Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.     

蛋白质结构的“限域下最低能量结构片段”假说与蛋白质进化的“石器时代”

蛋白质折叠问题被称为第二遗传密码,至今未破译;蛋白质序列的天书仍然是"句读之不知,惑之不解"。在最近工作的基础上,我们提出了蛋白质结构的"限域下最低能量结构片段"假说。这一假说指出,蛋白质中存在一些关键的长程强相互作用位点,这些位点相当于标点符号,将蛋白质序列的天书变成可读的句子(多肽片段)。这些片段的天然结构是在这些强长程相互作用位点限域下的能量最低状态。完整的蛋白质结构由这些"限域下最低能量结构片段"拼合而成,而蛋白质整体结构并不一定是全局性的能量最低状态。在蛋白质折叠过程中,局部片段的天然结构倾向性为强长程相互作用的形成提供主要基于焓效应的驱动力,而天然强长程相互作用的形成为局部片段的天然结构提供主要基于熵效应的稳定性。在蛋白质进化早期,可能存在一个"石器时代",即依附不同界面(比如岩石)的限域作用而稳定的多肽片段先进化出来,后由这些片段逐步进化(包括拼合)而成蛋白质。     

Metal sensor proteins: nature's metalloregulated allosteric switches

Metalloregulatory proteins control the expression of genes that allow organisms to quickly adapt to chronic toxicity or deprivation of both biologically essential metal ions and heavy metal pollutants found in their microenvironment. Emerging evidence suggests that metal ion homeostasis and resistance defines an important tug-of-war in human host-bacterial pathogen interactions. This adaptive response originates with the formation of "metal receptor" complexes of exquisite selectivity. In this perspective, we summarize consensus structural features of metal sensing coordination complexes and the evolution of distinct metal selectivities within seven characterized metal sensor protein families. In addition, we place recent efforts to understand the structural basis of metal-induced allosteric switching of these metalloregulatory proteins in a thermodynamic framework, and review the degree to which coordination chemistry drives changes in protein structure and dynamics in selected metal sensor systems. New insights into how metal sensor proteins function in the complex intracellular milieu of the cytoplasm of cells will require a more sophisticated understanding of the "metallome" and will benefit greatly from ongoing collaborative efforts in bioinorganic, biophysical and analytical chemistry, structural biology and microbiology.     

Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives of Sinorhizobium meliloti strain 2011

Sinorhizobium meliloti was studied by proteomic analysis to investigate the contribution made by plasmid-encoded functions on the intracellular regulation of this bacterium. Protein profiles of strain 2011 were compared with those from its mutant strains which were either cured of their pRme2011a (also called pSyma) plasmid (strain 818), or contained an extensive deletion of this plasmid (strain SmA146). Plasmid pSyma contains the nodulation and nitrogen fixation genes and is 1.4 Mbp with an estimated coding potential of 1,400 proteins. However, under the growth conditions used we could detect 60 differences between the parent strain and its pSyma-cured derivative, strain 818. While the majority of these differences were due to regulatory changes, such as up- and downregulation, some proteins were totally missing in some strains. These 60 proteins were classified into 21 subgroups, A to U, based on their measured protein levels when the cells were grown in the presence or absence of luteolin. Comparisons were made between the different strains to assess the possible interactions of the different proteins of the subgroups and plasmid pSyma. These results suggest that pSyma has a role in the regulation of the expression of genes from the other replicons (3.5 Mbp chromosome and the 1.7 Mbp pSymB plasmid) present in the S. meliloti cells. Proteome analysis provides a sensitive tool to examine the functional organisation of the S. meliloti genome and the intracellular gene interactions between replicons and will provide a powerful analytical tool to complement the genome sequencing of strain 1021.     

Rooteomics: the challenge of discovering plant defense-related proteins in roots

In recent years, a strong emphasis has been given in deciphering the function of genes unraveled by the completion of several genome sequencing projects. In plants, functional genomics has been massively used in order to search for gene products of agronomic relevance. As far as root-pathogen interactions are concerned, several genes are recognized to provide tolerance/resistance against potential invaders. However, very few proteins have been identified by using current proteomic approaches. One of the major drawbacks for the successful analysis of root proteomes is the inherent characteristics of this tissue, which include low volume content and high concentration of interfering substances such as pigments and phenolic compounds. The proteome analysis of plant-pathogen interactions provides important information about the global proteins expressed in roots in response to biotic stresses. Moreover, several pathogenic proteins superimpose the plant proteome and can be identified and used as targets for the control of viruses, bacteria, fungi and nematode pathogens. The present review focuses on advances in different proteomic strategies dedicated to the challenging analysis of plant defense proteins expressed during bacteria-, fungi- and nematode-root interactions. Recent developments, limitations of the current techniques, and technological perspectives for root proteomics aiming at the identification of resistance-related proteins are discussed.     

Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake

Delivery and toxicity are critical issues facing nanomedicine research. Currently, there is limited understanding and connection between the physicochemical properties of a nanomaterial and its interactions with a physiological system. As a result, it remains unclear how to optimally synthesize and chemically modify nanomaterials for in vivo applications. It has been suggested that the physicochemical properties of a nanomaterial after synthesis, known as its "synthetic identity", are not what a cell encounters in vivo. Adsorption of blood components and interactions with phagocytes can modify the size, aggregation state, and interfacial composition of a nanomaterial, giving it a distinct "biological identity". Here, we investigate the role of size and surface chemistry in mediating serum protein adsorption to gold nanoparticles and their subsequent uptake by macrophages. Using label-free liquid chromatography tandem mass spectrometry, we find that over 70 different serum proteins are heterogeneously adsorbed to the surface of gold nanoparticles. The relative density of each of these adsorbed proteins depends on nanoparticle size and poly(ethylene glycol) grafting density. Variations in serum protein adsorption correlate with differences in the mechanism and efficiency of nanoparticle uptake by a macrophage cell line. Macrophages contribute to the poor efficiency of nanomaterial delivery into diseased tissues, redistribution of nanomaterials within the body, and potential toxicity. This study establishes principles for the rational design of clinically useful nanomaterials.     

Computational prediction of miRNA/mRNA duplexomes at the whole human genome scale reveals functional subnetworks of interacting genes with embedded miRNA annealing motifs

Perfect annealing between microRNAs (miRNAs) and messenger RNAs (mRNAs) was computationally searched at a broad scale in the human genome to determine whether theoretical pairing is restrictively represented in functional subnetworks or is randomly distributed. Massive RNA interference (RNAi) pairing motifs in genes constitute a remarkable subnetwork that displays highly genetically and biochemically interconnected genes. These analyses show unexpected repertoires of genes defined by their congruence in comatching with miRNAs at numerous sites and by their interconnection based on protein/protein interactions or proteins regulating the activity of others. This offers insights into the putatively coregulated homeostasis of large networks of genes by RNAi, whereas other networks seem to be independent of this regulatory mode. Genes accordingly defined by theoretical RNAi pairing cluster mainly in subnetworks related to cellular, metabolic and developmental processes and their regulation. Indeed, genes harboring numerous potential sites of hybridization with miRNAs are highly enriched with GO terms depicting the abovementioned processes and are grouped in a subnetwork of genes that are significantly more highly connected than they would be according to a random distribution. The significant number of interacting genes that present numerous potential comatches with miRNAs suggests that they may be under the control of the integrative and concerted action of multiple miRNAs.     

Expanding the utility of proteins as platforms for coordination chemistry

Whether for constructing advanced materials and complex biological devices or for building sophisticated coordination complexes with diverse metal-based functions, proteins are nature's favorite building blocks. Yet, our ability to control the assembly of proteins or to use them as ligand platforms for inorganic chemistry has been somewhat limited. In this review, we highlight our work from the past four years, which has aimed to exploit the utility of a protein scaffold in both regards. First, by considering proteins as “simple” ligand platforms and controlling the metal coordination chemistry on their surfaces, we show how their self-assembly can be readily dictated by metal binding. Second, we show how metal-mediated protein self-assembly leads to novel metal centers buried within protein interfaces. While on one hand our studies have pointed out the challenges of using proteins as ligands, they have also revealed how the extensive, chemically-rich protein surfaces can be exploited to form a network of covalent and non-covalent interactions around interfacial metal centers, providing a powerful handle to control their coordination chemistry.     

Advanced analytical tools in proteomics

Proteomics deals with the study of proteins, their structures, localizations, posttranslational modifications, functions and interactions with other proteins. The mapping of protein structure-function holds the key to a better understanding of cellular functions under both normal and disease states, which is critical for modern drug discovery. However, the study of human proteome presents scientists with a task much more daunting than the human genome project. In fact, the estimated >100,000 different proteins expressed from 30,000 to 40,000 human genes make it extremely challenging, if not impossible with existing protein analysis techniques, to map the entire cellular functions at the translational level. Consequently, there have been rapid advances in the techniques and methods capable of large-scale proteomic studies. Among them, the recently developed high-throughput screening methods have enabled scientists to analyze proteins quickly and efficiently at an organism-wide scale. Herein, we overview some of these emerging tools for high-throughput protein analysis. In particular, we focus on recent advances in the bioassay development, which has provided sensitive and selective tools for high-throughput identification and characterizations of enzymes. Finally, the recently developed bioimaging techniques to visualize and quantify proteins in living cells are also discussed.     

Vertebrate 2xRBD hnRNP proteins: a comparative analysis of genome, mRNA and protein sequences

hnRNP proteins are involved in many cell functions, primarily in pre-mRNA processing. We report here a comparative analysis of the genes of the 2xRBD members of the hnRNP family and of their expression products. Starting from the seven well characterized hnRNP members of human and murine origin (A0, A1, A2/B1, A3, AB, D and DL) and the three MuSashI-like proteins with related RBD tandems (MSI1, MSI2 and DAZAP1), we identified through BLAST search 12 homologous genes in the genome of Danio rerio and 10 in the genome of Takifugu rubripes, which can be divided into three subgroups, each with its highly conserved exon/intron structure, matching perfectly the exon/intron structures found in human and mouse genes. An exception is the gene of hnRNP A0, which is intronless consistently in all the four species. The analysis has been supported also at the level of cDNA and EST databases and extended in this respect to other vertebrate species, namely chicken, Xenopus laevis and Silurana tropicalis. PHYLIP 3.62 package (SEQBOOT, PROTDIST/DNADIST, NEIGHBOR, CONSENSE) was used for all the proteins and their CDSs and human RBDs I and II to infer relevant aspects of the phylogenesis of these proteins. Some clues to the evolution of introns in these genes have come from the analysis of their distribution in homologous genes of other eukaryotes, namely Ciona, Drosophila, Caenorhabditis, Saccharomyces and Arabidopsis.     

Analysis of aging-related protein interactome and cross-network module comparisons across tissues provide new insights into aging

Delaying the human aging process and thus eliminating the risk factors for age-related diseases is one of the prime objectives. While various aging-associated genes and proteins have been characterized, which provide a significant understanding of the human aging process, a significant success in regulating aging is not achieved yet. Understanding how aging proteins interact with each other and also with other proteins could provide important insights into the underlying mechanisms governing the aging process. Therefore, in this work, information of gene expression was included to the static aging-related protein interactome to understand the network-based relationships among aging-related essential (AE) proteins, aging-related non-essential (ANE) proteins, and housekeeping-proteins that could regulate or influence aging. Comprehensive analyses provided various systems-level insights into the regulatory characteristics of aging; for example, (i) network-based correlation analysis predicted functional relationships among AE proteins and ANE proteins; (ii) network variability analysis predicted aging to affect different tissues in strikingly different ways by differentially regulating various regulatory interactions; (iii) cross-network comparisons identified two aging-related modules to be significantly conserved across most of the tissues. Overall, the findings obtained during this study could be helpful for researchers to delay, prevent, or even reverse various aspects of the aging.     

The importance of questioning scientific assumptions: some lessons from f element chemistry

As scientists, we know that we should constantly question the assumptions upon which our research is based. We also know that we do not do this often enough. The recent results in f element chemistry described here should serve to remind us not to take the traditional boundaries of any area of chemistry for granted including topics as fundamental as redox chemistry and bond-length generalizations. New ways of doing reductive chemistry in the f element area as well as the synthesis of "long bond organometallics" that have unconventional bond distances and reactivity demonstrate how the "rules" in this area, thought to be true for decades, have been recently overturned. The synthetic chemistry that made these advances possible has generated additional unexpected opportunities in f element chemistry that are also described here. Overall, these results should stimulate researchers in all areas to challenge their assumptions.     

Repair of Iron Center Proteins—A Different Class of Hemerythrin-like Proteins

Repair of Iron Center proteins (RIC) form a family of di-iron proteins that are widely spread in the microbial world. RICs contain a binuclear nonheme iron site in a four-helix bundle fold, two basic features of hemerythrin-like proteins. In this work, we review the data on microbial RICs including how their genes are regulated and contribute to the survival of pathogenic bacteria. We gathered the currently available biochemical, spectroscopic and structural data on RICs with a particular focus on Escherichia coli RIC (also known as YtfE), which remains the best-studied protein with extensive biochemical characterization. Additionally, we present novel structural data for Escherichia coli YtfE harboring a di-manganese site and the protein’s affinity for this metal. The networking of protein interactions involving YtfE is also described and integrated into the proposed physiological role as an iron donor for reassembling of stress-damaged iron-sulfur centers.     

Molecular Determinants of Bim(BH3) Peptide Binding to Pro-Survival Proteins

Proteins of the Bcl-2 family regulate apoptosis through the formation of heterodimers between antiapoptotic or pro-survival proteins and proapoptotic or pro-death proteins. Overexpression of antiapoptotic proteins not only contributes to the progression of many cancers, but also confers resistance to the chemo- and radiotherapeutic treatments. It has been demonstrated that peptides containing the BH3 domain of proapoptotic Bcl-2 family members are able to bind and inhibit antiapoptotic proteins. For this reason, the design of small molecules mimicking the BH3 domain of proapoptotic proteins has emerged as a promising therapeutic strategy for cancer treatment during the last years. However, BH3 domains exhibit different affinities for binding to antiapoptotic proteins; whereas Bim(BH3) and Puma(BH3) are able to bind all antiapoptotic proteins, others like Bad(BH3) and Bmf(BH3) show preference for some proteins over others. Consequently, the ability of a BH3-mimetic to kill tumor cells will depend on the BH3 peptide used as template and thus will have a selective or pan-inhibition effect. Recently, it has been suggested that this last approach could be interesting. Therefore, the present work is aimed to elucidate how the nonselective peptide Bim(BH3) is able to bind to all of the Bcl-2 family antiapoptotic proteins. To unravel the molecular determinants of this pan-inhibition, we used the MM-PB/GBSA approaches to calculate the binding free energy of the different complexes studied and to determine which residues of the peptide have the largest contribution to complex formation. Results obtained in the present work show that the binding of Bim(BH3) to pro-survival proteins is mainly hydrophobic and that specific interactions are fully distributed along the peptide sequence.     

Advancing formaldehyde cross-linking towards quantitative proteomic applications

Formaldehyde is a key fixation reagent. This review explores its application in combination with qualitative and quantitative mass spectrometry (MS). Formalin-fixed and paraffin-embedded (FFPE) tissues form a large reservoir of biologically valuable samples and their investigation by MS has only recently started. Furthermore, formaldehyde can be used to stabilise protein-protein interactions in living cells. Because formaldehyde is able to modify proteins, performing MS analysis on these samples can pose a challenge. Here we discuss the chemistry of formaldehyde cross-linking, describe the problems of and progress in these two applications and their common aspects, and evaluate the potential of these methods for the future.     

Engineering a beta-sheet protein toward the folding speed limit

Recent experimental studies have shown that alpha-helical proteins can approach the folding "speed limit", where folding switches from an activated to a downhill process in free energy. beta-sheet proteins are generally thought to fold more slowly than helix bundles. However, based on studies of hairpins, folding should still be able to approach the microsecond time scale. Here we demonstrate how the hPin1 WW domain, a triple-stranded beta-sheet protein with a sharp thermodynamic melting transition, can be engineered toward the folding "speed limit" without a significant loss in thermal denaturation cooperativity.     

From Valleys to Ridges: Exploring the Dynamic Energy Landscape of Single Membrane Proteins

Membrane proteins are involved in essential biological processes such as energy conversion, signal transduction, solute transport and secretion. All biological processes, also those involving membrane proteins, are steered by molecular interactions. Molecular interactions guide the folding and stability of membrane proteins, determine their assembly, switch their functional states or mediate signal transduction. The sequential steps of molecular interactions driving these processes can be described by dynamic energy landscapes. The conceptual energy landscape allows to follow the complex reaction pathways of membrane proteins while its modifications describe why and how pathways are changed. Single‐molecule force spectroscopy (SMFS) detects, quantifies and locates interactions within and between membrane proteins. SMFS helps to determine how these interactions change with temperature, point mutations, oligomerization and the functional states of membrane proteins. Applied in different modes, SMFS explores the co‐existence and population of reaction pathways in the energy landscape of the protein and thus reveals detailed insights into local mechanisms, determining its structural and functional relationships. Here we review how SMFS extracts the defining parameters of an energy landscape such as the barrier position, reaction kinetics and roughness with high precision.