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Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications., Competing Interests: AB, LS, LD, AP, FS, EH, CE, HJ, HY, GF, DC No competing interests declared, EG Now works for Boehringer Ingelheim Pharma GmbH & Co KG, MP Board member of Tartan Cell Technologies Ltd, JS Board member of Tartan Cell Technologies Ltd and Glencoe Software Ltd, AL Board member of Tartan Cell Technologies Ltd and Platinum Informatics Ltd, (© 2024, Brenes et al.)

Background: Neutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19), but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery., Methods: Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96 h of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale., Results: Neutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified, with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature, but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism, immunosuppression and pattern recognition, while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins, chemokine and leukotriene receptors, integrins and inhibitory receptors., Conclusions: SARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines., Competing Interests: Conflict of interest: H.R. Keir has received speaker fees from Insmed. A. Shoemark has received grant funding from AstraZeneca. A.J. Brenes has received support for attending meetings from the British Society for Proteome research. J.D. Chalmers has received research grants from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Gilead Sciences, Grifols, Novartis, Insmed and Trudell, and received consultancy or speaker fees from Antabio, AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Insmed, Janssen, Novartis, Pfizer, Trudell and Zambon. All other authors declare no conflicts of interest., (Copyright ©The authors 2024.)

The surprising decision by Novo Nordisk Foundation (NNF) to discontinue funding for the Center for Protein Research in Copenhagen should prompt discussions about public and private commitment to support basic research., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459., Competing Interests: Conflict of interest The Institute of Cancer Research has a commercial interest in HSP90 and HSF1 inhibitors and operates a reward to discoverers scheme from which employees may benefit. P. W. received funding from Vernalis for the discovery of HSP90 inhibitors, and intellectual property for this program was licensed to Vernalis Ltd and Novartis. P. W. was previously involved in a research collaboration with AstraZeneca in the area of the HSF1 pathway, and intellectual property was licensed to Sixth Element Capital/Pioneer Fund and Nuvectis Pharma. P. W. has been/is a consultant/advisory board member to Alterome Therapeutics, Astex Therapeutics, Black Diamond Therapeutics, CHARM Therapeutics, CV6 Therapeutics, EpiCombi Therapeutics, Novartis, STORM Therapeutics, and Vividion Therapeutics (acquired by Bayer AG) and is a Science Partner for Nextech Invest. P. W. is a Non-Executive Board member and holds stock in STORM Therapeutics and also holds stock in Alterome Therapeutics, Black Diamond Therapeutics, Chroma Therapeutics, EpiCombi Therapeutics, Nextech Invest, and Nuvectis Pharma. P. W. is the Executive Director of the non-profit Chemical Probes Portal. P. W. and P. A. C. received research funding from Merck KGaA and Astex Therapeutics, and P. W. received research funding from AstraZeneca, Battle Against Cancer Investment Trust (BACIT), and CRT Pioneer Fund/Sixth Element Capital). P. W. is a former employee of AstraZeneca. B. A. L. is a former employee of The Institute of Cancer Research which operates reward to inventors program and a former employee of Inpharmatica Ltd (later acquired by Galapagos). B. A. L. has financial interest and/or acts/acted as a consultant or a Scientific Advisory Board member for Exscientia AI, AstraZeneca, Astex Pharmaceuticals, GSK, Astellas Pharma, and Definiens AG (member of AstraZeneca group). B. A. L. is the Director of Informatics for the non-profit Chemical Probes Portal., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

To overcome oxidative, inflammatory, and metabolic stress, cells have evolved cytoprotective protein networks controlled by nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and its negative regulator, Kelch-like ECH associated protein 1 (Keap1). Here, using high-resolution mass spectrometry we characterize the proteomes of macrophages with altered Nrf2 status revealing significant differences among the genotypes in metabolism and redox homeostasis, which were validated with respirometry and metabolomics. Nrf2 affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity. Notably, Nrf2 activation promoted mitochondrial fusion. The Keap1 inhibitor, 4-octyl itaconate remodeled the inflammatory macrophage proteome, increasing redox and suppressing type I interferon (IFN) response. Similarly, pharmacologic or genetic Nrf2 activation inhibited the transcription of IFN-β and its downstream effector IFIT2 during LPS stimulation. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages., Competing Interests: A.T.D.K. is a member of the scientific advisory board of Evgen Pharma., (© 2022 The Author(s).)

Tissue-resident intestinal intraepithelial T lymphocytes (T-IEL) patrol the gut and have important roles in regulating intestinal homeostasis. T-IEL include both induced T-IEL, derived from systemic antigen-experienced lymphocytes, and natural T-IEL, which are developmentally targeted to the intestine. While the processes driving T-IEL development have been elucidated, the precise roles of the different subsets and the processes driving activation and regulation of these cells remain unclear. To gain functional insights into these enigmatic cells, we used high-resolution, quantitative mass spectrometry to compare the proteomes of induced T-IEL and natural T-IEL subsets, with naive CD8 + T cells from lymph nodes. This data exposes the dominant effect of the gut environment over ontogeny on T-IEL phenotypes. Analyses of protein copy numbers of >7000 proteins in T-IEL reveal skewing of the cell surface repertoire towards epithelial interactions and checkpoint receptors; strong suppression of the metabolic machinery indicating a high energy barrier to functional activation; upregulated cholesterol and lipid metabolic pathways, leading to high cholesterol levels in T-IEL; suppression of T cell antigen receptor signalling and expression of the transcription factor TOX, reminiscent of chronically activated T cells. These novel findings illustrate how T-IEL integrate multiple tissue-specific signals to maintain their homeostasis and potentially function., Competing Interests: AB, OJ, HW, JH, LS, DD, AL, MS None, MV none, (© 2021, Brenes et al.)

Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long ultracentrifugation to collecting and analyzing the fractions. Each of these steps in the SDG workflow can introduce potential technical variation that affects the reproducibility of gradient profiles between samples. To address these limitations, we have developed a flexible, alternative approach for analyzing polysomes and ribosomal subunits based on size-exclusion chromatography (SEC), termed 'Ribo Mega-SEC.' In comparison with the SDG method, Ribo Mega-SEC involves a single step using ultra-high-performance liquid chromatography (uHPLC). The entire workflow, from injecting the lysate to collecting the fractions, can be performed in as little as 15 min, with high reproducibility. By varying the pore size of the SEC column, polysomes and ribosomal subunits can be separated using extracts from either human or mouse cultured cell lines or from tissue samples, Drosophila embryos, or budding yeast. The resulting separated fractions are suitable for analysis using a wide range of subsequent analytical techniques including mass spectrometry (MS)-based proteomics, RNA-Seq, electron microscopy (EM), and multiple biochemical assays., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

X chromosome inactivation (XCI) is a dosage compensation mechanism in female mammals whereby transcription from one X chromosome is repressed. Analysis of human induced pluripotent stem cells (iPSCs) derived from female donors identified that low levels of XIST RNA correlated strongly with erosion of XCI. Proteomic analysis, RNA sequencing (RNA-seq), and polysome profiling showed that XCI erosion resulted in amplified RNA and protein expression from X-linked genes, providing a proteomic characterization of skewed dosage compensation. Increased protein expression was also detected from autosomal genes without an mRNA increase, thus altering the protein-RNA correlation between the X chromosome and autosomes. XCI-eroded lines display an ∼13% increase in total cell protein content, with increased ribosomal proteins, ribosome biogenesis and translation factors, and polysome levels. We conclude that XCI erosion in iPSCs causes a remodeling of the proteome, affecting the expression of a much wider range of proteins and disease-linked loci than previously realized., Competing Interests: Declaration of interests D.S. now works for GSK., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Human disease phenotypes are driven primarily by alterations in protein expression and/or function. To date, relatively little is known about the variability of the human proteome in populations and how this relates to variability in mRNA expression and to disease loci. Here, we present the first comprehensive proteomic analysis of human induced pluripotent stem cells (iPSC), a key cell type for disease modelling, analysing 202 iPSC lines derived from 151 donors, with integrated transcriptome and genomic sequence data from the same lines. We characterised the major genetic and non-genetic determinants of proteome variation across iPSC lines and assessed key regulatory mechanisms affecting variation in protein abundance. We identified 654 protein quantitative trait loci (pQTLs) in iPSCs, including disease-linked variants in protein-coding sequences and variants with trans regulatory effects. These include pQTL linked to GWAS variants that cannot be detected at the mRNA level, highlighting the utility of dissecting pQTL at peptide level resolution., Competing Interests: BM, DS, DB, AB, MB, HK, OS, AL No competing interests declared, (© 2020, Mirauta et al.)

Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin-dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (∼3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts., Competing Interests: * The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Griesser et al.)

Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.

Background: Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin ( FLG ) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG . Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term 'axon guidance'.      Conclusions: This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Elias MS et al.)

Quantitative mass spectrometry reveals how CD4 + and CD8 + T cells restructure proteomes in response to antigen and mammalian target of rapamycin complex 1 (mTORC1). Analysis of copy numbers per cell of >9,000 proteins provides new understanding of T cell phenotypes, exposing the metabolic and protein synthesis machinery and environmental sensors that shape T cell fate. We reveal that lymphocyte environment sensing is controlled by immune activation, and that CD4 + and CD8 + T cells differ in their intrinsic nutrient transport and biosynthetic capacity. Our data also reveal shared and divergent outcomes of mTORC1 inhibition in naïve versus effector T cells: mTORC1 inhibition impaired cell cycle progression in activated naïve cells, but not effector cells, whereas metabolism was consistently impacted in both populations. This study provides a comprehensive map of naïve and effector T cell proteomes, and a resource for exploring and understanding T cell phenotypes and cell context effects of mTORC1.

Background : Cellular proteins vary significantly in both abundance and turnover rates. These parameters depend upon their rates of synthesis and degradation and it is useful to have access to data on protein turnover rates when, for example, designing genetic knock-down experiments or assessing the potential usefulness of covalent enzyme inhibitors. Little is known about the nature and regulation of protein turnover in Trypanosoma brucei , the etiological agent of human and animal African trypanosomiasis. Methods : To establish baseline data on T. brucei proteome turnover, a Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based mass spectrometry analysis was performed to reveal the synthesis and degradation profiles for thousands of proteins in the bloodstream and procyclic forms of this parasite. Results : This analysis revealed a slower average turnover rate of the procyclic form proteome relative to the bloodstream proteome. As expected, many of the proteins with the fastest turnover rates have functions in the cell cycle and in the regulation of cytokinesis in both bloodstream and procyclic forms. Moreover, the cellular localization of T. brucei proteins correlates with their turnover, with mitochondrial and glycosomal proteins exhibiting slower than average turnover rates. Conclusions : The intention of this study is to provide the trypanosome research community with a resource for protein turnover data for any protein or group of proteins. To this end, bioinformatic analyses of these data are made available via an open-access web resource with data visualization functions., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Tinti M et al.)

Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, tandem mass tags (TMT) in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We identified a significant inflation rate of protein missing values as multiple batches are integrated and show that this pattern is aggravated at the peptide level. We also show that without normalization strategies to address the batch effects, the high precision of quantitation within a single multiplexed TMT batch is not reproduced when data from multiple TMT batches are integrated.Further, the incidence of false positives was studied by using Y chromosome peptides as an internal control. The iPSC lines quantified in this data set were derived from both male and female donors, hence the peptides mapped to the Y chromosome should be absent from female lines. Nonetheless, these Y chromosome-specific peptides were consistently detected in the female channels of all TMT batches. We then used the same Y chromosome specific peptides to quantify the level of ion coisolation as well as the effect of primary and secondary reporter ion interference. These results were used to propose solutions to mitigate the limitations of multi-batch TMT analyses. We confirm that including a common reference line in every batch increases precision by facilitating normalization across the batches and we propose experimental designs that minimize the effect of cross population reporter ion interference., (© 2019 Brenes et al.)

Background: Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin ( FLG ) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG . Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term 'axon guidance'.      Conclusions: This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Elias MS et al.)

Background: Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear., Objectives: We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease., Methods: We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples., Results: Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein., Conclusion: Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary: The Encyclopedia of Proteome Dynamics (EPD) 'KinoViewer' is an interactive data visualization tool designed for analysis and exploration of both protein and transcript data, showing expression of kinase genes in either human or mouse cells and tissues. The KinoViewer provides a comprehensive, updated graphical display of all human/mouse kinases and an open access analysis tool for the community with a user-friendly graphical interface., Availability and Implementation: The KinoViewer is based on a manually drawn SVG, which is utilized with D3.js to create a dynamic visualization. It can be accessed at: https://peptracker.com/epd/analytics/. The KinoViewer is currently only accessible through the EPD, it is open access and can be used either to view internal datasets, or used to upload and visualize external user datasets., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

Immune activated T lymphocytes modulate the activity of key metabolic pathways to support the transcriptional reprograming and reshaping of cell proteomes that permits effector T cell differentiation. The present study uses high resolution mass spectrometry and metabolic labelling to explore how murine T cells control the methionine cycle to produce methyl donors for protein and nucleotide methylations. We show that antigen receptor engagement controls flux through the methionine cycle and RNA and histone methylations. We establish that the main rate limiting step for protein synthesis and the methionine cycle is control of methionine transporter expression. Only T cells that respond to antigen to upregulate and sustain methionine transport are supplied with methyl donors that permit the dynamic nucleotide methylations and epigenetic reprogramming that drives T cell differentiation. These data highlight how the regulation of methionine transport licenses use of methionine for multiple fundamental processes that drive T lymphocyte proliferation and differentiation., Competing Interests: LS, AH, AB, LS, JH, AM, XL, ST, PT, JR, JL, AL, DC No competing interests declared, (© 2019, Sinclair et al.)

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 ( SMN1 ) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated its composition in Drosophila melanogaster Using transgenic flies that exclusively express Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes ( CG14164 , CG31950 and CG2371 ) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster , CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes ( CG32783 and CG32786 ). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability., (Copyright © 2019 Matera et al.)

Proteomes are highly dynamic and can respond rapidly to environmental and cellular signals. Within cells, proteins often form distinct pools with different functions and properties. However, in quantitative proteomics studies it is common to measure averaged values for proteins that do not reflect variations that may occur between different protein isoforms, different subcellular compartments, or in cells at different cell cycle stages and so on. Here we review experimental approaches that can be used to enhance the signal from specific pools of protein that may otherwise be obscured through averaging across protein populations. This signal enhancement can help to reveal functions associated with specific protein pools, providing insight into the regulation of cellular processes. We review different strategies for proteomic signal enhancement, with a focus on the analysis of protein pools in different subcellular locations. We describe how MS-based proteome analyses can be combined with a general physico-chemical cell fractionation procedure that can be applied to many cultured cell lines., (Copyright © 2018. Published by Elsevier Ltd.)

We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies., Competing Interests: HY, ML, DH, RS, TL, TO, AL No competing interests declared, (© 2018, Yoshikawa et al.)

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Natural killer (NK) cells are lymphocytes with important anti-tumour functions. Cytokine activation of NK cell glycolysis and oxidative phosphorylation (OXPHOS) are essential for robust NK cell responses. However, the mechanisms leading to this metabolic phenotype are unclear. Here we show that the transcription factor cMyc is essential for IL-2/IL-12-induced metabolic and functional responses in mice. cMyc protein levels are acutely regulated by amino acids; cMyc protein is lost rapidly when glutamine is withdrawn or when system L-amino acid transport is blocked. We identify SLC7A5 as the predominant system L-amino acid transporter in activated NK cells. Unlike other lymphocyte subsets, glutaminolysis and the tricarboxylic acid cycle do not sustain OXPHOS in activated NK cells. Glutamine withdrawal, but not the inhibition of glutaminolysis, results in the loss of cMyc protein, reduced cell growth and impaired NK cell responses. These data identify an essential role for amino acid-controlled cMyc for NK cell metabolism and function.

We describe a single-step centrifugal elutriation method to produce synchronous Gap1 (G1)-phase procyclic trypanosomes at a scale amenable for proteomic analysis of the cell cycle. Using ten-plex tandem mass tag (TMT) labeling and mass spectrometry (MS)-based proteomics technology, the expression levels of 5325 proteins were quantified across the cell cycle in this parasite. Of these, 384 proteins were classified as cell-cycle regulated and subdivided into nine clusters with distinct temporal regulation. These groups included many known cell cycle regulators in trypanosomes, which validates the approach. In addition, we identify 40 novel cell cycle regulated proteins that are essential for trypanosome survival and thus represent potential future drug targets for the prevention of trypanosomiasis. Through cross-comparison to the TrypTag endogenous tagging microscopy database, we were able to validate the cell-cycle regulated patterns of expression for many of the proteins of unknown function detected in our proteomic analysis. A convenient interface to access and interrogate these data is also presented, providing a useful resource for the scientific community. Data are available via ProteomeXchange with identifier PXD008741 (https://www.ebi.ac.uk/pride/archive/)., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background : Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome. Methods : Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A) cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs). We used pulse-SILAC MS ( Boisvert et al ., 2012), to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins. Results : Src-induced transformation changed the expression and/or turnover levels of ~3% of proteins, affecting ~1.5% of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1) are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database ( www.peptracker.com/epd). Conclusions : We present the first comprehensive analysis measuring how protein expression and protein turnover is affected by cell transformation, providing a detailed picture at the protein level of the consequences of activation of an oncogene., Competing Interests: No competing interests were disclosed.

Ribosome-associated mRNA quality control mechanisms ensure the fidelity of protein translation 1,2 . Although these mechanisms have been extensively studied in yeast, little is known about their role in mammalian tissues, despite emerging evidence that stem cell fate is controlled by translational mechanisms 3,4 . One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes 5 . Here we show that Pelo is required for mammalian epidermal homeostasis. Conditional deletion of Pelo in mouse epidermal stem cells that express Lrig1 results in hyperproliferation and abnormal differentiation of these cells. By contrast, deletion of Pelo in Lgr5-expressing stem cells has no effect and deletion in Lgr6-expressing stem cells induces only a mild phenotype. Loss of Pelo results in accumulation of short ribosome footprints and global upregulation of translation, rather than affecting the expression of specific genes. Translational inhibition by rapamycin-mediated downregulation of mTOR (mechanistic target of rapamycin kinase) rescues the epidermal phenotype. Our study reveals that the ribosome-rescue machinery is important for mammalian tissue homeostasis and that it has specific effects on different stem cell populations.

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human  SMN1 Expression of a duplicate gene ( SMN2 ) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although  SMN2  exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a  Drosophila  model system and label-free proteomics to identify the SCF Slmb  ubiquitin E3 ligase complex as a novel SMN binding partner. SCF Slmb  interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCF Slmb  binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7 S270A , but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers., (© 2018 Gray et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Driven by improvements in speed and resolution of mass spectrometers (MS), the field of proteomics, which involves the large-scale detection and analysis of proteins in cells, tissues and organisms, continues to expand in scale and complexity. There is a resulting growth in datasets of both raw MS files and processed peptide and protein identifications. MS-based proteomics technology is also used increasingly to measure additional protein properties affecting cellular function and disease mechanisms, including post-translational modifications, protein-protein interactions, subcellular and tissue distributions. Consequently, biologists and clinicians need innovative tools to conveniently analyse, visualize and explore such large, complex proteomics data and to integrate it with genomics and other related large-scale datasets. We have created the Encyclopedia of Proteome Dynamics (EPD) to meet this need (https://peptracker.com/epd/). The EPD combines a polyglot persistent database and web-application that provides open access to integrated proteomics data for >30 000 proteins from published studies on human cells and model organisms. It is designed to provide a user-friendly interface, featuring graphical navigation with interactive visualizations that facilitate powerful data exploration in an intuitive manner. The EPD offers a flexible and scalable ecosystem to integrate proteomics data with genomics information, RNA expression and other related, large-scale datasets., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A disproportionate number of predicted proteins from the genome sequence of the protozoan parasite Trypanosoma brucei , an important human and animal pathogen, are hypothetical proteins of unknown function. This paper describes a protein correlation profiling mass spectrometry approach, using two size exclusion and one ion exchange chromatography systems, to derive sets of predicted protein complexes in this organism by hierarchical clustering and machine learning methods. These hypothesis-generating proteomic data are provided in an open access online data visualization environment (http://134.36.66.166:8083/complex_explorer). The data can be searched conveniently via a user friendly, custom graphical interface. We provide examples of both potential new subunits of known protein complexes and of novel trypanosome complexes of suggested function, contributing to improving the functional annotation of the trypanosome proteome. Data are available via ProteomeXchange with identifier PXD005968., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA - promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Starvation causes comprehensive metabolic changes, which are still not fully understood. Here, we used quantitative proteomics and RNA sequencing to examine the temporal starvation responses in wild-type Caenorhabditis elegans and animals lacking the transcription factor HLH-30. Our findings show that starvation alters the abundance of hundreds of proteins and mRNAs in a temporal manner, many of which are involved in central metabolic pathways, including lipoprotein metabolism. We demonstrate that premature death of hlh-30 animals under starvation can be prevented by knockdown of either vit-1 or vit-5, encoding two different lipoproteins. We further show that the size and number of intestinal lipid droplets under starvation are altered in hlh-30 animals, which can be rescued by knockdown of vit-1. Taken together, this indicates that survival of hlh-30 animals under starvation is closely linked to regulation of intestinal lipid stores. We provide the most detailed poly-omic analysis of starvation responses to date, which serves as a resource for further mechanistic studies of starvation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This corrects the article DOI: 10.1038/nature22403.

Asymmetric cell divisions are required for cellular diversity and defects can lead to altered daughter cell fates and numbers. In a genetic screen for C. elegans mutants with defects in dopaminergic head neuron specification or differentiation, we isolated a new allele of the transcription factor HAM-1 [HSN (Hermaphrodite-Specific Neurons) Abnormal Migration]. Loss of both HAM-1 and its target, the kinase PIG-1 [PAR-1(I)-like Gene], leads to abnormal dopaminergic head neuron numbers. We identified discrete genetic relationships between ham-1, pig-1 and apoptosis pathway genes in dopaminergic head neurons. We used an unbiased, quantitative mass spectrometry-based proteomics approach to characterise direct and indirect protein targets and pathways that mediate the effects of PIG-1 kinase loss in C. elegans embryos. Proteins showing changes in either abundance, or phosphorylation levels, between wild-type and pig-1 mutant embryos are predominantly connected with processes including cell cycle, asymmetric cell division, apoptosis and actomyosin-regulation. Several of these proteins play important roles in C. elegans development. Our data provide an in-depth characterisation of the C. elegans wild-type embryo proteome and phosphoproteome and can be explored via the Encyclopedia of Proteome Dynamics (EPD) - an open access, searchable online database.

Improved sample processing workflows enable rapid, deep analysis of human cellular and tissue proteomes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation-mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N-terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1-PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1-PP1 protects the origin-binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1-depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1-targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Effective network analysis of protein data requires high-quality proteomic datasets. Here, we report a near doubling in coverage of the C. elegans adult proteome, identifying >11,000 proteins in total with ∼9,400 proteins reproducibly detected in three biological replicates. Using quantitative mass spectrometry, we identify proteins whose abundances vary with age, revealing a concerted downregulation of proteins involved in specific metabolic pathways and upregulation of cellular stress responses with advancing age. Among these are ∼30 peroxisomal proteins, including the PRX-5/PEX5 import protein. Functional experiments confirm that protein import into the peroxisome is compromised in vivo in old animals. We also studied the behavior of the set of age-variant proteins in chronologically age-matched, long-lived daf-2 insulin/IGF-1-pathway mutants. Unexpectedly, the levels of many of these age-variant proteins did not scale with extended lifespan. This indicates that, despite their youthful appearance and extended lifespans, not all aspects of aging are reset in these long-lived mutants., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754)., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP-HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy.

Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-associated factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd), an open access, searchable database resource., (© 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)