PubMed Health⌕ Search

Biomedical subjects

Anna Shevchenko

Publications and source records attributed to Anna Shevchenko.

28 records · Page 2Linked to original sources
PubMed2003

MultiTag: multiple error-tolerant sequence tag search for the sequence-similarity identification of proteins by mass spectrometry.

The characterization of proteomes by mass spectrometry is largely limited to organisms with sequenced genomes. To identify proteins from organisms with unsequenced genomes, database sequences from related species must be employed for sequence-similarity protein identifications. Peptide sequence tags (Mann, 1994) have been used successfully for the identification of proteins in sequence databases using partially interpreted tandem mass spectra of tryptic peptides. We have extended the ability of sequence tag searching to the identification of proteins whose sequences are yet unknown but are homologous to known database entries. The MultiTag method presented here assigns statistical significance to matches of multiple error-tolerant sequence tags to a database entry and ranks alignments by their significance. The MultiTag approach has the distinct advantage over other sequence-similarity approaches of being able to perform sequence-similarity identifications using only very short (2-4) amino acid residue stretches of peptide sequences, rather than complete peptide sequences deduced by de novo interpretation of tandem mass spectra. This feature facilitates the identification of low abundance proteins, since noisy and low-intensity tandem mass spectra can be utilized.

Alcohol Dehydrogenase↗
PubMed2002

High conservation of the Set1/Rad6 axis of histone 3 lysine 4 methylation in budding and fission yeasts.

Histone 3 lysine 4 (H3 Lys(4)) methylation in Saccharomyces cerevisiae is mediated by the Set1 complex (Set1C) and is dependent upon ubiquitinylation of H2B by Rad6. Mutually exclusive methylation of H3 at Lys(4) or Lys(9) is central to chromatin regulation; however, S. cerevisiae lacks Lys(9) methylation. Furthermore, a different H3 Lys(4) methylase, Set 7/9, has been identified in mammals, thereby questioning the relevance of the S. cerevisiae findings for eukaryotes in general. We report that the majority of Lys(4) methylation in Schizosaccharomyces pombe, like in S. cerevisiae, is mediated by Set1C and is Rad6-dependent. S. pombe Set1C mediates H3 Lys(4) methylation in vitro and contains the same eight subunits found in S. cerevisiae, including the homologue of the Drosophila trithorax Group protein, Ash2. Three additional features of S. pombe Set1C each involve PHD fingers. Notably, the Spp1 subunit is dispensable for H3 Lys(4) methylation in budding yeast but required in fission yeast, and Sp_Set1C has a novel proteomic hyperlink to a new complex that includes the homologue of another trithorax Group protein, Lid (little imaginal discs). Thus, we infer that Set1C is highly conserved in eukaryotes but observe that its links to the proteome are not.

DNA-Binding Proteins↗
PubMed2002

Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit.

Sur2 is a metazoan Mediator subunit that interacts with the adenovirus E1A protein and functions in a mitogen-activated protein kinase pathway required for vulva development in Caenorhabditis elegans. We generated sur2-/- embryonic stem cells to analyze its function as a mammalian Mediator component. Our results show that Sur2 forms a subcomplex of the Mediator with two other subunits, TRAP/Med100 and 95. Knock-out of Sur2 prevents activation by E1A-CR3 and the mitogen-activated protein kinase-regulated ETS transcription factor Elk-1, but not by multiple other transcription factors. These results imply that specific activation domains stimulate transcription by binding to distinct Mediator subunits. Activation by E1A and Elk-1 requires recruitment of Mediator to a promoter by binding to its Sur2 subunit.

Adenovirus E1A Proteins↗
PubMed2002

Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective.

We employed a combination of tandem affinity purification and mass spectrometry for deciphering protein complexes and the protein interaction network in budding yeast. 53 genes were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates. 38 baits pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. We identified four proteins shared between complexes of different functionality thus charting segments of a protein interaction network. Concordance with the results of genome-wide two-hybrid screening was poor (14% of identified interactors overlapped) suggesting that the two approaches may provide complementary views on physical interactions within the proteome.

Chromatography, Affinity↗
PubMed2002

Charting the protein complexome in yeast by mass spectrometry.

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks.

Cell Cycle Proteins↗
PubMed2002

Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery.

During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-alpha 1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.

Adenovirus E1B Proteins↗
PubMed2002

Saccharomyces cerevisiae Bzz1p is implicated with type I myosins in actin patch polarization and is able to recruit actin-polymerizing machinery in vitro.

In Saccharomyces cerevisiae, the WASP (Wiskott-Aldrich syndrome protein) homologue Las17p (also called Bee1p) is an important component of cortical actin patches. Las17p is part of a high-molecular-weight protein complex that regulates Arp2/3 complex-dependent actin polymerization at the cell cortex and that includes the type I myosins Myo3p and Myo5p and verprolin (Vrp1p). To identify other factors implicated with this complex in actin regulation, we isolated proteins that bind to Las17p by two-hybrid screening and affinity chromatography. Here, we report the characterization of Lsb7/Bzz1p (for Las seventeen binding protein 7), an Src homology 3 (SH3) domain protein that interacts directly with Las17p via a polyproline-SH3 interaction. Bzz1p coimmunoprecipitates in a complex with Las17p, Vrp1p, Myo3/5p, Bbc1p, Hsp70p, and actin. It colocalizes with cortical actin patches and with Las17p. This localization is dependent on Las17p, but not on F-actin. Bzz1p interacts physically and genetically with type I myosins. While deletion of BZZ1 shows no obvious phenotype, simultaneous deletion of the BZZ1, MYO3, and MYO5 genes is lethal. Overexpression of Bzz1p inhibits cell growth, and a bzz1Delta myo5Delta double mutant is unable to restore actin polarity after NaCl stress. Finally, Bzz1p in vitro is able to recruit a functional actin polymerization machinery through its SH3 domains. Its interactions with Las17p, Vrp1p, and the type I myosins are essential for this process. This suggests that Bzz1p could be implicated in the regulation of actin polymerization.

Actins↗
PubMed2002

"De novo" sequencing of peptides recovered from in-gel digested proteins by nanoelectrospray tandem mass spectrometry.

Proteins separated by one-dimensional or two-dimensional gel electrophoresis can be digested in-gel with trypsin and the recovered peptides can be sequenced de novo using triple quadrupole or hybrid quadrupole time-of-flight instruments equipped with a nanoelectrospray ion source. The peptide sequences determined provide useful information for identification of proteins by homology searching for cloning of the cognate genes by PCR based approaches.

Animals↗
PubMedDate not supplied

Sequence similarity-based proteomics in insects: characterization of the larvae venom of the Brazilian moth Cerodirphia speciosa.

Using a combination of tandem mass spectrometric sequencing and sequence similarity searches, we characterized the larvae venom of the moth Cerodirphia speciosa, which belongs to the Saturniidae family of the Lepidoptera order. Despite the paucity of available database sequence resources, the approach enabled us to identify 48 out of 58 attempted spots on its two-dimensional gel electrophoresis map, which represented 37 unique proteins, whereas it was only possible to identify 13 proteins by conventional non-error tolerant database searching methods. The majority of cross-species hits were made to proteins from the phylogenetically related Lepidoptera organism, the silk worm Bombyx mori. The protein composition of the venom suggested that envenoming by C. speciosa toxins might proceed through the contact with its hemolymph, similarly to another toxic Lepidoptera organism, Lonomia obliqua.

Animals↗