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The proteomes of neurotransmitter receptor complexes form modular networks with distributed functionality underlying plasticity and behaviour.

Neuronal synapses play fundamental roles in information processing, behaviour and disease. Neurotransmitter receptor complexes, such as the mammalian N-methyl-D-aspartate receptor complex (NRC/MASC) comprising 186 proteins, are major components of the synapse proteome. Here we investigate the organisation and function of NRC/MASC using a systems biology approach. Systematic annotation showed that the complex contained proteins implicated in a wide range of cognitive processes, synaptic plasticity and psychiatric diseases. Protein domains were evolutionarily conserved from yeast, but enriched with signalling domains associated with the emergence of multicellularity. Mapping of protein-protein interactions to create a network representation of the complex revealed that simple principles underlie the functional organisation of both proteins and their clusters, with modularity reflecting functional specialisation. The known functional roles of NRC/MASC proteins suggest the complex co-ordinates signalling to diverse effector pathways underlying neuronal plasticity. Importantly, using quantitative data from synaptic plasticity experiments, our model correctly predicts robustness to mutations and drug interference. These studies of synapse proteome organisation suggest that molecular networks with simple design principles underpin synaptic signalling properties with important roles in physiology, behaviour and disease.

Animals↗

A proteomic biosensor for enteropathogenic E. coli.

The study of proteins and the molecules with which they interact on an organismwide scale is critical to understanding basic biology, and understanding and improving human health. New platform technologies allowing label-free, quantitative array-based analysis of proteins are particularly desirable. We have developed an analytical technology, reflective interferometry (RI), which provides specific, rapid, and label-free optical detection of biomolecules in complex mixtures. In order to evaluate the suitability of RI for proteomics, we have prepared a series of arrays bearing the extracellular domain of the secreted enteropathogenic Escherichia coli (EPEC) protein Translocated Intimin Receptor (Tir). These arrays are able to selectively detect the extracellular domain of the protein Intimin, Tir's natural binding partner. Furthermore, we demonstrate the use of RI and Tir-functionalized arrays for the selective detection of EPEC directly from culture.

Animals↗

Modeling depression: social dominance-submission gene expression patterns in rat neocortex.

Gene expression profiles in the cortex of adult Long-Evans rats as a function of a stressful social loss and victory in inter-male fighting encounters were examined. This social dominance and subordination model has been postulated to simulate early changes in the onset of depression in the losers. Microarrays were fabricated containing 45mer oligonucleotides spotted in quadruplicate and representing 1178 brain-associated genes. Dynamic range, discrimination power, accuracy and reproducibility were determined with standard mRNA "spiking" studies. Gene expression profiles in dominant and subordinate animals were compared using a "universal" reference design [Churchill GA (2002) Fundamentals of experimental design for cDNA microarrays. Nat Genet 32 (Suppl):490-495]. Data were analyzed by significance analysis of microarrays using rank scores [Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116-5121; van de Wiel MA (2004) Significance analysis of microarrays using rank scores. Kwantitatieve Methoden 71:25-37]. Ontological analyses were then performed using the GOMiner algorithm [Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein JN (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 4(4):R28]. And finally, genes of special interest were further studied using quantitative reverse transcriptase polymerase chain reaction. Twenty-two transcripts were statistically significantly differentially expressed in the neocortex between dominant and subordinate animals. Ontological analyses revealed that significant gene changes were clustered primarily into functional neurochemical pathways associated with protein biosynthesis and cytoskeletal dynamics. The most robust of these were the increased expression of interleukin-18, heat shock protein 27, beta3-tubulin, ribosome-associated membrane protein 4 in subordinate animals. Interleukin-18 has been found to be over-expressed in human depression and panic disorder as well as other physiological stress paradigms [Takeuchi M, Okura T, Mori T, Akita K, Ohta T, Ikeda M, Ikegami H, Kurimoto M (1999) Intracellular production of interleukin-18 in human epithelial-like cell lines is enhanced by hyperosmotic stress in vitro. Cell Tissue Res 297(3):467-473] and heat shock proteins have been shown to be involved in the pathogenesis of many neurodegenerative and psychiatric disorders [Iwamoto K, Kakiuchi C, Bundo M, Ikeda K, Kato T (2004) Molecular characterization of bipolar disorder by comparing gene expression profiles of postmortem brains of major mental disorders. Mol Psychiatry 9(4):406-416; Pongrac JL, Middleton FA, Peng L, Lewis DA, Levitt P, Mirnics K (2004) Heat shock protein 12A shows reduced expression in the prefrontal cortex of subjects with schizophrenia. Biol Psychiatry 56(12):943-950]. Thus, the gene expression changes that we have observed here are consistent with and extend the observations found in the clinical literature and link them to the animal model used here thereby reinforcing its use to better understand the genesis of depression and identify novel therapeutic targets for its treatment.

Animals↗

Nova regulates brain-specific splicing to shape the synapse.

Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2-/- mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.

Alternative Splicing↗

A community resource benchmarking predictions of peptide binding to MHC-I molecules.

Recognition of peptides bound to major histocompatibility complex (MHC) class I molecules by T lymphocytes is an essential part of immune surveillance. Each MHC allele has a characteristic peptide binding preference, which can be captured in prediction algorithms, allowing for the rapid scan of entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, and chimpanzee MHC class I alleles. We use this data to establish a set of benchmark predictions with one neural network method and two matrix-based prediction methods extensively utilized in our groups. In general, the neural network outperforms the matrix-based predictions mainly due to its ability to generalize even on a small amount of data. We also retrieved predictions from tools publicly available on the internet. While differences in the data used to generate these predictions hamper direct comparisons, we do conclude that tools based on combinatorial peptide libraries perform remarkably well. The transparent prediction evaluation on this dataset provides tool developers with a benchmark for comparison of newly developed prediction methods. In addition, to generate and evaluate our own prediction methods, we have established an easily extensible web-based prediction framework that allows automated side-by-side comparisons of prediction methods implemented by experts. This is an advance over the current practice of tool developers having to generate reference predictions themselves, which can lead to underestimating the performance of prediction methods they are not as familiar with as their own. The overall goal of this effort is to provide a transparent prediction evaluation allowing bioinformaticians to identify promising features of prediction methods and providing guidance to immunologists regarding the reliability of prediction tools.

Animals↗

[Nutrition genomics].

The importance of nutrition for human health and its influence on the onset and course of many diseases are nowadays considered as proven. Only the recent development of molecular biology and biochemical methods allows the elucidation of the molecular mechanisms of diet constituent actions and their subsequent effect on homeostatic mechanisms in health and disease states. The availability of the draft human genome sequence as well as the genome sequences of model organisms, combined with the functional and integrative genomics approaches of systems biology, bring about the possibility to identify alleles and haplotypes responsible for specific reaction to the dietary challenge in susceptible individuals. Such complex interactions are studied within the newly conceived field, the nutrition genomics (nutrigenomics). Using the tools of highly parallel analyses of transcriptome, proteome and metabolome, the nutrition genomics pursues its ultimate goal, i.e. the individualized diet, respecting not only quantitative and qualitative nutritional needs and the actual health status, but also the genetic predispositions of an individual. This approach should lead to prevention of the onset of such diseases as obesity, hypertension or type 2 diabetes, or enhance the efficiency of their therapy.

Animals↗

The Karyote physico-chemical genomic, proteomic, metabolic cell modeling system.

Modeling approaches to the dynamics of a living cell are presented that are strongly based on its underlying physical and chemical processes and its hierarchical spatio-temporal organization. Through the inclusion of a broad spectrum of processes and a rigorous analysis of the multiple scale nature of cellular dynamics, we are attempting to advance cell modeling and its applications. The presentation focuses on our cell modeling system, which integrates data archiving and quantitative physico-chemical modeling and information theory to provide a seamless approach to the modeling/data analysis endeavor. Thereby the rapidly growing mess of genomic, proteomic, metabolic, and cell physiological data can be automatically used to develop and calibrate a predictive cell model. The discussion focuses on the Karyote cell modeling system and an introduction to the CellX and VirusX models. The Karyote software system integrates three elements: (1) a model-building and data archiving module that allows one to define a cell type to be modeled through its reaction network, structure, and transport processes as well as to choose the surrounding medium and other parameters of the phenomenon to be modeled; (2) a genomic, proteomic, metabolic cell simulator that solves the equations of metabolic reaction, transcription/translation polymerization and the exchange of molecules between parts of the cell and with the surrounding medium; and (3) an information theory module (ITM) that automates model calibration and development, and integrates a variety of data types with the cell dynamic computations. In Karyote, reactions may be fast (equilibrated) or slow (finite rate), and the special effects of enzymes and other minority species yielding steady-state cycles of arbitrary complexities are accounted for. These features of the dynamics are handled via rigorous multiple scale analysis. A user interface allows for an automated generation and solution of the equations of multiple timescale, compartmented dynamics. Karyote is based on a fixed intracellular structure. However, cell response to changes in the host medium, damage, development or transformation to abnormality can involve dramatic changes in intracellular structure. As this changes the nature of the cellular dynamics, a new model, CellX, is being developed based on the spatial distribution of concentration and other variables. This allows CellX to capture the self-organizing character of cellular behavior. The self-assembly of organelles, viruses, and other subcellular bodies is being addressed in a second new model, VirusX, that integrates molecular mechanics and continuum theory. VirusX is designed to study the influence of a host medium on viral self-assembly, structural stability, infection of a single cell, and transmission of disease.

Animals↗

Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis.

It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.

Cell Membrane↗

Protein profile changes in the human breast cancer cell line MCF-7 in response to SEL1L gene induction.

The ectopic expression of the gene SEL1L in the human breast carcinoma cell line MCF-7 resulted in a reduction of the aggressive behaviour of these cells in vitro. In addition, in vivo analysis on a series of primary breast carcinomas revealed an association between the SEL1L protein levels and the patient's overall survival. We aimed to find those proteins, associated with SEL1L, which may be involved in modulating the aggressive or invasive behaviour of breast cancer cells. For this purpose, we used both the proteomic and microarray approaches. Image analysis of two-dimensional electropherograms revealed the presence of 27 qualitative and 35 quantitative variations between the MCF7-SEL1L expressing cells compared to control. Mass spectrometry identified 32 changing proteins mostly involved in cytoskeletal and metabolic activities, stress response and protein folding, selenoprotein synthesis and cellular proliferation. Five of these also showed changes in transcript levels, as assessed by Affymetrix microarray analysis. Interestingly, seven proteins: carbonic anhydrase (CA) II, ovarian/breast septin, S100A16 calcium binding protein, 14-3-3 protein sigma, proteasome subunit beta type 6, Hsp60 and protein disulphide-isomerase A3 merit particular attention since they are known to be involved in cancer, in response to cellular stress and in protein folding.

Adenocarcinoma↗

Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.

Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.

Animals↗

Genomics and proteomics in cancer.

Cancer development is driven by the accumulation of DNA changes in the approximately 40000 chromosomal genes. In solid tumours, chromosomal numerical/structural aberrations are common. DNA repair defects may lead to genome-wide genetic instability, which can drive further cancer progression. The genes code the actual players in the cellular processes, the 100000-10 million proteins, which in (pre)malignant cells can also be altered in a variety of ways. Over the past decade, our knowledge of the human genome and Genomics (the study of the human genome) in (pre)malignancies has increased enormously and Proteomics (the analysis of the protein complement of the genome) has taken off as well. Both will play an increasingly important role. In this article, a short description of the essential molecular biological cell processes is given. Important genomic and proteomic research methods are described and illustrated. Applications are still limited, but the evidence so far is exciting. Will genomics replace classical diagnostic or prognostic procedures? In breast cancers, the gene expression array is stronger than classical criteria, but in endometrial hyperplasia, quantitative morphological features are more cost-effective than genetic testing. It is still too early to make strong statements, the more so because it is expected that genomics and proteomics will expand rapidly. However, it is likely that they will take a central place in the understanding, diagnosis, monitoring and treatment of (pre)cancers of many different sites.

Cell Transformation, Neoplastic↗

Biophysical characterization of proteins in the post-genomic era of proteomics.

Proteomics focuses on the high throughput study of the expression, structure, interactions, and, to some extent, function of large numbers of proteins. A true understanding of the functioning of a living cell also requires a quantitative description of the stoichiometry, kinetics, and energetics of each protein complex in a cellular pathway. Classical molecular biophysical studies contribute to understanding of these detailed properties of proteins on a smaller scale than does proteomics in that individual proteins are usually studied. This perspective article deals with the role of biophysical methods in the study of proteins in the proteomic era. Several important physical biochemical methods are discussed briefly and critiqued from the standpoint of information content and data acquisition. The focus is on conformational changes and macromolecular assembly, the utility of dynamic and static structural data, and the necessity to combine experimental approaches to obtain a full functional description. The conclusions are that biophysical information on proteins is a useful adjunct to "standard" proteomic methods, that data can be obtained by high throughput technology in some instances, but that hypothesis-driven experimentation may frequently be required.

Biophysical Phenomena↗

Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide-induced cell differentiation/apoptosis of promyelocytic leukemia.

Understanding the complexity and dynamics of cancer cells in response to effective therapy requires hypothesis-driven, quantitative, and high-throughput measurement of genes and proteins at both spatial and temporal levels. This study was designed to gain insights into molecular networks underlying the clinical synergy between retinoic acid (RA) and arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), which results in a high-quality disease-free survival in most patients after consolidation with conventional chemotherapy. We have applied an approach integrating cDNA microarray, 2D gel electrophoresis with MS, and methods of computational biology to study the effects on APL cell line NB4 treated with RA, ATO, and the combination of the two agents and collected in a time series. Numerous features were revealed that indicated the coordinated regulation of molecular networks from various aspects of granulocytic differentiation and apoptosis at the transcriptome and proteome levels. These features include an array of transcription factors and cofactors, activation of calcium signaling, stimulation of the IFN pathway, activation of the proteasome system, degradation of the PML-RARalpha oncoprotein, restoration of the nuclear body, cell-cycle arrest, and gain of apoptotic potential. Hence, this investigation has provided not only a detailed understanding of the combined therapeutic effects of RA/ATO in APL but also a road map to approach hematopoietic malignancies at the systems level.

Antineoplastic Agents↗

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I.

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.

Animals↗

Activation of human T lymphocytes under conditions similar to those that occur during exposure to microgravity: a proteomics study.

A number of experiments, conducted under microgravity conditions, i.e. in space shuttle biolaboratories or in ground based systems simulating the conditions occurring in microgravity, show that in hypogravity, in vitro human lymphocyte activation is severely impaired. However, very early stimulation steps of T lymphocytes are not compromised, since CD69 receptor, the earliest membrane activation marker, is expressed by T cells at a level comparable to that observed on 1 g activated lymphocytes. Since CD69 engagement, together with submitogenic doses of phorbol esters, transduces an activation signal to T lymphocytes, we undertook a comparative study on the stimulation mediated through this receptor on human CD3+ cells cultured under conditions similar to those which occur during exposure to microgravity, i.e. in clinorotation, or at 1 g. During the early hours of activation, increased levels of intracellular calcium and increased mitochondrial membrane potential were detectable in clinorotating as well as in 1 g cells. However, after 48 hours clinorotation, interleukin 2 production by T lymphocytes was significantly reduced and cell proliferation was greatly decreased. By means of a differential proteomics approach on T cells activated in clinorotation or at 1 g for 48 hours, we were able to detect statistically significant quantitative protein alterations. Seven proteins with modified expression values were identified; they are involved in nucleic acids processing, proteasome regulation and cytoskeleton structure.

Antigens, CD↗

Multiresolution image registration for two-dimensional gel electrophoresis.

In proteomic research, two-dimensional electrophoresis (2-D) is an important tool for investigating differential patterns of qualitative and quantitative protein expression. The strength of the technique is due to its unrivalled power of being able to separate simultaneously thousands of proteins. The key to the comparison of 2-D protein profiles, however, lies in the use of a fast and robust image matching process which is essential to the subsequent quantification procedure. To satisfy the growing demand for a robust and fully automatic method of matching 2-D gel protein separation profiles, we describe in this paper a novel registration technique based on image intensity distribution rather than selected features. The method uses a multiresolution representation of the gel profiles and exploits the fact that coarse approximations to the optimal matching can be extracted efficiently from low-resolution images. This permits the removal of misalignments at different scales in a systematic manner and the strength of the new method has been confirmed by a double blind trial of 111 2-D gel pairs. The proposed method requires neither landmarks nor an a priori image alignment, and takes about five seconds for processing a typical gel pair on a standard personal computer.

Algorithms↗

Proteomic analysis of eIF-5A in lung adenocarcinomas.

We examined the eIF-5A protein expression in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis with identification by mass spectrometry and 2-D Western blots. The same tissue samples were examined for the eIF-5A mRNA expression using oligonucleotide microarrays, and the cellular localization of the eIF-5A protein was examined using immunohistochemical analysis on tissue arrays. Higher eIF-5A protein expression was present in tumors showing poor differentiation, 12/13(th) codon K-ras mutations, p53 nuclear accumulation, and tumors with positive lymphocytic response. The eIF-5A mRNA was also significantly increased in lung adenocarcinomas compared to normal lung, but the eIF-5A protein expression was not correlated to its mRNA levels indicating that the increase in the eIF-5A protein expression in lung tumors is post-transcriptionally/translationally/post-translationally regulated. Patients having a higher eIF-5A protein expression showed a relatively poorer survival suggesting the use of eIF-5A as prognostic marker in lung adenocarcinoma. Moreover, the investigation on agents that inhibit eIF-5A function is encouraged.

Adenocarcinoma↗

"Reverse-staining" of biomolecules in electrophoresis gels: analytical and micropreparative applications.

Negative or reverse staining using imidazole and zinc salts for protein detection in electrophoresis gels was originally introduced in 1990. The method is based on the selective precipitation of zinc imidazolate in the gel except in the zones where proteins are located. The method was later adapted to allow high-sensitivity negative detection of nucleic acids and bacterial lipopolysaccharides. It provides a practically quantitative recovery of intact biomolecules and is a method of choice for micropreparative applications of gel electrophoresis to proteomics and similar structural studies. Zinc-mediated protein fixation in the gel is fully reversible and the eluted biomolecules are neither chemically modified nor contaminated with organic dyes. Here we present a detailed compilation of practical methods for implementing these techniques with emphasis in their analytical or micropreparative applications.

DNA↗