Biomedical subjects

John R Yates

Publications and source records attributed to John R Yates.

At least 55 records · Page 3Linked to original sources

PubMed2006

Proteomic analysis of mature melanosomes from the retinal pigmented epithelium.

The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks.

Animals↗

PubMed2006

Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway.

Protein phosphorylation has become a focus of many proteomic studies due to the central role that it plays in biology. We combine peptide-based gel-free isoelectric focusing and immobilized metal affinity chromatography to enhance the detection of phosphorylation events within complex protein samples using LC-MS. This method is then used to carry out a quantitative phosphoproteomic analysis of the tumor necrosis factor (TNF) pathway using HeLa cells metabolically labeled with 15N-containing amino acids, where 145 phosphorylation sites were found to be up-regulated upon the activation of the TNF pathway.

Chromatography, Affinity↗

PubMed2006

Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies.

A growing number of biological processes have been found to be regulated by the covalent attachment of the ubiquitin-like protein SUMO to key cellular targets. A critical step in the process of analyzing the role of SUMO in regulating the activity of these proteins is the identification of the lysine residues that are targeted by this modification. Unfortunately, current methods aimed at mapping these attachment-sites are laborious and often ineffective. We report here the development of a platform that combines the use of different C-terminal SUMO mutants with different protease digestion strategies to enable the rapid and efficient identification of SUMO attachment sites. We successfully apply this approach to several model SUMO substrates as well as to a mixture of SUMO conjugates purified from Saccharomyces cerevisiae. Although we specifically employ this strategy for the identification of SUMO attachment sites in yeast, this general approach can easily be adapted to map the sites of conjugation for other ubiquitin-like proteins from a wide range of organisms.

Amino Acid Sequence↗

PubMed2006

Maspin and tumor metastasis.

For most cancer cell types, the acquisition of metastatic activity leads to clinically incurable disease. Improvements in surgery and radiotherapy, and the development of new chemotherapeutic agents or their use in new combinations, have, so far, only incrementally improved patient survival. Despite the obvious importance of metastasis, the process remains incompletely characterized at the molecular and biochemical levels. Tumor metastasis is a complex process and requires multiple cellular functions over time. From cellular invasion, extravasation from the primary tumor, intravasation to the secondary organs, to successful colonization, tumor cells utilize many cellular or biochemical mechanisms to complete the metastatic spread. During the process of metastasis, there are consistent changes in gene expression. Studies of genes that are reduced or silenced have yielded surprising insights into in vivo mechanisms of regulating tumor metastasis. This review describes a tumor suppressor gene, Maspin, which is often silenced in cancer cells and exhibits suppressing activity against tumor growth and metastasis. Maspin has been shown to be involved in processes that are important to both tumor growth and metastasis such as cell invasion, angiogenesis, and more recently apoptosis. Hence, many efforts have been devoted to deciphering the molecular mechanism of maspin. While some insights have come from the protease inhibitory effect of maspin, more perceptive results on how maspin may function in suppressing tumor metastasis have come from studies of gene manipulation, protein interactions and global protein profiling.

Apoptosis↗

PubMed2006

Host cell factor and an uncharacterized SANT domain protein are stable components of ATAC, a novel dAda2A/dGcn5-containing histone acetyltransferase complex in Drosophila.

Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells.

Amino Acid Sequence↗

PubMed2006

The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex.

Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.

Carrier Proteins↗

PubMed2005

Phosphorylation of the chromosomal passenger protein Bir1 is required for localization of Ndc10 to the spindle during anaphase and full spindle elongation.

The Saccharomyces cerevisiae inhibitor of apoptosis (IAP) repeat protein Bir1 localizes as a chromosomal passenger. A deletion analysis of Bir1 identified two regions important for function. The C-terminal region is essential for growth, binds Sli15, and is necessary and sufficient for the localization of Bir1 as a chromosomal passenger. The middle region is not essential but is required to localize the inner kinetochore protein Ndc10 to the spindle during anaphase and to the midzone at telophase. In contrast, precise deletion of the highly conserved IAP repeats conferred no phenotype and did not alter the cell cycle delay caused by loss of cohesin. Bir1 is phosphorylated in a cell cycle-dependent manner. Mutation of all nine CDK consensus sites in the middle region of Bir1 significantly decreased the level of phosphorylation and blocked localization of Ndc10 to the spindle at anaphase. Moreover, immunoprecipitation of Ndc10 with Bir1 was dependent on phosphorylation. The loss of Ndc10 from the anaphase spindle prevented elongation of the spindle beyond 7 microm. We conclude that phosphorylation of the middle region of Bir1 is required to bring Ndc10 to the spindle at anaphase, which is required for full spindle elongation.

Anaphase↗

PubMed2005

Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides.

Stable isotope-enriched molecules are used as internal standards and as tracers of in vivo substrate metabolism. The accurate conversion of measured ratios in the mass spectrometer to mole ratios is complicated because a polyatomic molecule containing enriched atoms will result in a combinatorial distribution of isotopomers depending on the enrichment and number of "labeled" atoms. This effect could potentially cause a large error in the mole ratio measurement depending on which isotope peak or peaks were used to determine the ratio. We report a computational method that predicts isotope distributions over a range of enrichments and compares the predicted distributions to experimental peptide isotope distributions obtained by Fourier transform ion cyclotron resonance mass spectrometry. Our approach is accurate with measured enrichments within 1.5% of expected isotope distributions. The method is also precise with 4.9, 2.0, and 0.8% relative standard deviations for peptides containing 59, 79, and 99 atom % excess (15)N, respectively. The approach is automated making isotope enrichment calculations possible for thousands of peptides in a single muLC-FTICR-MS experiment.

Animals↗

PubMed2005

Replication-independent histone deposition by the HIR complex and Asf1.

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.

Cell Cycle Proteins↗

PubMed2005

Glial membranes at the node of Ranvier prevent neurite outgrowth.

Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.

Animals↗

PubMed2005

A role for the P-body component GW182 in microRNA function.

In animals, the majority of microRNAs regulate gene expression through the RNA interference (RNAi) machinery without inducing small-interfering RNA (siRNA)-directed mRNA cleavage. Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies. Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations that prevent Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a siRNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.

Argonaute Proteins↗

PubMed2005

Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT).

In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (MudPIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture.

Biomarkers, Tumor↗

PubMed2005

A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.

Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1-depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

Anaphase↗

PubMed2005

Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

Amino Acid Motifs↗

PubMed2005

Proteomic comparison of two fractions derived from the transsynaptic scaffold.

A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin-based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF.

Animals↗

PubMed2005

Isolation and angiogenesis by endothelial progenitors in the fetal liver.

Endothelial progenitor cells (EPCs) have significant therapeutic potential. However, the low quantity of such cells available from bone marrow and their limited capacity to proliferate in culture make their use difficult. Here, we present the first definitive demonstration of the presence of true EPCs in murine fetal liver capable of forming blood vessels in vivo connected to the host's vasculature after transplantation. This population is particularly interesting because it can be obtained at high yield and has a high angiogenic capacity as compared with bone marrow-derived EPCs. The EPC capacity is contained within the CD31+Sca1+ cell subset. We demonstrate that these cells are dependent for survival and proliferation on a feeder cell monolayer derived from the fetal liver. In addition, we describe a novel and easy method for the isolation and ex vivo proliferation of these EPCs. Finally, we used gene expression profiling and tandem mass spectrometry proteomics to examine the fetal liver endothelial progenitors and the feeder cells to identify possible proangiogenic growth factor and endothelial differentiation-associated genes.

Animals↗

PubMed2005

Molecular analysis of kinetochore architecture in fission yeast.

Kinetochore composition and structure are critical for understanding how kinetochores of different types perform similar functions in chromosome segregation. We used affinity purification to investigate the kinetochore composition and assembly in Schizosaccharomyces pombe. We identified a conserved DASH complex that functions to ensure precise chromosome segregation. Unlike DASH in budding yeast that is localized onto kinetochores throughout the cell cycle, SpDASH is localized onto kinetochores only in mitosis. We also identified two independent groups of kinetochore components, one of which, the Sim4 complex, contains several novel Fta proteins in addition to known kinetochore components. DASH is likely to be associated with the Sim4 complex via Dad1 protein. The other group, Ndc80-MIND-Spc7 complex, contains the conserved Ndc80 and MIND complexes and Spc7 protein. We propose that fission yeast kinetochore is comprised of at least two major structural motifs that are biochemically separable. Our results suggest a high degree of conservation between the kinetochores of budding yeast and fission yeast even though many individual protein subunits do not have a high degree of sequence similarity.

Amino Acid Sequence↗

PubMed2005

TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium.

Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G protein-coupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.

Amino Acid Sequence↗