Negative feedback via RSK modulates Erk-dependent progression from naïve pluripotency.

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK-responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in RSK family genes. Genotypes that included homozygous null mutations in Rps6ka1, encoding RSK1, resulted in elevated ERK phosphorylation. These RSK-depleted ES cells exhibit altered kinetics of transition into differentiation, with accelerated downregulation of naïve pluripotency factors, precocious expression of transitional epiblast markers and early onset of lineage specification. We further show that chemical inhibition of RSK increases ERK phosphorylation and expedites ES cell transition without compromising multilineage potential. These findings demonstrate that the ERK activation profile influences the dynamics of pluripotency progression and highlight the role of signalling feedback in temporal control of cell state transitions.

Tracking the embryonic stem cell transition from ground state pluripotency.

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

Prophossi: automating expert validation of phosphopeptide-spectrum matches from tandem mass spectrometry.

MOTIVATION: Complex patterns of protein phosphorylation mediate many cellular processes. Tandem mass spectrometry (MS/MS) is a powerful tool for identifying these post-translational modifications. In high-throughput experiments, mass spectrometry database search engines, such as MASCOT provide a ranked list of peptide identifications based on hundreds of thousands of MS/MS spectra obtained in a mass spectrometry experiment. These search results are not in themselves sufficient for confident assignment of phosphorylation sites as identification of characteristic mass differences requires time-consuming manual assessment of the spectra by an experienced analyst. The time required for manual assessment has previously rendered high-throughput confident assignment of phosphorylation sites challenging. RESULTS: We have developed a knowledge base of criteria, which replicate expert assessment, allowing more than half of cases to be automatically validated and site assignments verified with a high degree of confidence. This was assessed by comparing automated spectral interpretation with careful manual examination of the assignments for 501 peptides above the 1% false discovery rate (FDR) threshold corresponding to 259 putative phosphorylation sites in 74 proteins of the Trypanosoma brucei proteome. Despite this stringent approach, we are able to validate 80 of the 91 phosphorylation sites (88%) positively identified by manual examination of the spectra used for the MASCOT searches with a FDR < 15%. CONCLUSIONS: High-throughput computational analysis can provide a viable second stage validation of primary mass spectrometry database search results. Such validation gives rapid access to a systems level overview of protein phosphorylation in the experiment under investigation. AVAILABILITY: A GPL licensed software implementation in Perl for analysis and spectrum annotation is available in the supplementary material and a web server can be assessed online at http://www.compbio.dundee.ac.uk/prophossi.

Application of electrospray mass spectrometry to the structural determination of glycosylphosphatidylinositol membrane anchors.

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins is an important posttranslational modification in eukaryotic cells. The complete structural elucidation of GPI anchors is a complex process that requires relatively large amounts of starting material. In this paper, we assess the degree of structural information that can be obtained by applying electrospray mass spectrometry and tandem mass spectrometry to permethylated GPI glycans prepared from a well-characterized GPI-anchored glycoprotein, the variant surface glycoprotein from Trypanosoma brucei. All GPI glycans contain a non-N-acetylated glucosamine residue, and permethylation leads to the formation of a fixed positive charge on the glycans, in the form of a quaternary amine. The permethylated glycans were detected as [M +- Na](2+-) ions, and tandem mass spectrometry of these ions produced substantial, albeit incomplete, structural information on the branching patterns and linkage types for various GPI glycoforms of the variant surface glycoprotein.

Improved tricyclic inhibitors of trypanothione reductase by screening and chemical synthesis.

Trypanothione reductase (TryR) is a key validated enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes and leishmania parasites. This system is absent in humans, being replaced with glutathione and glutathione reductase, and as such offers a target for selective inhibition. As part of a program to discover antiparasitic drugs, the LOPAC1280 library of 1266 compounds was screened against TryR and the top hits evaluated against glutathione reductase and T. brucei parasites. The top hits included a number of known tricyclic neuroleptic drugs along with other new scaffolds for TryR. Three novel druglike hits were identified and SAR studies on one of these using information from the tricyclic neuroleptic agents led to the discovery of a competitive inhibitor (K(i)=330 nM) with an improved potency against T. brucei (EC(50)=775 nM).

The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness.

The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.

Identification and specific localization of tyrosine-phosphorylated proteins in Trypanosoma brucei.

Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in T. brucei, a principle well known from higher eukaryotes.