Biomedical subjects

John R Yates

Publications and source records attributed to John R Yates.

At least 37 records · Page 2Linked to original sources

PubMed2006

Global analysis of protein palmitoylation in yeast.

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.

Acetyltransferases↗

PubMed2006

Sws1 is a conserved regulator of homologous recombination in eukaryotic cells.

Rad52-dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM-type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Delta and rqh1Delta mutants and rescues the inviability of srs2Delta rqh1Delta cells. Sws1 functions at an early step of recombination in a pro-recombinogenic complex with Rlp1 and Rdl1, two RecA-like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2-RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells.

Amino Acid Sequence↗

PubMed2006

Protein disulfide isomerase serves as a molecular chaperone to maintain estrogen receptor alpha structure and function.

The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.

Cell Line, Tumor↗

PubMed2006

Rtn1p is involved in structuring the cortical endoplasmic reticulum.

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Delta cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

Endoplasmic Reticulum↗

PubMed2006

The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres.

In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H-I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H-I complex proteins, indicating that the CENP-H-I complex may function, in part, as a marker directing CENP-A deposition to centromeres.

Animals↗

PubMed2006

The human CENP-A centromeric nucleosome-associated complex.

The basic element for chromosome inheritance, the centromere, is epigenetically determined in mammals. The prime candidate for specifying centromere identity is the array of nucleosomes assembled with CENP-A, the centromere-specific histone H3 variant. Here, we show that CENP-A nucleosomes directly recruit a proximal CENP-A nucleosome associated complex (NAC) comprised of three new human centromere proteins (CENP-M, CENP-N and CENP-T), along with CENP-U(50), CENP-C and CENP-H. Assembly of the CENP-A NAC at centromeres is dependent on CENP-M, CENP-N and CENP-T. Facilitates chromatin transcription (FACT) and nucleophosmin-1 (previously implicated in transcriptional chromatin remodelling and as a multifunctional nuclear chaperone, respectively) are absent from histone H3-containing nucleosomes, but are stably recruited to CENP-A nucleosomes independent of CENP-A NAC. Seven new CENP-A-nucleosome distal (CAD) centromere components (CENP-K, CENP-L, CENP-O, CENP-P, CENP-Q, CENP-R and CENP-S) are identified as assembling on the CENP-A NAC. The CENP-A NAC is essential, as disruption of the complex causes errors of chromosome alignment and segregation that preclude cell survival despite continued centromere-derived mitotic checkpoint signalling.

Amino Acid Sequence↗

PubMed2006

Cross-correlation algorithm for calculation of peptide molecular weight from tandem mass spectra.

We have developed an approach to identify the molecular weight of a peptide ion directly from its corresponding tandem mass spectrum using a cross-correlation function. We have shown that the monoisotopic molecular weight can be calculated for approximately 90% of tandem mass spectra identified from tryptic digests of complex protein mixtures. The accuracy of the calculated monoisotopic masses was dependent on the resolution and mass accuracy of the spectra analyzed, but was typically <0.25 amu for linear ion trap mass spectra. The ability to calculate accurate monoisotopic molecular weights for low-resolution ion trap data should significantly improve both the speed and performance of database searches for which typical mass accuracies of approximately 3 amu are employed. In addition, this strategy can also be used to identify the precursor ion for tandem mass spectra acquired using large ion selection windows in data-independent collision-activated dissociation and has the potential to identify multiplexed tandem mass spectra.

Algorithms↗

PubMed2006

PMS2 mutations in childhood cancer.

Until recently, the PMS2 DNA mismatch repair gene has only rarely been implicated as a cancer susceptibility locus. New studies have shown, however, that earlier analyses of this gene have had technical limitations and also that the genetic behavior of mutant PMS2 alleles is unusual, in that, unlike MLH1 or MSH2 mutations, PMS2 mutations show low heterozygote penetrance. As a result, a dominantly inherited cancer predisposition has not been a feature reported in families with PMS2 mutations. Such families have instead been ascertained through childhood-onset cancers in homozygotes or through apparently sporadic colorectal cancer in heterozygotes. We present further information on the phenotype associated with homozygous PMS2 deficiency in 13 patients from six families of Pakistani origin living in the United Kingdom. This syndrome is characterized by café-au-lait skin pigmentation and a characteristic tumor spectrum, including leukemias, lymphomas, cerebral malignancies (such as supratentorial primitive neuroectodermal tumors, astrocytomas, and glioblastomas), and colorectal neoplasia with an onset in early adult life. We present evidence for a founder effect in five families, all of which carried the same R802-->X mutation (i.e., arginine-802 to stop) in PMS2. This cancer syndrome can be mistaken for neurofibromatosis type 1, with important management implications including the risk of the disorder occurring in siblings and the likelihood of tumor development in affected individuals.

Adenosine Triphosphatases↗

PubMed2006

Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis.

Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption-ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)-tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.

Amino Acid Sequence↗

PubMed2006

Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways.

In plants, animals, and fungi, members of the Dicer family of RNase III-related enzymes process double-stranded RNA (dsRNA) to initiate small-RNA-mediated gene-silencing mechanisms. To learn how C. elegans Dicer, DCR-1, functions in multiple distinct silencing mechanisms, we used a mass-spectrometry-based proteomics approach to identify DCR-1-interacting proteins. We then generated and characterized deletion alleles for the corresponding genes. The interactors are required for production of three species of small RNA, including (1) small interfering RNAs (siRNAs), derived from exogenous dsRNA triggers (exo-siRNAs); (2) siRNAs derived from endogenous triggers (endo-siRNAs); and (3) developmental regulatory microRNAs (miRNAs). One interactor, the conserved RNA-phosphatase homolog PIR-1, is required for the processing of a putative amplified DCR-1 substrate. Interactors required for endo-siRNA production include ERI-1 and RRF-3, whose loss of function enhances RNAi. Our findings provide a first glimpse at the complex biochemical niche of Dicer and suggest that competition exists between DCR-1-mediated small-RNA pathways.

Amino Acid Sequence↗

PubMed2006

Performance of a linear ion trap-Orbitrap hybrid for peptide analysis.

Proteomic analysis of digested complex protein mixtures has become a useful strategy to identify proteins involved in biological processes. We have evaluated the use of a new mass spectrometer that combines a linear ion trap and an Orbitrap to create a hybrid tandem mass spectrometer. A digested submandibular/sublingual saliva sample was used for the analysis. We find the instrument is capable of mass resolution in excess of 40,000 and mass measurement accuracies of less than 2 ppm for the analysis of complex peptide mixtures. Such high mass accuracy allowed the elimination of virtually any false positive peptide identifications, suggesting that peptides that do not match the specificity of the protease used in the digestion of the sample should not automatically be considered as false positives. Tandem mass spectra from the linear ion trap and from the Orbitrap have very similar ion abundance ratios. We conclude this instrument will be well suited for shotgun proteomic types of analyses.

Humans↗

PubMed2006

Cip1 and Cip2 are novel RNA-recognition-motif proteins that counteract Csx1 function during oxidative stress.

Eukaryotic cells reprogram their global patterns of gene expression in response to stress. Recent studies in Schizosaccharomyces pombe showed that the RNA-binding protein Csx1 plays a central role in controlling gene expression during oxidative stress. It does so by stabilizing atf1(+) mRNA, which encodes a subunit of a bZIP transcription factor required for gene expression during oxidative stress. Here, we describe two related proteins, Cip1 and Cip2, that were identified by multidimensional protein identification technology (MudPIT) as proteins that coprecipitate with Csx1. Cip1 and Cip2 are cytoplasmic proteins that have RNA recognition motifs (RRMs). Neither protein is essential for viability, but a cip1Delta cip2Delta strain grows poorly and has altered cellular morphology. Genetic epistasis studies and whole genome expression profiling show that Cip1 and Cip2 exert posttranscriptional control of gene expression in a manner that is counteracted by Csx1. Notably, the sensitivity of csx1Delta cells to oxidative stress and their inability to induce expression of Atf1-dependent genes are partially rescued by cip1Delta and cip2Delta mutations. This study emphasizes the importance of a modulated mRNA stability in the eukaryotic stress response pathways and adds new information to the role of RNA-binding proteins in the oxidative stress response.

Amino Acid Motifs↗

PubMed2006

The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p.

Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Central limit theorem as an approximation for intensity-based scoring function.

In this paper, we present an intensity-based probability function to identify peptides from tandem mass spectra and amino acid sequence databases. The function is an approximation to the central limiting theorem, and it explicitly depends on the cumulative product ion intensities, number of product ions of a peptide, and expectation value of the cumulative intensity. We compare the results of database searches using the new scoring function and scoring functions from earlier algorithms, which implement hypergeometric probability, Poisson's model, and cross-correlation scores. For a standard protein mixture (tandem mass spectra generated from the mixture of five known proteins), we generate receiver operating curves with all scoring schemes. The receiver operating curves show that the shared peaks count-based probability methods (like Poisson and hypergeometric models) are the most specific for matching high-quality tandem mass spectra. The intensity-based (central limit model) and intensity-modeled (cross-correlation) methods are more sensitive when matching low-quality tandem mass spectra, where the number of shared peaks is insufficient to correctly identify a peptide. Cross-correlation methods show a small advantage over the intensity-based probability method.

Algorithms↗

PubMed2006

Differential protein expression in the metal-reducing bacterium Geobacter sulfurreducens strain PCA grown with fumarate or ferric citrate.

Geobacter sulfurreducens, generally considered to be a strict anaerobe, is a predominant microbe in subsurface environments, where it utilizes available metals as electron acceptors. To better understand the metabolic processes involved in the metal-reduction capability of this microbe, the proteins expressed by cells grown anaerobically with either fumarate or ferric citrate as electron acceptor were compared. Proteins were separated by 2-DE under denaturing or nondenaturing conditions, and proteins varying in abundance with a high level of statistical significance (p<0.0001) were identified by peptide mass analysis. Denaturing 2-DE revealed significant differences in the relative abundance of the membrane proteins OmpA and peptidoglycan-associated lipoprotein, several metabolic enzymes, and, in addition, superoxide dismutase and rubredoxin oxidoreductase. Nondenaturing 2-DE revealed elevated catalase in cells grown with ferric citrate. These results suggest that, in addition to adjustments in membrane transport and specific metabolic pathways in response to these two different electron acceptors, distinct differences exist in the oxidative environment within the cell when fumarate or soluble ferric citrate is provided as electron acceptor. Although an anaerobe, G. sulfurreducens appears to have alternate mechanisms for dealing with reactive oxygen species in response to increased intracellular soluble iron.

Anaerobiosis↗

PubMed2006

Simultaneous identification and verification of Bacillus anthracis.

Specific identification of Bacillus anthracis (B. anthracis) is vital for the accurate treatment of afflicted personnel during biological warfare situations and civilian terrorist attacks. In order to accomplish this, we have subjected the lysates from B. anthracis to affinity purification using monoclonal antibodies for the selected antigenic protein present in the bacteria. The bound antigenic protein was identified by multi-dimensional protein identification technology (MudPIT) to be a surface layer protein EA1. The same antigen was identified from the lysates from a few strains of B. anthracis demonstrating the observation to be common for B. anthracis strains. Hence, this presents an effective pathway for the identification of the bacteria present in unknown samples of various origins. Generation of a database containing the EA1 protein has been found to be useful in the database search of unknown samples.

Affinity Labels↗

PubMed2006

Phospho-regulation of the Cdc14/Clp1 phosphatase delays late mitotic events in S. pombe.

In eukaryotes, exit from mitosis occurs through the inactivation of the Cdk1-cyclin B kinase complex and the reversal of its phosphorylation events. These late mitotic events are tightly regulated to occur only after the onset of anaphase and prior to cytokinesis. Central to this regulation is the conserved Cdc14 family of protein phosphatases, whose activity reverses Cdk-dependent phosphorylation events. S. cerevisiae Cdc14 activity is restrained from dephosphorylating Cdk substrates and inactivating Cdk1 through its nucleolar sequestration prior to anaphase. Here, we describe a unique mode of Cdc14 regulation that operates prior to anaphase in fission yeast. Cdk1 phosphorylates and inhibits the catalytic activity of the Cdc14 family member, Clp1/Flp1. As Cdk1 activity declines during anaphase progression, Clp1/Flp1 autocatalytically reverses these phosphorylation events to stimulate its own activity. These findings point to a simple regulatory circuit that couples Cdk1 activation with its inactivation mediated through phosphorylation-dependent regulation of Clp1/Flp1 phosphatase activity.

CDC2 Protein Kinase↗

PubMed2006

Proteomic identification of palmitoylated proteins.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

Alkylation↗