[Show abstract][Hide abstract] ABSTRACT: PHD1 belongs to a family of prolyl hydroxylases (PHDs), which are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of a critical centrosomal protein Cep192, establishing a link between oxygen sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, 4 and 6 at Serine 130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of HIF protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, while phosphorylation of PHD1 at Serine 130 lowers its activity towards HIF1α, this modification increases PHD1's activity towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.
Full-text · Article · Dec 2015 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: 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 C. elegans, a eukaryote composed of 959 somatic cells and ∼1,500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼ 3,000 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. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.
No preview · Article · Nov 2015 · Nature Immunology
[Show abstract][Hide abstract] ABSTRACT: We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plas-mid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.
[Show abstract][Hide abstract] ABSTRACT: Despite many recent advances in instrumentation, the sheer complexity of biological samples remains a major challenge in large-scale proteomics experiments, reflecting both the large number of protein isoforms and the wide dynamic range of their expression levels. However, while the dynamic range of expression levels for different components of the proteome is estimated to be ∼107–8, the equivalent dynamic range of LC–MS is currently limited to ∼106. Sample pre-fractionation has therefore become routinely used in large-scale proteomics to reduce sample complexity during MS analysis and thus alleviate the problem of ion suppression and undersampling. There is currently a wide range of chromatographic techniques that can be applied as a first dimension separation. Here, we systematically evaluated the use of hydrophilic interaction liquid chromatography (HILIC), in comparison with hSAX, as a first dimension for peptide fractionation in a bottom-up proteomics workflow. The data indicate that in addition to its role as a useful pre-enrichment method for PTM analysis, HILIC can provide a robust, orthogonal and high-resolution method for increasing the depth of proteome coverage in large-scale proteomics experiments. The data also indicate that the choice of using either HILIC, hSAX, or other methods, is best made taking into account the specific types of biological analyses being performed.
No preview · Article · Aug 2015 · International Journal of Mass Spectrometry
[Show abstract][Hide abstract] ABSTRACT: The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.Oncogene advance online publication, 13 April 2015; doi:10.1038/onc.2015.104.
[Show abstract][Hide abstract] ABSTRACT: The proteome is a dynamic system in which each protein has interconnected properties - dimensions - that together contribute to the phenotype of a cell. Measuring these properties has proved challenging owing to their diversity and dynamic nature. Advances in mass spectrometry-based proteomics now enable the measurement of multiple properties for thousands of proteins, including their abundance, isoform expression, turnover rate, subcellular localization, post-translational modifications and interactions. Complementing these experimental developments are new data analysis, integration and visualization tools as well as data-sharing resources. Together, these advances in the multidimensional analysis of the proteome are transforming our understanding of various cellular and physiological processes.
[Show abstract][Hide abstract] ABSTRACT: Fluorescence microscopy is a powerful technique that has become central in the study of the structure and function of biological specimens. This is due in large part to its specificity and versatility. Although an understanding of structure-typically through high-resolution imaging of fixed material-has proved an important tool to understanding function, fluorescence microscopy also offers a mechanism to interrogate cells in the living state, providing a means to explore dynamic process within the specimen over long time periods at high temporal resolution. The cell nucleus is a highly compartmented environment whose components are often highly motile and in a constant state of flux. The ability to monitor the dynamic behavior of nuclear bodies by live-cell imaging provides the researcher with important information regarding underlying mechanistic processes relating to their formation and maintenance. Two techniques have proved particularly valuable to our study of cellular dynamics and molecular mobility, namely, time-lapse imaging and fluorescence recovery after photobleaching (FRAP). Time-lapse microscopy allows for qualitative and quantitative analysis of a wide range of events at the cellular and subcellular level. FRAP provides a mechanism to study the mobility of a population of proteins in a range of conditions within discrete areas of the biological specimen. Therefore, fluorescence microscopy is unique in its ability to provide data at high temporal resolution and in such exquisite detail.
No preview · Article · Jan 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/), an online, searchable resource.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription
and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly
in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would
be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504
drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed “madrasin,” i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome
assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell
cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs
in both HeLa and HEK293 cells.
Full-text · Article · Oct 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney and co-workers (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2 fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.
[Show abstract][Hide abstract] ABSTRACT: Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001
[Show abstract][Hide abstract] ABSTRACT: Cajal bodies are nuclear structures involved in snRNP and snoRNP biogenesis, telomere maintenance and histone mRNA processing. Recently, the SUMO isopeptidase USPL1 was identified as a Cajal body component essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its relation to Cajal bodies. We demonstrate that the levels of RNAPII-transcribed snRNAs are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalizes with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus our data indicate that USPL1 plays a key role in the process of RNAPII-mediated snRNA transcription.
No preview · Article · Jan 2014 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: During the past 15 years mass spectrometry (MS)-based analyses have become established as the method of choice for direct protein identification and measurement. Owing to the remarkable improvements in the sensitivity and resolution of MS instruments, this technology has revolutionised the opportunities available for the system-wide characterisation of proteins, with wide applications across virtually the whole of cell biology. In this article we provide a perspective on the current state of the art and discuss how the future of cell biology research may benefit from further developments and applications in the field of MS and proteomics, highlighting the major challenges ahead for the community in organising the effective sharing and integration of the resulting data mountain.
No preview · Article · Nov 2013 · Trends in cell biology
[Show abstract][Hide abstract] ABSTRACT: Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.
[Show abstract][Hide abstract] ABSTRACT: Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we have combined native size-exclusion chromatography (SEC) with high throughput proteomic analysis to characterise soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1- MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific GO terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data sharing resource available to the community.