Effect of IAPP on the proteome of cultured Rin-5F cells.

Background: Islet amyloid polypeptide (IAPP) or amylin deposits can be found in the islets of type 2 diabetes patients. The peptide is suggested to be involved in the etiology of the disease through formation of amyloid deposits and destruction of β islet cells, though the underlying molecular events leading from IAPP deposition to β cell death are still largely unknown.

Results: We used OFFGEL™ proteomics to study how IAPP exposure affects the proteome of rat pancreatic insulinoma Rin-5F cells.

Archetypal transcriptional blocks underpin yeast gene regulation in response to changes in growth conditions.

The transcriptional responses of yeast cells to diverse stresses typically include gene activation and repression. Specific stress defense, citric acid cycle and oxidative phosphorylation genes are activated, whereas protein synthesis genes are coordinately repressed. This view was achieved from comparative transcriptomic experiments delineating sets of genes whose expression greatly changed with specific stresses.

Dynamic changes in eIF4F-mRNA interactions revealed by global analyses of environmental stress responses.

Background: Translation factors eIF4E and eIF4G form eIF4F, which interacts with the messenger RNA (mRNA) 5' cap to promote ribosome recruitment and translation initiation. Variations in the association of eIF4F with individual mRNAs likely contribute to differences in translation initiation frequencies between mRNAs. As translation initiation is globally reprogrammed by environmental stresses, we were interested in determining whether eIF4F interactions with individual mRNAs are reprogrammed and how this may contribute to global environmental stress responses.

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800Saccharomyces cerevisiaeproteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study.

Integrated multi-omics analyses reveal the pleiotropic nature of the control of gene expression by Puf3p.

The PUF family of RNA-binding proteins regulate gene expression post-transcriptionally. Saccharomyces cerevisiae Puf3p is characterised as binding nuclear-encoded mRNAs specifying mitochondrial proteins. Extensive studies of its regulation of COX17 demonstrate its role in mRNA decay.

The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation.

Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood.

Global mRNA selection mechanisms for translation initiation.

Background: The selection and regulation of individual mRNAs for translation initiation from a competing pool of mRNA are poorly understood processes. The closed loop complex, comprising eIF4E, eIF4G and PABP, and its regulation by 4E-BPs are perceived to be key players. Using RIP-seq, we aimed to evaluate the role in gene regulation of the closed loop complex and 4E-BP regulation across the entire yeast transcriptome.

The yeast La related protein Slf1p is a key activator of translation during the oxidative stress response.

The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins.

mRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave.

The relocalization of translationally repressed mRNAs to mRNA processing bodies Pbodies is a key consequence of cellular stress across many systems. Pbodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to Pbodies is poorly understood. We used the MS2GFP system to follow the movement of specific endogenous mRNAs in live Saccharomyces cerevisiae cells after nutritional stress.

Puf3p induces translational repression of genes linked to oxidative stress.

In response to stress, the translation of many mRNAs in yeast can change in a fashion discordant with the general repression of translation. Here, we use machine learning to mine the properties of these mRNAs to determine specific translation control signals. We find a strong association between transcripts acutely translationally repressed under oxidative stress and those associated with the RNA-binding protein Puf3p, a known regulator of cellular mRNAs encoding proteins targeted to mitochondria.

The use of selected reaction monitoring in quantitative proteomics.

Selected reaction monitoring (SRM) has a long history of use in the area of quantitative MS. In recent years, the approach has seen increased application to quantitative proteomics, facilitating multiplexed relative and absolute quantification studies in a variety of organisms. This article discusses SRM, after introducing the context of quantitative proteomics (specifically primarily absolute quantification) where it finds most application, and considers topics such as the theory and advantages of SRM, the selection of peptide surrogates for protein quantification, the design of optimal SRM co-ordinates and the handling of SRM data.

Software for analysing ion mobility mass spectrometry data to improve peptide identification.

The development of ion mobility (IM) MS instruments has the capability to provide an added dimension to peptide analysis pipelines in proteomics, but, as yet, there are few software tools available for analysing such data. IM can be used to provide additional separation of parent ions or product ions following fragmentation. In this work, we have created a set of software tools that are capable of converting three dimensional IM data generated from analysis of fragment ions into a variety of formats used in proteomics.

A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance.

Selected reaction monitoring mass spectrometry has been combined with the use of an isotopically labelled synthetic protein, made up of proteotypic tryptic peptides selected from parasite proteins of interest. This allows, for the first time, absolute quantification of proteins from Plasmodium falciparum. This methodology is demonstrated to be of sufficient sensitivity to quantify, even within whole cell extracts, proteins of low abundance from the folate pathway as well as more abundant "housekeeping" proteins.

Glucose depletion inhibits translation initiation via eIF4A loss and subsequent 48S preinitiation complex accumulation, while the pentose phosphate pathway is coordinately up-regulated.

Cellular stress can globally inhibit translation initiation, and glucose removal from yeast causes one of the most dramatic effects in terms of rapidity and scale. Here we show that the same rapid inhibition occurs during yeast growth as glucose levels diminish. We characterize this novel regulation showing that it involves alterations within the 48S preinitiation complex.

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum.

Background: The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes.

Changed genome heterochromatinization upon prolonged activation of the Raf/ERK signaling pathway.

The Raf/ERK (Extracellular Signal Regulated Kinase) signal transduction pathway controls numerous cellular processes, including growth, differentiation, cellular transformation and senescence. ERK activation is thought to involve complex spatial and temporal regulation, to achieve a high degree of specificity, though precisely how this is achieved remains to be confirmed. We report here that prolonged activation of a conditional form of c-Raf-1 (BXB-ER) leads to profound changes in the level and distribution of a heterochromatic histone mark.

A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry.

Background: Plasmodium species are difficult to study using proteomic technology because they contain large amounts of haemoglobin-derived products (HDP), generated by parasite breakdown of host haemoglobin. HDP are known to interfere with isoelectric focussing, a cornerstone of fractionation strategies for the identification of proteins by mass spectrometry. In addition to the challenge presented by this material, as in most proteomes, there exists in this parasite a considerable dynamic range between proteins of high and low abundance.

Fusel alcohols regulate translation initiation by inhibiting eIF2B to reduce ternary complex in a mechanism that may involve altering the integrity and dynamics of the eIF2B body.

Recycling of eIF2-GDP to the GTP-bound form constitutes a core essential, regulated step in eukaryotic translation. This reaction is mediated by eIF2B, a heteropentameric factor with important links to human disease. eIF2 in the GTP-bound form binds to methionyl initiator tRNA to form a ternary complex, and the levels of this ternary complex can be a critical determinant of the rate of protein synthesis.

Characterisation of the bifunctional dihydrofolate synthase-folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition.

Unusually for a eukaryote, the malaria parasite Plasmodium falciparum expresses dihydrofolate synthase (DHFS) and folylpolyglutamate synthase (FPGS) as a single bifunctional protein. The two activities contribute to the essential pathway of folate biosynthesis and modification. The DHFS activity of recombinant PfDHFS-FPGS exhibited non-standard kinetics at high co-substrate (glutamate and ATP) concentrations, being partially inhibited by increasing concentrations of its principal substrate, dihydropteroate (DHP).

Reduced expression of lamin A/C results in modified cell signaling and metabolism coupled with changes in expression of structural proteins.

Nuclear lamins are intermediate filament proteins that define the shape and stability of nuclei in mammalian cells. In addition to this dominant structural role, recent studies have suggested that the lamin proteins also regulate fundamental aspects of nuclear function. In order to understand different roles played by lamin proteins, we used RNA interference to generate a series of HeLa cell lines to study loss-of-function phenotypes associated with depletion of lamin protein expression.

Interaction with checkpoint kinase 1 modulates the recruitment of nucleophosmin to chromatin.

The Checkpoint kinase 1 (Chk1) plays a central role in the cellular response to DNA damage and also contributes to the efficacy of DNA replication in the absence of genomic stress. However, we have only limited knowledge regarding the molecular mechanisms that regulate differential Chk1 function in the absence and presence of DNA damage. To address this, we used vertebrate cells with compromised Chk1 function to analyze how altered Chk1 activity influences protein interactions in chromatin.

Lamin B1 maintains the functional plasticity of nucleoli.

The dynamic ability of genomes to interact with discrete nuclear compartments appears to be essential for chromatin function. However, the extent to which structural nuclear proteins contribute to this level of organization is largely unresolved. To test the links between structure and function, we evaluated how nuclear lamins contribute to the organization of a major functional compartment, the nucleolus.

Plasmodium falciparum: a paradigm for alternative folate biosynthesis in diverse microorganisms?

Folates have a key role in metabolism, and the folate-dependent generation of DNA precursors in the form of deoxythymidine 5'-phosphate is particularly important for the replication of malaria parasites. Although Plasmodium falciparum can synthesize folate derivatives de novo, a long-standing mystery has been the apparent absence of a key enzyme, dihydroneopterin aldolase, in the classical folate biosynthetic pathway of this organism. The discovery that a different enzyme, pyruvoyltetrahydropterin synthase, can produce the necessary substrate for the subsequent step in folate synthesis raises the question of whether this solution is unique to P.

An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites.

Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate.

Microscale solution isoelectric focusing as an effective strategy enabling containment of hemeoglobin-derived products for high-resolution gel-based analysis of the Plasmodium falciparum proteome.

The high hemeozoin (beta-hemeatin) content of Plasmodium falciparum lysates imposes severe limitations on the analysis of the malarial proteome, in particular compromising the loading capacities of two-dimensional gels. Here we report on the adaptation of a recently developed solution-phase isoelectric focusing-based fractionation technique as a prefractionation strategy for efficient containment of hemeoglobin-derived products and complexity reduction, to facilitate the high-resolution gel-based quantitative analysis of plasmodial lysates.