive solution to the problem of characterizing the activation energetics, the analysis is equivocal because of inherent assumptions about reaction mechanism and the magnitude of the transmission coefficient. In view of those uncertainties it is suggested that a preferable course of action entails reversion to the empirical Arrhenius analysis with regard to the energy of activation and a preexponential factor. The former is essentially equivalent to the enthalpy of activation, whereas the magnitude of the latter indicates directly the extent of disparity between the frequency of product formation and the universal frequency factor (temperature multiplied by the ratio of the Boltzmann and Planck constants) and hence the likelihood of a more complicated kinetic mechanism than that encompassed by the Eyring transition-state theory. (C) 2004 Elsevier Inc. All rights reserved.

Comparative proteomic analysis to study molecular events during gonad development in mice

Sex determination represents a critical bifurcation in the road of embryonic development. It is based on a finely regulated network of gene activity, as well as protein-protein interactions and activation or silencing of signaling pathways. Despite the identification of a number of critical genes, many aspects of the molecular cascade that drives the differentiation of the embryonic gonad into either a testis or an ovary remain poorly understood. To identify new proteins involved in this cascade, we employed two-dimensional gel electrophoresis and mass spectrometry to compare the protein expression profiles of fetal mouse testes and ovaries. Three proteins, hnRPA1, TRA1, and HSC71, were found to be expressed in a male-specific manner and this expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Moreover, HSC71 was found to be hyperphosphorylated in male compared to female gonads, emphasizing the advantage of the proteomic approach in allowing the detection of posttranslational modifications.

Interpretation of the temperature dependence of equilibrium and rate constants

The objective of this review is to draw attention to potential pitfalls in attempts to glean mechanistic information from the magnitudes of standard enthalpies and entropies derived from the temperature dependence of equilibrium and rate constants for protein interactions. Problems arise because the minimalist model that suffices to describe the energy differences between initial and final states usually comprises a set of linked equilibria, each of which is characterized by its own energetics. For example, because the overall standard enthalpy is a composite of those individual values, a positive magnitude for AHO can still arise despite all reactions within the subset being characterized by negative enthalpy changes: designation of the reaction as being entropy driven is thus equivocal. An experimenter must always bear in mind the fact that any mechanistic interpretation of the magnitudes of thermodynamic parameters refers to the reaction model rather than the experimental system For the same reason there is little point in subjecting the temperature dependence of rate constants for protein interactions to transition-state analysis. If comparisons with reported values of standard enthalpy and entropy of activation are needed, they are readily calculated from the empirical Arrhenius parameters. Copyright (c) 2006 John Wiley & Sons, Ltd.

Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking

We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 x 10(11) possible conformations. While cross-links represent only limited structural constraints, the combination of only three experimental cross-links with very basic molecular docking was sufficient to determine the complex structure. The crystal structure of the complex between latexin and carboxypeptidase A4 determined recently allowed us to assess the success of this structure determination approach. Our structure was shown to be within 4 angstrom r.m.s. deviation of C alpha atoms of the crystal structure. The study demonstrates that cross-linking in combination with mass spectrometry can lead to efficient and accurate structural modelling of protein complexes.