Biomedical subjects

John R Yates

Publications and source records attributed to John R Yates.

At least 91 records · Page 5Linked to original sources

PubMed2005

Chlamydial GroEL autoregulates its own expression through direct interactions with the HrcA repressor protein.

In the pathogenic bacterium Chlamydia trachomatis, a transcriptional repressor, HrcA, regulates the major heat shock operons, dnaK and groE. Cellular stress causes a transient increase in transcription of these heat shock operons through relief of HrcA-mediated repression, but the pathway leading to derepression is unclear. Elevated temperature alone is not sufficient, and it is hypothesized that additional chlamydial factors play a role. We used DNA affinity chromatography to purify proteins that interact with HrcA in vivo and identified a higher-order complex consisting of HrcA, GroEL, and GroES. This endogenous HrcA complex migrated differently than recombinant HrcA, but the complex could be disrupted, releasing native HrcA that resembled recombinant HrcA. In in vitro assays, GroEL increased the ability of HrcA to bind to the CIRCE operator and to repress transcription. Other chlamydial heat shock proteins, including the two additional GroEL paralogs present in all chlamydial species, did not modulate HrcA activity.

Bacterial Proteins↗

PubMed2005

Identification of novel RPGR ORF15 mutations in X-linked progressive cone-rod dystrophy (XLCORD) families.

PURPOSE: To test the incidence of mutations in RPGR ORF15 in six families with X-linked progressive retinal degeneration (cone-rod dystrophy [XLCORD], macular or cone dystrophy) and to undertake a detailed phenotypic assessment of families in whom ORF15 mutations were identified. METHODS: To amplify and sequence ORF15 in its entirety, a cloning strategy was developed. Families with mutations in ORF15 underwent electrophysiological testing, color vision assessment, color fundus photography, and fundus autofluorescence (AF) imaging. RESULTS: Novel protein truncation mutations were identified in two families. In family A, a 2-bp mutation was identified in ORF15+A1094C G1095T, predicted to result in a truncated protein (E364D/E365X). In family B, a G-to-T transversion (ORF15+1176G>T) resulted in a nonsense mutation (G392X). Characteristics of phenotype in both families included progressive deterioration of central vision and subsequently night vision, mild photophobia, and moderate to high myopia. Ophthalmoscopic abnormalities were generally confined to the macula. A parafoveal ring of increased AF was observed, and electrophysiological evidence of a greater generalized abnormality in cone than rod responses were consistent with a cone-rod dystrophy phenotype. CONCLUSIONS: The cloning strategy for ORF15 facilitated comprehensive sequence analysis in patients. Two families were identified with nonsense mutations, and clinical evaluation revealed them both to have a similar phenotype. The presence of a parafoveal ring of increased AF was an early indicator of affected status in these families. No disease-causing mutations in ORF15 were detected in four other families, suggesting that ORF15 mutations may not be the most common cause of XLCORD.

Adult↗

PubMed2005

Identification of novel integral membrane proteins of the nuclear envelope with potential disease links using subtractive proteomics.

Lamin A and some integral membrane proteins of the nuclear envelope (NE) have been linked to human diseases, mostly dystrophies. To comprehensively identify integral membrane proteins specific to the nuclear envelope, we have carried out a subtractive proteomics analysis of NEs isolated from rodent liver using Multidimensional Protein Identification Technology (MudPIT). An NE fraction and a nucleus-depleted membrane fraction were separately analyzed by MudPIT and proteins appearing in both fractions were 'subtracted' from the NE fraction. This identified 67 novel putative NE transmembrane proteins in addition to the 13 that had been previously characterized. Most or all of the new proteins we identified are likely to be bona fide NE Transmembrane proteins (NETs), since all eight of the first group of proteins we tested in a cell transfection assay target to the NE. Moreover, five of the eight NETs remained associated with the nuclear periphery after extraction with Triton-X100, suggesting an association with the nuclear lamin polymer. 27 of the proteins occur in chromosomal regions where 18 different human dystrophies have been mapped, making these proteins disease candidates. We have analysed the expression of these proteins using transcriptome databases, providing direction for future functional analysis of these novel proteins.

Animals↗

PubMed2004

Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast.

The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs. The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms. Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway. Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273. Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro. Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF (Skp1-Cdc53-F box) ubiquitin ligases. Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth. Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity.

Amino Acid Sequence↗

PubMed2004

The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1.

Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. Here, we show that PINCH, a protein required for integrin-dependent cell adhesion and actin-membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, we identify RSU-1, a regulator of Ras signaling in mammalian cells, as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. Our results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development.

Actins↗

PubMed2004

Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions.

Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. Here, we demonstrate that the Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. Thus, these data reveal a previously unknown mechanism for selective histone exchange that uses the concerted action of two distinct chromatin-remodeling enzymes within the same multiprotein complex.

Acetyl Coenzyme A↗

PubMed2004

Strategies for shotgun identification of post-translational modifications by mass spectrometry.

The global identification of post-translationally modified proteins is a difficult challenge that is currently being addressed by many researchers in the field of mass spectrometry (MS)-based proteomics. The ability to identify thousands of proteins by shotgun-based strategies has made the mere idea of a global analysis of a particular protein modification seem reasonable. There has been much progress in the development of methods that make use of shotgun-based protein identification in the analysis of a wide variety of protein modifications, some of which will be discussed here.

Mass Spectrometry↗

PubMed2004

Actin-binding proteins in a postsynaptic preparation: Lasp-1 is a component of central nervous system synapses and dendritic spines.

CNS synapses are complex sites of cell-cell communication. Identification and characterization of the protein components of synapses will lead to a better understanding of the mechanisms of neurotransmission and plasticity. We applied multidimensional protein identification technology (MudPIT) to purified, guanidine-solubilized postsynaptic fractions to identify novel synaptically localized molecules. We identified several actin-associated proteins known to regulate actin polymerization and control cell motility in nonneural cells that have not previously been associated with CNS synaptic function. One of these is lasp-1, an actin-associated LIM and SH3 domain-containing protein. We show that lasp-1 is strongly expressed by CNS neurons and is concentrated at synaptic sites. Overall, the preponderance of actin-associated proteins in postsynaptic density fractions, and specifically those involved in actin reorganization, suggests that there are many modes by which the state of synaptic F-actin polymerization and, hence, synaptic physiology are affected.

Actins↗

PubMed2004

The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector.

Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.

14-3-3 Proteins↗

PubMed2004

Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra.

To take advantage of the potential quantitative benefits offered by tandem mass spectrometry, we have modified the method in which tandem mass spectrum data are acquired in 'shotgun' proteomic analyses. The proposed method is not data dependent and is based on the sequential isolation and fragmentation of precursor windows (of 10 m/z) within the ion trap until a desired mass range has been covered. We compared the quantitative figures of merit for this method to those for existing strategies by performing an analysis of the soluble fraction of whole-cell lysates from yeast metabolically labeled in vivo with (15)N. To automate this analysis, we modified software (RelEx) previously written in the Yates lab to generate chromatograms directly from tandem mass spectra. These chromatograms showed improvements in signal-to-noise ratio of approximately three- to fivefold over corresponding chromatograms generated from mass spectrometry scans. In addition, to demonstrate the utility of the data-independent acquisition strategy coupled with chromatogram reconstruction from tandem mass spectra, we measured protein expression levels in two developmental stages of Caenorhabditis elegans.

Algorithms↗

PubMed2004

A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension.

Kinetochores play an essential role in chromosome segregation by forming dynamic connections with spindle microtubules. Here, we identify a set of 10 copurifying kinetochore proteins from Caenorhabditis elegans, seven of which were previously uncharacterized. Using in vivo assays to monitor chromosome segregation, kinetochore assembly, and the mechanical stability of chromosome-microtubule attachments, we show that this copurifying protein network plays a central role at the kinetochore-microtubule interface. In addition, our analysis suggests that the network is comprised of three groups of proteins that contribute in distinct ways to this interface: KNL proteins act after the assembly of centromeric chromatin to generate the core of the microtubule-binding interface, MIS proteins control the rate and extent of formation of this interface, and NDC proteins are necessary to sustain tension during interactions with spindle microtubules. We also purify a similar set of associated proteins from human cells that includes four novel proteins and has recognizable homologs from each functional class. Thus, this protein network is a conserved constituent of the outer kinetochore, and the functions defined by our analysis in C. elegans are likely to be widely relevant.

Amino Acid Sequence↗

PubMed2004

Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo.

Cancer research depends on the use of human cell lines for both the in vitro (culture) and in vivo (xenograft) analysis of tumor progression and treatment. However, the extent to which cultured preparations of human cancer lines display similar properties in vivo, where important host factors may influence tumor biology, remains unclear. Here, we address this question by conducting a functional proteomic analysis of the human breast cancer line MDA-MB-231 grown in culture and as orthotopic xenograft tumors in the mammary fad pad of immunodeficient mice. Using a suite of activity-based chemical probes, we identified carcinoma (human) enzyme activities that were expressed selectively in culture or in xenograft tumors. Likewise, distinct groups of stromal (mouse) enzyme activities were found that either infiltrated or were excluded from xenograft tumors, indicating a contribution by specific host components to breast cancer development. MDA-MB-231 cells isolated from tumors exhibited profound differences in their enzyme activity profiles compared with the parent cell line, including the dramatic posttranscriptional up-regulation of the serine proteases urokinase plasminogen activator and tissue plasminogen activator and down-regulation of the glycolytic enzyme phosphofructokinase. These altered enzyme activity profiles correlated with significantly greater tumor growth rates and metastases for xenograft-derived MDA-MB-231 cells upon reintroduction into mice. Collectively, these data indicate that the in vivo environment of the mouse mammary fat pad cultivates the growth of human breast cancer cells with elevated tumorigenic properties and highlight the value of activity-based protein profiling for identifying proteomic signatures that depict such changes in cancer cell biology.

Amino Acid Sequence↗

PubMed2004

Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis.

To quantify proteins on a global level from mammalian tissue, a method was developed to metabolically introduce 15N stable isotopes into the proteins of Rattus norvegicus for use as internal standards. The long-term metabolic labeling of rats with a diet enriched in 15N did not result in adverse health consequences. The average 15N amino acid enrichments reflected the relative turnover rates in the different tissues and ranged from 74.3 mpe in brain to 92.2 mpe in plasma. Using the 15N-enriched liver as a quantitative internal standard, changes in individual protein levels in response to cycloheximide treatment were measured for 310 proteins. These measurements revealed 127 proteins with altered protein level (p < 0.05). Most proteins with altered level have previously reported functions involving xenobiotic metabolism and protein-folding machinery of the endoplasmic reticulum. This approach is a powerful tool for the global quantitation of proteins, is capable of measuring proteome-wide changes in response to a drug, and will be useful for studying animal models of disease.

Animals↗

PubMed2004

Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis.

The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1+3) holds replicated sister chromatids together until mitosis, condensin (Smc2+4) acts in chromosome condensation, and Smc5+6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5+6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5+6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5+6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5+6 complex is important for replication fork stability.

Amino Acid Sequence↗

PubMed2004

Global analysis of protein sumoylation in Saccharomyces cerevisiae.

Although the modification of cellular factors by SUMO is an essential process in Saccharomyces cerevisiae, the identities of the substrates remain largely unknown. Using a mass spectrometry-based approach, we have identified 271 new SUMO targets. These substrates play roles in a diverse set of biological processes and greatly expand the scope of SUMO regulation in eukaryotic cells. Transcription appears to be the most prevalent process associated with sumoylation with novel SUMO substrates found in basal transcription machinery for RNA polymerases I, II, and III, pol II transcriptional elongation complexes, and a variety of chromatin remodeling, chromatin modifying, and chromatin silencing complexes. Additionally, our global analysis has revealed a number of interesting biological patterns in the list of SUMO targets including a clustering of sumoylation targets within macromolecular complexes.

Amino Acid Sequence↗

PubMed2004

A novel estrogen receptor alpha-associated protein alters receptor-deoxyribonucleic acid interactions and represses receptor-mediated transcription.

Estrogen receptor alpha (ER alpha) serves as a ligand-activated transcription factor, turning on transcription of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with the receptor to influence ER alpha-mediated transactivation. In this study, we have identified pp32, which interacts with the DNA binding domain of ER alpha when the receptor is free, but not when it is bound to an estrogen response element. Coimmunoprecipitation experiments demonstrate that endogenously expressed pp32 and ER alpha from MCF-7 breast cancer cells interact. Although pp32 substantially enhances the association of the receptor with estrogen response element-containing DNA, overexpression of pp32 in MCF-7 cells decreases transcription of an estrogen-responsive reporter plasmid. pp32 Represses p300-mediated acetylation of ER alpha and histones in vitro and inhibits acetylation of ER alpha in vivo. pp32 Also binds to other nuclear receptors and inhibits thyroid hormone receptor beta-mediated transcription. Taken together, our studies provide evidence that pp32 plays a role in regulating transcription of estrogen-responsive genes by modulating acetylation of histones and ER alpha and also influences transcription of other hormone-responsive genes as well.

Acetylation↗

PubMed2004

Automatic quality assessment of peptide tandem mass spectra.

MOTIVATION: A powerful proteomics methodology couples high-performance liquid chromatography (HPLC) with tandem mass spectrometry and database-search software, such as SEQUEST. Such a set-up, however, produces a large number of spectra, many of which are of too poor quality to be useful. Hence a filter that eliminates poor spectra before the database search can significantly improve throughput and robustness. Moreover, spectra judged to be of high quality, but that cannot be identified by database search, are prime candidates for still more computationally intensive methods, such as de novo sequencing or wider database searches including post-translational modifications. RESULTS: We report on two different approaches to assessing spectral quality prior to identification: binary classification, which predicts whether or not SEQUEST will be able to make an identification, and statistical regression, which predicts a more universal quality metric involving the number of b- and y-ion peaks. The best of our binary classifiers can eliminate over 75% of the unidentifiable spectra while losing only 10% of the identifiable spectra. Statistical regression can pick out spectra of modified peptides that can be identified by a de novo program but not by SEQUEST. In a section of independent interest, we discuss intensity normalization of mass spectra.

Algorithms↗

PubMed2004

Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture.

Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations. We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells. Forty-one percent of proteins expressed in vivo are not detected in vitro. Statistical analysis measuring reproducibility reveals that seven to ten MudPIT measurements are necessary to achieve > or =95% confidence of analytical completeness with current ion trap equipment. Large-scale mapping of the proteome of vascular endothelial cell surface in vivo, as demonstrated here, is advisable because distinct protein expression is apparently regulated by the tissue microenvironment that cannot yet be duplicated in standard cell culture.

Animals↗