Global effects of RAB3GAP1 dysexpression on the proteome of mouse cortical neurons

Mounting studies have demonstrated that RAB3GAP1 expression is modified in brain diseases with multiple neurobiological functions and processes and acts as a potentially significant target. However, the cellular and molecular events arising from RAB3GAP1 dysexpression are still incompletely understood. In this work, underexpression and overexpression of RAB3GAP1 were first induced into cultured mouse cortical neurons by transfection with lentivirus plasmids. Then we globally explored the effects of RAB3GAP1 dysexpression on the proteome of the neurons through the use of isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics with bioinformatics. A total of 364 proteins in the RAB3GAP1-underexpression group and 314 proteins in the RAB3GAP1-overexpression group were identified to be differentially expressed. Subsequent bioinformatics analysis indicated that the proteome functional expression profiles induced by RAB3GAP1 underexpression and overexpression were different, suggesting the potential differences in biological processes and cellular effects. Subsequent intergroup cross-comparison revealed some candidate target proteins regulated directly by RAB3GAP1. Further parallel reaction monitoring (PRM) analysis illustrated that Sub1, Ssrp1, and Top1 proteins might serve as new potentially important linkers in the RAB3GAP1-mediated autophagy pathway in the cortical neurons. Collectively, the current proteomics data furnished new valuable insights to better understand the regulatory molecular mechanism of neuronal RAB3GAP1.

     

Global analysis of a "simple" proteome: Methanococcus jannaschii

The completed genome of Methanococcus jannaschii, including the main chromosome and two extra-chromosomal elements, predicts a proteome comprised of 1783 proteins. How many of those proteins are expressed at any given time and the relative abundance of the expressed proteins, however, cannot be predicted solely from the genome sequence. Two-dimensional gel electrophoresis coupled with peptide mass spectrometry is being used to identify the proteins expressed by M. jannaschii cells grown under different conditions as part of an effort to correlate protein expression with regulatory mechanisms. Here we describe the identification of 170 of the most abundant proteins found in total lysates of M. jannaschii grown under optimal fermentation conditions. To optimize the number of proteins detected, two different protein specific stains (Coomassie Blue R250 or silver nitrate) and two different first dimension separation methods (isoelectric focusing or nonequilibrium pH gradient electrophoresis) were used. Thirty-two percent of the proteins identified are annotated as hypothetical (21% conserved hypothetical and 11% hypothetical), 21% are enzymes involved in energy metabolism, 12% are proteins required for protein synthesis, and the remainder include proteins necessary for intermediary metabolism, cell division, and cell structure. Evidence of post-translational modification of numerous M. jannaschii proteins has been found, as well as indications of incomplete dissociation of protein-protein complexes. These results demonstrate the complexity of proteome analysis even when dealing with a relatively simple genome.     

Preparation of dissociated mouse cortical neuron cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.     

Global strategies to integrate the proteome and metabolome

A fundamental goal of proteomics is to assign physiological functions to all proteins encoded by eukaryotic and prokaryotic genomes. Of the many activities performed by proteins, the chemical transformations catalyzed by enzymes form the basis for most, if not all, metabolic and signaling pathways. Elucidation of these pathways and their integration into larger cellular networks require new strategies to rapidly and systematically identify physiological substrates of enzymes. Here, we review emerging technologies that aim to assign endogenous biochemical functions to enzymes by profiling the metabolome.     

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

Analysis of the form of cortical neuron reaction to acetylcholine

Reaction patterns of 90 cortical neurons to acetylcholine approximated by two parabolas have been divided on simple and complex. The participation of different types of cholinoreceptors in complex reactions to transmitter is proposed.     

Global analysis of the Ralstonia metallidurans proteome: prelude for the large-scale study of heavy metal response

A proteome map of Ralstonia metallidurans strain CH34 was constructed using two-dimensional (2-D) gel electrophoresis in combination with automated Edman degradation and mass spectrometry (MS). R. metallidurans CH34 is the type-strain of a family of highly related strains characterized by their multiple resistance to millimolar amounts of heavy metals, conferred by two large plasmids. The protein content of this bacterium grown in minimal medium was separated by 2-D gel electrophoresis using various pH gradients. Protein identification was carried out via N-terminal amino acid sequencing, matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) and tandem MS. So far, 224 different proteins were characterized from 352 protein spots. Although the proteome map is still not complete, one could appraise the importance of proteomics for genome analyses through (i). the identification of previously undetected open reading frames, (ii). the identification of proteins not encoded by the already sequenced genome fragments, (iii). the characterization of protein-encoding genes spanning two different contigs, enabling their merging, and (iv). the precise delineation of the N-terminus of several proteins. Finally, this map will prove a useful tool in the identification of proteins differentially expressed in the presence of different heavy metals.     

Novel genetic tools facilitate the study of cortical neuron migration

Key facets of mammalian forebrain cortical development include the radial migration of projection neurons and subsequent cellular differentiation into layer-specific subtypes. Inappropriate regulation of these processes can lead to a number of congenital brain defects in both mouse and human, including lissencephaly and intellectual disability. The genes regulating these processes are still not all identified, suggesting genetic analyses will continue to be a powerful tool in mechanistically studying the development of the cerebral cortex. Reelin is a molecule which we have understood to be critical for proper cortical development for many years. The precise mechanism of Reelin, however, is not fully understood. To address both of these unresolved issues, we report here the creation of a novel conditional allele of the Reelin gene and showcase the use of an Etv1-GFP transgenic line highlighting a subpopulation of the cortex: layer V pyramidal neurons. Together, these represent genetic tools which may facilitate the study of cortical development in a number of different ways.     

Plant proteome analysis

Proteome analysis is becoming a powerful tool in the functional characterization of plants. Due to the availability of vast nucleotide sequence information and based on the progress achieved in sensitive and rapid protein identification by mass spectrometry, proteome approaches open up new perspectives to analyze the complex functions of model plants and crop species at different levels. In this review, an overview is given on proteome studies performed to analyze whole plants or specific tissues with particular emphasis on important physiological processes such as germination. The chapter on subcellular proteome analysis of plants focuses on the progress achieved for plastids and mitochondria but also mentions the difficulties associated with membrane-bound proteins of these organelles. Separate chapters are dedicated to the challenging analysis of woody plants and to the use of proteome approaches to investigate the interaction of plants with pathogens or with symbiotic organisms. Limitations of current techniques and recent conceptual and technological perspectives for plant proteomics are briefly discussed in the final chapter.     

Evaluation of forms of cortical neuron response to acetylcholine

Analysis was carried out of the form of excitatory components of the reaction of 92 neurones in rats sensorimotor cortical area to electrophoretically applied acetylcholine. The used method of approximation of neuronal reaction form by two polynomes of the second degree allowed to identify one- and two-component types of responses to the transmitter. It is suggested that in genesis of the excitatory component of neuronal reaction to acetylcholine one or two types of cholinoreceptors may participate.     

Global impact of oncogenic Src on a phosphotyrosine proteome

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.     

Analysis of the simultaneous discharges of neuron pairs in the brain cortical microstructures

Dynamics of simultaneous discharges of pairs of cortical neurones was studied in three behavioural conditions. Parameters of the stimulation applied was shown to be encoded in trains of simultaneous unit discharges.     

Characteristics of human cortical pyramidal neuron development during the second gestational trimester

Prenatal ontogeny of the human neocortex exhibits specific characteristics that make its organization unique. Therefore, experimental data obtained on animal models cannot be extrapolated to human cortex morphogenesis during the middle and late gestational periods. Characteristics of the development of cortical pyramidal neurons of the human brain were studied in the brains of eight fetuses at gestational ages between 16 and 26 weeks. Immunohistochemical labeling of neurons was performed using antibodies against microtubule associated protein 2 (MAP2), a structural protein of microtubules. Expression of this protein marks the beginning of dendrogenesis. MAP2 is mainly located in the neuron body and dendrites, which allowed the neuron morphotype and location in specific cortical layers to be determined. It was shown that MAP2-immunopositive neurons were identifiable in embryonic cortical layer eV as early as the 18th gestational week. By the 25th gestational week, two populations of pyramidal neurons were discernible in the cortical plate, one of them located in layer eV and the other, in layer eIII, which developed later. Since differentiating neurons are known to be more vulnerable than neuroblasts and mature neurons, these results suggest that critical periods for corticofugal and corticocortical populations of pyramidal cells occur at different stages of the second gestational trimester.     

Towards complete analysis of the platelet proteome

Platelets exert a crucial function in haemostasis, wound repair, and the formation of vascular plugs, underlying thrombotic diseases such as stroke and myocardial infarction. Analysis of platelet biochemistry is largely dependent on protein analysis as platelets are anucleated cells providing little analytical target for DNA or RNA based strategies. Here we present data from our analysis of the human platelet proteome, the entire set of proteins building a platelet at a given point in time. Proteins were separated by two-dimensional electrophoresis (2-DE) using broad and narrow range pH gradients in the isoelectric focusing step. Consequently, a high-resolution 2-DE proteome map has been generated that comprises approximately 2300 different protein features. From the 536 protein features detected in the 4-5 pI range 284 features were identified by electrospray ionisation time of flight tandem mass spectrometry. These 284 proteins originate from 123 different open reading frames. This includes the five human proteins KIAA0193, KIAA0573, KIAA0830, WUGSC:H_DJ0777O23 protein, and cytokine receptor related protein 4, all isolated for the first time. The data are discussed with regard to proteome characteristics, protein function, and the high prevalence of signalling molecules. This study contributes to a more thorough and holistic understanding of platelet biology, helping to build the basis for future identification of new drug targets and therapeutic strategies.     

Preliminary analysis of the cauliflower mitochondrial proteome

Purified cauliflower (Brassica oleracea var. botrytis) mitochondrial proteins fractionated into soluble, membrane, integral membrane and peripheral membrane samples were analyzed by 2D- PAGE (isoelectric focusing/ SDS polyacrylamide gel electrophoresis). 2D gels patterns were compared using the Imager Master 2D Elite software. 561 silver stained protein spots were resolved after electrophoresis under standard conditions of a whole protein extract. In the soluble fraction a prevalent number of more intense protein spots was observed. The cauliflower protein 2D patterns resembled Arabidopsis thaliana 2D patterns. The two protein spots selected which occupied a similar isoelectric point positions on both gels represented the same proteins as revealed by ESI-MS analysis of cauliflower proteins. The third selected spot belongs to unidentified proteins. The comparative analysis of mitochondrial suborganellar fractions proved the usefulness of this approach.