Use of functional proteomics to investigate PKC epsilon-mediated cardioprotection: the signaling module hypothesis.
(73/6804)
The characterization of biological processes on the basis of alterations in the cellular proteins, or "proteomic" analysis, is a powerful approach that may be adopted to decipher the signaling mechanisms that underlie various pathophysiological conditions, such as ischemic heart disease. This review represents a prospectus for the implementation of proteomic analyses to delineate the myocardial intracellular signaling events that evoke cardioprotection against ischemic injury. In concert with this, the manifestation of a protective phenotype has recently been shown to involve dynamic modulation of protein kinase C-epsilon (PKC epsilon) signaling complexes (Ping P, Zhang J, Pierce WM Jr, and Bolli R. Circ Res 88: 59--62, 2001). Accordingly, "the signaling module hypothesis" is formulated as a plausible mechanism by which multipurpose stress-activated proteins and signaling kinases may function collectively to facilitate the genesis of cardioprotection. (+info)
The cohesin complex: sequence homologies, interaction networks and shared motifs.
(74/6804)
BACKGROUND: Cohesin is a macromolecular complex that links sister chromatids together at the metaphase plate during mitosis. The links are formed during DNA replication and destroyed during the metaphase-to-anaphase transition. In budding yeast, the 14S cohesin complex comprises at least two classes of SMC (structural maintenance of chromosomes) proteins - Smc1 and Smc3 - and two SCC (sister-chromatid cohesion) proteins - Scc1 and Scc3. The exact function of these proteins is unknown. RESULTS: Searches of protein sequence databases have revealed new homologs of cohesin proteins. In mouse, Mmip1 (Mad member interacting protein 1) and Smc3 share 99% sequence identity and are products of the same gene. A phylogenetic tree of SMC homologs reveals five families: Smc1, Smc2, Smc3, Smc4 and an ancestral family that includes the sequences from the Archaea and Eubacteria. This ancestral family also includes sequences from eukaryotes. A cohesion interaction network, comprising 17 proteins, has been constructed using two proteomic databases. Genes encoding six proteins in the cohesion network share a common upstream region that includes the MluI cell-cycle box (MCB) element. Pairs of the proteins in this network share common sequence motifs that could represent common structural features such as binding sites. Scc2 shares a motif with Chk1 (kinase checkpoint protein), that comprises part of the serine/threonine protein kinase motif, including the active-site residue. CONCLUSIONS: We have combined genomic and proteomic data into a comprehensive network of information to reach a better understanding of the function of the cohesin complex. We have identified new SMC homologs, created a new SMC phylogeny and identified shared DNA and protein motifs. The potential for Scc2 to function as a kinase - a hypothesis that needs to be verified experimentally - could provide further evidence for the regulation of sister-chromatid cohesion by phosphorylation mechanisms, which are currently poorly understood. (+info)
Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics.
(75/6804)
Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes. (+info)
Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance.
(76/6804)
Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding. (+info)
Proteomic analysis of the bacterial cell cycle.
(77/6804)
A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control. (+info)
Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine.
(78/6804)
Development of noninvasive methods for the diagnosis of transitional cell carcinoma (TCC) of the bladder remains a challenge. A ProteinChip technology (surface enhanced laser desorption/ionization time of flight mass spectrometry) has recently been developed to facilitate protein profiling of biological mixtures. This report describes an exploratory study of this technology as a TCC diagnostic tool. Ninety-four urine samples from patients with TCC, patients with other urogenital diseases, and healthy donors were analyzed. Multiple protein changes were reproducibly detected in the TCC group, including five potential novel TCC biomarkers and seven protein clusters (mass range, 3.3 to 133 kd). One of the TCC biomarkers (3.4 kd) was also detected in bladder cancer cells procured from bladder barbotage and was identified as defensin. The TCC detection rates provided by the individual markers ranged from 43 to 70% and specificities from 70 to 86%. Combination of the protein biomarkers and clusters, increased significantly the sensitivity for detecting TCC to 87% with a specificity of 66%. Interestingly, this combinatorial approach provided sensitivity of 78% for detecting low-grade TCC compared to only 33% of voided urine or bladder-washing cytology. Collectively these results support the potential of this proteomic approach for the development of a highly sensitive urinary TCC diagnostic test. (+info)
PartsList: a web-based system for dynamically ranking protein folds based on disparate attributes, including whole-genome expression and interaction information.
(79/6804)
As the number of protein folds is quite limited, a mode of analysis that will be increasingly common in the future, especially with the advent of structural genomics, is to survey and re-survey the finite parts list of folds from an expanding number of perspectives. We have developed a new resource, called PartsList, that lets one dynamically perform these comparative fold surveys. It is available on the web at http://bioinfo.mbb.yale.edu/partslist and http://www.partslist.org. The system is based on the existing fold classifications and functions as a form of companion annotation for them, providing 'global views' of many already completed fold surveys. The central idea in the system is that of comparison through ranking; PartsList will rank the approximately 420 folds based on more than 180 attributes. These include: (i) occurrence in a number of completely sequenced genomes (e.g. it will show the most common folds in the worm versus yeast); (ii) occurrence in the structure databank (e.g. most common folds in the PDB); (iii) both absolute and relative gene expression information (e.g. most changing folds in expression over the cell cycle); (iv) protein-protein interactions, based on experimental data in yeast and comprehensive PDB surveys (e.g. most interacting fold); (v) sensitivity to inserted transposons; (vi) the number of functions associated with the fold (e.g. most multi-functional folds); (vii) amino acid composition (e.g. most Cys-rich folds); (viii) protein motions (e.g. most mobile folds); and (ix) the level of similarity based on a comprehensive set of structural alignments (e.g. most structurally variable folds). The integration of whole-genome expression and protein-protein interaction data with structural information is a particularly novel feature of our system. We provide three ways of visualizing the rankings: a profiler emphasizing the progression of high and low ranks across many pre-selected attributes, a dynamic comparer for custom comparisons and a numerical rankings correlator. These allow one to directly compare very different attributes of a fold (e.g. expression level, genome occurrence and maximum motion) in the uniform numerical format of ranks. This uniform framework, in turn, highlights the way that the frequency of many of the attributes falls off with approximate power-law behavior (i.e. according to V(-b), for attribute value V and constant exponent b), with a few folds having large values and most having small values. (+info)
Desorption/ionization on silicon (DIOS): a diverse mass spectrometry platform for protein characterization.
(80/6804)
Since the advent of matrix-assisted laser desorption/ionization and electrospray ionization, mass spectrometry has played an increasingly important role in protein functional characterization, identification, and structural analysis. Expanding this role, desorption/ionization on silicon (DIOS) is a new approach that allows for the analysis of proteins and related small molecules. Despite the absence of matrix, DIOS-MS yields little or no fragmentation and is relatively tolerant of moderate amounts of contaminants commonly found in biological samples. Here, functional assays were performed on an esterase, a glycosidase, a lipase, as well as exo- and endoproteases by using enzyme-specific substrates. Enzyme activity also was monitored in the presence of inhibitors, successfully demonstrating the ability of DIOS to be used as an inhibitor screen. Because DIOS is a matrix-free desorption technique, it also can be used as a platform for multiple analyses to be performed on the same protein. This unique advantage was demonstrated with acetylcholine esterase for qualitative and quantitative characterization and also by its subsequent identification directly from the DIOS platform. (+info)