Biomedical subjects

Judit Villén

Publications and source records attributed to Judit Villén.

12 recordsLinked to original sources

PubMed2006

Inhibition of homologous recombination by a cohesin-associated clamp complex recruited to the rDNA recombination enhancer.

Silencing within the yeast ribosomal DNA (rDNA) repeats protects the integrity of this highly repetitive array by inhibiting hyperrecombination and repressing transcription from foreign promoters. Using affinity purification combined with highly sensitive mixture mass spectrometry, we have analyzed the protein interaction network involved in suppressing homologous recombination within the rDNA locus. We show that the Net1 and Sir2 subunits of the RENT (regulator of nucleolar silencing and telophase exit) silencing complex, and Fob1, which recruits RENT to the nontranscribed spacer I (NTS1) region of rDNA, are physically associated with Tof2. In addition to RENT components and Fob1, Tof2 copurified with a two-subunit complex composed of Lrs4 and Csm1. Tof2, Lrs4, and Csm1 are recruited to the NTS1 region by Fob1 and are specifically required for silencing at this rDNA region. Moreover, Lrs4 and Csm1 act synergistically with Sir2 to suppress unequal crossover at the rDNA and are released from the nucleolus during anaphase. Together with previous observations showing that Csm1 physically associates with cohesin, these findings suggest a possible model in which RENT, Tof2, and Lrs4/Csm1 physically clamp rDNA to the cohesin ring, thereby restricting the movement of rDNA sister chromatids relative to each other to inhibit unequal exchange.

Cell Cycle Proteins↗

PubMed2006

A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

Algorithms↗

PubMed2006

A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span.

Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. Here, we demonstrate that the protein kinase MST1 mediates oxidative-stress-induced cell death in primary mammalian neurons by directly activating the FOXO transcription factors. MST1 phosphorylates FOXO proteins at a conserved site within the forkhead domain that disrupts their interaction with 14-3-3 proteins, promotes FOXO nuclear translocation, and thereby induces cell death in neurons. We also extend the MST-FOXO signaling link to nematodes. Knockdown of the C. elegans MST1 ortholog CST-1 shortens life span and accelerates tissue aging, while overexpression of cst-1 promotes life span and delays aging. The cst-1-induced life-span extension occurs in a daf-16-dependent manner. The identification of the FOXO transcription factors as major and evolutionarily conserved targets of MST1 suggests that MST kinases play important roles in diverse biological processes including cellular responses to oxidative stress and longevity.

14-3-3 Proteins↗

PubMed2006

Optimization and use of peptide mass measurement accuracy in shotgun proteomics.

Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%.

Calibration↗

PubMed2006

Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon.

ClpXP, a bacterial AAA+ protease, controls intracellular levels of many stress-response proteins. To investigate substrate profile changes caused by a specific environmental stress, quantitative mass spectrometry (SILAC) was used to analyze proteins trapped by ClpXP(trap) before and after DNA damage. The abundance of half of the trapped proteins changed more than 3-fold after damage. Overrepresented substrates included the DNA-repair proteins RecN and UvrA. Among SOS-response proteins, 25% were ClpXP substrates and, importantly, nearly all of the highly induced regulon members were rapidly degraded. Other proteins, including the stress regulator sigma(S), were underrepresented in ClpXP(trap) after DNA damage; overproduction experiments suggest that simple substrate competition does not account for this reduced recognition. We conclude that damage-response proteins are an unusually rapidly degraded family and that ClpXP has substantial capacity to process the influx of newly synthesized substrates while maintaining the ability to degrade its other substrates in an environmentally responsive manner.

Adenosine Triphosphatases↗

PubMed2006

Rational dissection of binding surfaces for mimicking of discontinuous antigenic sites.

Peptide-based approaches to mimicking protein interactive regions have relied mainly on linear peptides; however, most binding sites are discontinuous and thus not easily reproducible by a linear sequence. Any attempt to replicate those sites by chemical means must not only integrate all residues involved in the recognition but also provide structural organization to native-like levels. Here we describe a surface mimic approach to the reconstruction of such complex molecular architectures, using as a model a discontinuous antigenic site of foot-and-mouth disease virus that is defined by residues belonging to three different capsid proteins. Our surface mimics are synthetic cyclic peptides, designed in silico, capable of binding antibodies directed to this site, and with demonstrated functional capabilities as vaccines in guinea pigs. Further, by saturation transfer difference NMR, we have determined several antibody binding residues on these peptides.

Animals↗

PubMed2005

A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation.

The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4(+)), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4(+), cmc1(+), cmc2(+) and rad24(+) results in a complete loss of silencing of a ura4(+) reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

Cell Cycle Proteins↗

PubMed2004

Towards a multi-site synthetic vaccine to foot-and-mouth disease: addition of discontinuous site peptide mimic increases the neutralization response in immunized animals.

Synthetic replicas of both antigenic sites A and D of foot-and-mouth disease virus have been tested as a first step towards a multicomponent peptide vaccine candidate. A first evaluation has been performed by neutralization assays on cells with serum mixtures from guinea pigs immunized independently with site A (A24) and site D (D8) peptides. The addition of site D antibodies to site A antibodies has a synergistic effect on neutralization. In a second group of experiments, guinea pigs have been immunized with a dendrimeric tetravalent (MAP) presentation of site A peptide, alone or in combination with D8, using the same total peptide dose. While the first inoculation gives a preferential response to site A-only antigen, specific response to site D and global neutralization levels significantly increase after reimmunization, reflecting a synergistic effect of site D.

Amino Acid Sequence↗

PubMed2004

Phosphoproteomic analysis of the developing mouse brain.

Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

Amino Acid Sequence↗

PubMed2004

Large-scale characterization of HeLa cell nuclear phosphoproteins.

Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

Amino Acid Sequence↗

PubMed2002

Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide.

Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulfide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194) (VP=viral protein). Guinea pig antisera to the peptides recognized the viral particle and competed with site D-specific monoclonal antibodies, while inoculation with a simple (not covalently joined to one another) admixture of the three VP1-VP3 sequences yielded no detectable virus-specific serum conversion. Similar results have been reproduced in two bovines. Antisera to the peptides also moderately neutralize FMDV in cell cultures and partially protect guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent, peptide-based vaccine against FMDV.

Amino Acid Sequence↗

PubMedDate not supplied

Synthetic approaches to multivalent lipopeptide dendrimers containing cyclic disulfide epitopes of foot-and-mouth disease virus.

The synthesis of a multiantigenic peptide dendrimer incorporating four copies of a cyclic disulfide epitope has been undertaken. Since standard chemoselective ligation procedures involving thioether formation are inadvisable in the presence of a preformed disulfide, conjugation through a peptide bond between the lipidated branched lysine scaffold and a suitably protected version of the cyclic disulfide has been used instead. Several synthetic approaches to the partially protected cyclic disulfide peptide have been explored. The most effective involves building a minimally protected version of the peptide by Boc solid phase synthesis, using fluorenyl-based anchorings and cysteine protecting groups. Peptide-resin cleavage and cysteine deprotection/oxidation are performed simultaneously by base-promoted elimination. The cyclic disulfide epitope is readily obtained in sufficient amounts by this procedure and subsequently incorporated to the lipidated lysine core by peptide bond formation in solution. A final acid deprotection step in anhydrous HF yields a peptide construction containing a maximum of three copies of the cyclic disulfide epitope, the lower substitution being attributable to steric constraints. This immunogen has been successfully used in an experimental vaccination trial against foot-and-mouth disease virus.

Animals↗