Biomedical subjects

James Wohlschlegel

Publications and source records attributed to James Wohlschlegel.

10 recordsLinked to original sources

PubMed2006

Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation.

The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb-Cdc28 inhibitor Sic1. In proliferating cells Cln-Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCF(Cdc4) and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCF(Cdc4) was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCF(Cdc4). In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.

Binding Sites↗

PubMed2006

SUMO-binding motifs mediate the Rad60-dependent response to replicative stress and self-association.

In fission yeast, the replication checkpoint is enforced by the kinase Cds1 (human Chk2), which regulates both cell cycle progression and DNA repair factors to ensure that the genome is faithfully duplicated prior to mitosis. Cds1 contains a forkhead-associated domain that mediates its interaction with phosphorylated residues in target proteins. One target of Cds1 is the essential nuclear protein Rad60, which contains the unique structural feature of tandem SUMO homology domains at its C terminus. Hypomorphic mutants of Rad60 cause profound defects in DNA repair and replication stress tolerance. To explore the physiological significance of the Cds1-Rad60 interaction, we have examined the phosphorylation of Rad60 by Cds1 in vitro and the in vivo phosphorylation of Rad60 in response to replication blocks. We find that the N terminus but not the SUMO-like domain of Rad60 is phosphorylated in both conditions. Three important Rad60 phosphorylation sites were identified: Thr(72), Ser(32), and Ser(34). Rad60 Thr(72) mediates the Cds1-Rad60 interaction and is required for the Cds1-dependent phosphorylation of Rad60 in response to replication arrest. Phosphorylation of Rad60 Ser(32) and Ser(34) in a putative SUMO-binding motif is critical for the survival of replication stress. In addition, mutation of Rad60 Ser(32) and Ser(34) to alanine is lethal in cells deleted for the RecQ DNA helicase Rqh1. Finally, we find that Rad60 self-associates via its C-terminal SUMO-like domain and putative SUMO-binding motifs.

Amino Acid Motifs↗

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

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

Constraining G1-specific transcription to late G1 phase: the MBF-associated corepressor Nrm1 acts via negative feedback.

G1-specific transcription in yeast depends upon SBF and MBF. We have identified Nrm1 (negative regulator of MBF targets 1), as a stable component of MBF. NRM1 (YNR009w), an MBF-regulated gene expressed during late G1 phase, associates with G1-specific promoters via MBF. Transcriptional repression upon exit from G1 phase requires both Nrm1 and MBF. Inactivation of Nrm1 results in prolonged expression of MBF-regulated transcripts and leads to hydroxyurea (HU) resistance and enhanced bypass of rad53Delta- and mec1Delta-associated lethality. Constitutive expression of a stabilized form of Nrm1 represses MBF targets and leads to HU sensitivity. The fission yeast homolog SpNrm1, encoded by the MBF target gene nrm1(+) (SPBC16A3.07c), binds to MBF target genes and acts as a corepressor. In both yeasts, MBF represses G1-specific transcription outside of G1 phase. A negative feedback loop involving Nrm1 bound to MBF leads to transcriptional repression as cells exit G1 phase.

Amino Acid Sequence↗

PubMed2006

p130-angiomotin associates to actin and controls endothelial cell shape.

Angiomotin, an 80 kDa protein expressed in endothelial cells, promotes cell migration and invasion, and stabilizes tube formation in vitro. Angiomotin belongs to a new protein family with two additional members, Amotl-1 and Amotl-2, which are characterized by conserved coiled-coil domains and C-terminal PDZ binding motifs. Here, we report the identification of a 130 kDa splice isoform of angiomotin that is expressed in different cell types including vascular endothelial cells, as well as cytotrophoblasts of the placenta. p130-Angiomotin consists of a cytoplasmic N-terminal extension that mediates its association with F-actin. Transfection of p130-angiomotin into endothelial cells induces actin fiber formation and changes cell shape. The p130-angiomotin protein remained associated with actin after destabilization of actin fibers with cytochalasin B. In contrast to p80-angiomotin, p130-angiomotin does not promote cell migration and did not respond to angiostatin. We propose that p80- and p130-angiomotin play coordinating roles in tube formation by affecting cell migration and cell shape, respectively.

Actins↗

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

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

Identification of ubiquitination sites and determination of ubiquitin-chain architectures by mass spectrometry.

The identification of protein modification sites is an important step toward understanding the biological role of covalent modifications. For example, the mapping of phosphorylation sites and analyses of phosphorylation site mutants have tremendously contributed to our knowledge of different cellular processes. Given the diverse functions of ubiquitination, similar studies with ubiquitin attachment site mutants are becoming increasingly important in understanding the molecular roles of ubiquitination. Relatively few studies to date have mapped ubiquitination sites, and in almost all cases the identification of the acceptor lysines were based on indirect evidence (Petroski and Deshaies, 2003; Scherer et al., 1995); that is, mutation of particular lysines to arginines blocked ubiquitination. Direct evidence for ubiquitin attachment sites has been obtained by mapping of hydroxylamine-derived peptides from ubiquitinated proteins (Chau et al., 1989); however, these experiments can be very challenging. Recent advances in protein mass spectrometry have enabled ubiquitinated lysine residues to be identified directly, thereby providing more convincing evidence for the exact location of the modification (Flick et al., 2004; Peng et al., 2003). In addition to mapping attachment sites, mass spectrometry can also be used to determine the type of ubiquitin chain linkage (Flick et al., 2004; Peng et al., 2003). In vivo evidence for the covalent attachment of the carboxyl terminus of one ubiquitin molecule to lysine residues in several locations in a different ubiquitin molecule demonstrates the complexity of ubiquitin biology (Peng et al., 2003). These different ubiquitin chain topologies can dramatically affect the molecular function of ubiquitin chains (Hoege et al., 2002; Spence et al., 1995), and, hence, the mass spectrometric determination of the ubiquitin chain architecture can provide important insight into the mechanisms of ubiquitin function. This chapter describes mass spectrometric approaches for identifying ubiquitin acceptor lysines on target proteins and analyzing the ubiquitin chain topology.

Amino Acid Sequence↗

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↗