Institute for Systems Biology

About: Institute for Systems Biology is a nonprofit organization based out in Seattle, Washington, United States. It is known for research contribution in the topics: Population & Proteomics. The organization has 1277 authors who have published 2777 publications receiving 353165 citations.

Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development

Abstract: In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4(+) T cells specialized in providing help to B lymphocytes in the induction of germinal centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)--a basic helix-loop-helix (bHLH) transcription factor--is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4(+) T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.

Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation

Abstract: We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.

Automated prediction of CASP-5 structures using the Robetta server.

Abstract: Robetta is a fully automated protein structure prediction server that uses the Rosetta fragment-insertion method. It combines template-based and de novo structure prediction methods in an attempt to produce high quality models that cover every residue of a submitted sequence. The first step in the procedure is the automatic detection of the locations of domains and selection of the appropriate modeling protocol for each domain. For domains matched to a homolog with an experimentally characterized structure by PSI-BLAST or Pcons2, Robetta uses a new alignment method, called K*Sync, to align the query sequence onto the parent structure. It then models the variable regions by allowing them to explore conformational space with fragments in fashion similar to the de novo protocol, but in the context of the template. When no structural homolog is available, domains are modeled with the Rosetta de novo protocol, which allows the full length of the domain to explore conformational space via fragment-insertion, producing a large decoy ensemble from which the final models are selected. The Robetta server produced quite reasonable predictions for targets in the recent CASP-5 and CAFASP-3 experiments, some of which were at the level of the best human predictions.

Solid-phase extraction of N-linked glycopeptides.

Abstract: Protein glycosylation is a common post-translational modification and has been increasingly recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. N-linked glycosylation is prevalent in proteins on the extracellular membrane, and many clinical biomarkers and therapeutic targets are glycoproteins. Here, we describe a protocol for solid-phase extraction of N-linked glycopeptides and subsequent identification of N-linked glycosylation sites (N-glycosites) by tandem mass spectrometry. The method oxidizes the carbohydrates in glycopeptides into aldehydes, which can be immobilized on a solid support. The N-linked glycopeptides are then optionally labeled with a stable isotope using deuterium-labeled succinic anhydride and the peptide moieties are released by peptide-N-glycosidase. In a single analysis, the method identifies hundreds of N-linked glycoproteins, the site(s) of N-linked glycosylation and the relative quantity of the identified glycopeptides.