Biomedical subjects

John R Yates

Publications and source records attributed to John R Yates.

At least 73 records · Page 4Linked to original sources

PubMed2005

Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans.

Nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory neurotransmission in neurons and muscles. To identify nAChR accessory proteins, which may regulate their expression or function, we performed tandem affinity purification of the levamisole-sensitive nAChR from Caenorhabditis elegans, mass spectrometry of associated components, and RNAi-based screening for effects on in vivo nicotine sensitivity. Among the proteins identified was the calcineurin A subunit TAX-6, which appeared to function as a negative regulator of nAChR activity. We also identified five proteins not previously linked to nAChR function, whose inactivation conferred nicotine resistance, implicating them as positive regulators of nAChR activity. Of these, the copine NRA-1 colocalized with the levamisole receptor at neuronal and muscle plasma membranes, and, when mutated, caused reduced synaptic nAChR expression. Loss of SOC-1, which acts in receptor tyrosine kinase (RTK) signaling, also reduced synaptic levamisole receptor levels, as did mutations in the fibroblast growth factor receptor EGL-15, and another RTK, CAM-1. Thus, tandem affinity purification is a viable approach to identify novel proteins regulating neurotransmitter receptor activity or expression in model systems like C. elegans.

Affinity Labels↗

PubMed2005

Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.

BACKGROUND: The centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known centriole proteins. RESULTS: We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (centrioles) isolated from the green alga Chlamydomonas reinhardtii. This analysis detected the majority of known centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated centriole candidate proteins in two classes. Members of the first class of proteins (BUG1-BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1-POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated centriole candidate proteins are encoded by orthologs of genes (OFD1, NPHP-4, and PACRG) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of centrioles and/or basal bodies. CONCLUSIONS: By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.

Amino Acid Sequence↗

PubMed2005

Polyglutamine-expanded spinocerebellar ataxia-7 protein disrupts normal SAGA and SLIK histone acetyltransferase activity.

Histone acetyltransferases have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous multisubunit histone acetyltransferase complexes that were originally identified in the yeast Saccharomyces cerevisiae. Here, we identify the protein Sgf73/Sca7 as a component of SAGA and SLIK, and a homologue of the human SCA7-encoded protein ataxin-7, which, in its polyglutamine expanded pathological form, is responsible for the neurodegenerative disease spinocerebellar ataxia 7 (SCA7). Our findings indicate that yeast Sca7 is necessary for the integrity and function of both SAGA and SLIK, and that the human ataxin-7 is able to compliment the loss of Sca7 in yeast. A polyglutamine-expanded version of ataxin-7 assembles a SAGA complex that is depleted of critical proteins that regulate the ability of SAGA to acetylate nucleosomes. These observations have significant implications for the function of the human Sca7 protein in disease pathogenesis.

Acetyltransferases↗

PubMed2005

Crystallin {gamma}B-I4F mutant protein binds to {alpha}-crystallin and affects lens transparency.

A new mouse mutant line, Clapper, identified from N-ethyl-N-nitrosurea (ENU)-mutagenized mice, develops a dominant lamellar cataract. The cataract blocks the image of retinal fundus and transmits a fuzzy fluorescein image of retinal vasculature during angiography. The cataractous lens opacity decreases as the mice age. The Clapper mutation has been identified to be a missense mutation of the gammaB-crystallin gene that replaces the 4th isoleucine residue with a phenylalanine (gammaB-I4F). Unlike wild type gammaB, the gammaB-I4F mutant protein binds to alpha-crystallin to form high molecular weight complexes in vivo and in vitro. Circular dichroism measurements indicate that gammaB-I4F protein is less stable than wild type gammaB at high temperature. Darkly stained aggregates, enlarged interfiber spaces, and disorganized and smaller inner mature fibers were found in the regions of the cataract in homozygous Clapper mutant lenses. Thus, the lamellar cataract is likely due to the light-scattering effects of the enlarged interfiber spaces and protein aggregates caused by gammaB-I4F mutant proteins interacting with alpha-crystallin in the lens.

Angiography↗

PubMed2005

Maspin alters the carcinoma proteome.

Maspin, a member of the serine protease inhibitor (serpin) family, is a tumor suppressor in breast and prostate cancer. To address molecular mechanisms underlying maspin's activity, we restored its expression in invasive carcinoma cells and analyzed the resulting changes by shotgun proteomics. Using a mass spectrometry-based multidimensional proteomic method, we observed changes to the expression of approximately 27% of the detectable proteome. In particular, we noted changes to the expression of proteins that regulate cytoskeletal architecture, cell death, and protein turnover. In each case, changes in protein expression were accompanied by measurable changes in tumor cell phenotype. Thus, maspin-expressing cells exhibit a more prominent actin cytoskeleton, a reduced invasive capacity, an increased rate of spontaneous apoptosis, and an altered proteasome function. These observations reveal for the first time the far reaching effects of maspin on multiple protein networks and a new hypothesis of maspin function based on the regulation of proteasome function.

Apoptosis↗

PubMed2005

The CENP-F-like proteins HCP-1 and HCP-2 target CLASP to kinetochores to mediate chromosome segregation.

During chromosome segregation, kinetochores form dynamic connections with spindle microtubules. In vertebrates, these attachments require the activities of a number of outer kinetochore proteins, including CENP-F [1, 2] and the widely conserved microtubule-associated protein CLASP [3]. Here, we investigate the functional relationship between HCP-1/2, two redundant CENP-F-like proteins, and CLASP(CLS-2) in Caenorhabditis elegans. HCP-1/2 and CLASP(CLS-2) localize transiently to mitotic C. elegans kinetochores with nearly identical kinetic profiles, and biochemical purifications demonstrate that they also associate physically. In embryos depleted of HCP-1/2, CLASP(CLS-2) no longer localizes to chromosomes, whereas CLASP(CLS-2) depletion does not prevent HCP-1/2 targeting. Consistent with the localization dependency and biochemical association, depletion of HCP-1/2 or CLASP(CLS-2) resulted in virtually identical defects in mitotic chromosome segregation characterized by a failure of sister-chromatid biorientation. This phenotype could be partially suppressed by disrupting the astral forces that pull spindle poles apart in the 1 cell embryo, indicating that CLASP(CLS-2) is required for biorientation when chromosome-spindle attachments are subjected to poleward force. Our results establish that the key role of HCP-1/2 is to target CLASP(CLS-2) to kinetochores, and they support the recently proposed model that CLASP functions to promote the polymerization of kinetochore bound microtubules [4].

Animals↗

PubMed2005

ZW10 links mitotic checkpoint signaling to the structural kinetochore.

The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80-Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1-Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived "stop anaphase" inhibitor.

Anaphase↗

PubMed2005

RNA-dependent cysteine biosynthesis in archaea.

Several methanogenic archaea lack cysteinyl-transfer RNA (tRNA) synthetase (CysRS), the essential enzyme that provides Cys-tRNA(Cys) for translation in most organisms. Partial purification of the corresponding activity from Methanocaldococcus jannaschii indicated that tRNA(Cys) becomes acylated with O-phosphoserine (Sep) but not with cysteine. Further analyses identified a class II-type O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS). SepRS specifically forms Sep-tRNA(Cys), which is then converted to Cys-tRNA(Cys) by SepCysS. Comparative genomic analyses suggest that this pathway, encoded in all organisms lacking CysRS, can also act as the sole route for cysteine biosynthesis. This was proven for Methanococcus maripaludis, where deletion of the SepRS-encoding gene resulted in cysteine auxotrophy. As the conversions of Sep-tRNA to Cys-tRNA or to selenocysteinyl-tRNA are chemically analogous, the catalytic activity of SepCysS provides a means by which both cysteine and selenocysteine may have originally been added to the genetic code.

Adenosine Triphosphate↗

PubMed2005

Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer.

To comprehensively identify proteins interacting with 14-3-3 sigma in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3 sigma in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3 sigma. 25 proteins were not previously assigned to any function and were named SIP2-26 (for 14-3-3 sigma-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3 sigma expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3 sigma could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3 sigma expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.

14-3-3 Proteins↗

PubMed2005

Global profiling of Shewanella oneidensis MR-1: expression of hypothetical genes and improved functional annotations.

The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.

Gene Expression Profiling↗

PubMed2005

Protein identification using 2D-LC-MS/MS.

Multidimensional liquid chromatography techniques have been coupled to tandem mass spectrometry to provide a robust method to identify proteins in complex mixtures. Data acquisition is interfaced directly with search algorithms for identification through cross-correlation with databases. This review describes the most recent advances in methodologies for protein identification by mass spectrometry and describes the limitations of the application of the technologies.

Algorithms↗

PubMed2005

Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation.

The specific post-translational modifications to histones influence many nuclear processes including gene regulation, DNA repair and replication. Recent studies have identified effector proteins that recognize patterns of histone modification and transduce their function in downstream processes. For example, histone acetyltransferases (HATs) have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. Yeast SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous and conserved multi-subunit HAT complexes, which preferentially acetylate histones H3 and H2B and deubiquitinate histone H2B. Here we identify the chromatin remodelling protein Chd1 (chromo-ATPase/helicase-DNA binding domain 1) as a component of SAGA and SLIK. Our findings indicate that one of the two chromodomains of Chd1 specifically interacts with the methylated lysine 4 mark on histone H3 that is associated with transcriptional activity. Furthermore, the SLIK complex shows enhanced acetylation of a methylated substrate and this activity is dependent upon a functional methyl-binding chromodomain, both in vitro and in vivo. Our study identifies the first chromodomain that recognizes methylated histone H3 (Lys 4) and possibly identifies a larger subfamily of chromodomain proteins with similar recognition properties.

Acetylation↗

PubMed2005

A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.

Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific. We observed posttranscriptional gene silencing through translational repression of messenger RNA during sexual development, and a 47-base 3' untranslated region motif is implicated in this process.

3' Untranslated Regions↗

PubMed2005

Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels.

2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation.

Acrylic Resins↗

PubMed2005

Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity.

Microtubules regulate actin-based processes such as cell migration and cytokinesis, but molecular mechanisms are not understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. Here, we identify tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During "new end take off" (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. Our results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly.

Actins↗

PubMed2005

A streamlined platform for high-content functional proteomics of primary human specimens.

Achieving information content of satisfactory breadth and depth remains a formidable challenge for proteomics. This problem is particularly relevant to the study of primary human specimens, such as tumor biopsies, which are heterogeneous and of finite quantity. Here we present a functional proteomics strategy that unites the activity-based protein profiling and multidimensional protein identification technologies (ABPP-MudPIT) for the streamlined analysis of human samples. This convergent platform involves a rapid initial phase, in which enzyme activity signatures are generated for functional classification of samples, followed by in-depth analysis of representative members from each class. Using this two-tiered approach, we identified more than 50 enzyme activities in human breast tumors, nearly a third of which represent previously uncharacterized proteins. Comparison with cDNA microarrays revealed enzymes whose activity, but not mRNA expression, depicted tumor class, underscoring the power of ABPP-MudPIT for the discovery of new markers of human disease that may evade detection by other molecular profiling methods.

Biomarkers, Tumor↗

PubMed2005

Proteomics of organelles and large cellular structures.

The mass-spectrometry-based identification of proteins has created opportunities for the study of organelles, transport intermediates and large subcellular structures. Traditional cell-biology techniques are used to enrich these structures for proteomics analyses, and such analyses provide insights into the biology and functions of these structures. Here, we review the state-of-the-art proteomics techniques for the analysis of subcellular structures and discuss the biological insights that have been derived from such studies.

Cell Fractionation↗

PubMed2005

Dim1p is required for efficient splicing and export of mRNA encoding lid1p, a component of the fission yeast anaphase-promoting complex.

Schizosaccharomyces pombe Dim1p is required for maintaining the steady-state level of the anaphase-promoting complex or cyclosome (APC/C) component Lid1p and thus for maintaining the steady-state level and activity of the APC/C. To gain further insight into Dim1p function, we have investigated the mechanism whereby Dim1p influences Lid1p levels. We show that S. pombe cells lacking Dim1p or Saccharomyces cerevisiae cells lacking its ortholog, Dib1p, are defective in generalized pre-mRNA splicing in vivo, a result consistent with the identification of Dim1p as a component of the purified yeast U4/U6.U5 tri-snRNP complex. Moreover, we find that Dim1p is part of a complex with the splicing factor Prp1p. However, although Dim1p is required for efficient splicing of lid1(+) pre-mRNA, circumventing the necessity for this particular function of Dim1p is insufficient for restoring normal Lid1p levels. Finally, we provide evidence that Dim1p also participates in the nuclear export of lid1(+) mRNA and that it is likely the combined loss of both of these two Dim1p functions which compromises Lid1p levels in the absence of proper Dim1p function. These data indicate that a mechanism acting at the level of mRNA impacts the functioning of the APC/C, a critical complex in controlling mitotic progression.

Anaphase-Promoting Complex-Cyclosome↗