Publications by authors named "Alex Montoya"

17 Publications

  • Page 1 of 1

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

Diabetologia 2021 Apr 25;64(4):850-864. Epub 2021 Jan 25.

Section of Cell Biology and Functional Genomics, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital, London, UK.

Aims/hypothesis: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B.

Methods: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry.

Results: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca. Binding partners for both included secretory-granule-localised PTPRN2/phogrin.

Conclusions/interpretation: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans.

Data Availability: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.
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http://dx.doi.org/10.1007/s00125-020-05350-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829492PMC
April 2021

Purified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction.

Mol Cell 2020 12 9;80(6):1039-1054.e6. Epub 2020 Dec 9.

Cell Cycle Group, MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN, UK. Electronic address:

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.
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http://dx.doi.org/10.1016/j.molcel.2020.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758880PMC
December 2020

TRF1 averts chromatin remodelling, recombination and replication dependent-break induced replication at mouse telomeres.

Elife 2020 Jan 14;9. Epub 2020 Jan 14.

Telomere Replication and Stability group, Medical Research Council - London Institute of Medical Sciences, London, United Kingdom.

Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that mouse telomeres lacking TRF1 undergo protein composition reorganisation associated with the recruitment of DNA damage response and chromatin remodellers. Surprisingly, mTRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for mTRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.
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http://dx.doi.org/10.7554/eLife.49817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986873PMC
January 2020

A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules.

Sci Adv 2019 11 27;5(11):eaay6804. Epub 2019 Nov 27.

Cell Cycle Group, Medical Research Council London Institute of Medical Sciences, Du Cane Road, London W12 0NN, UK.

Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a "permanent bridge" resisting forces over 80 pN and a force-sensitive "reversible bridge." The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. "Permanent" cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.
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http://dx.doi.org/10.1126/sciadv.aay6804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881171PMC
November 2019

FACT mediates cohesin function on chromatin.

Nat Struct Mol Biol 2019 10 3;26(10):970-979. Epub 2019 Oct 3.

Cell Cycle Group, MRC London Institute of Medical Sciences (LMS), London, UK.

Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.
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http://dx.doi.org/10.1038/s41594-019-0307-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779571PMC
October 2019

PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection.

Nucleic Acids Res 2019 11;47(20):10706-10727

Cell Cycle and Genome Stability Group, Institute of Functional Biology and Genomics (IBFG), Spanish National Research Council (CSIC). University of Salamanca (USAL), C/ Zacarías González 2, Salamanca 37007, Spain.

The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.
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http://dx.doi.org/10.1093/nar/gkz794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6846210PMC
November 2019

Spatial Regulation of Polo-Like Kinase Activity During Meiosis by the Nucleoplasmic HAL-2/HAL-3 Complex.

Genetics 2019 09 25;213(1):79-96. Epub 2019 Jul 25.

Departments of Developmental Biology and Genetics, Stanford University School of Medicine, California 94305

Proper partitioning of homologous chromosomes during meiosis relies on the coordinated execution of multiple interconnected events: Homologs must locate, recognize, and align with their correct pairing partners. Further, homolog pairing must be coupled to assembly of the synaptonemal complex (SC), a meiosis-specific tripartite structure that maintains stable associations between the axes of aligned homologs and regulates formation of crossovers between their DNA molecules to create linkages that enable their segregation. Here, we identify HAL-3 (Homolog Alignment 3) as an important player in coordinating these key events during meiosis. HAL-3, and the previously identified HAL-2, are interacting and interdependent components of a protein complex that localizes to the nucleoplasm of germ cells. (or ) mutants exhibit multiple meiotic prophase defects including failure to establish homolog pairing, inappropriate loading of SC subunits onto unpaired chromosome axes, and premature loss of synapsis checkpoint protein PCH-2. Further, loss of function results in misregulation of the subcellular localization and activity of Polo-like kinases (PLK-1 and PLK-2), which dynamically localize to different defined subnuclear sites during wild-type prophase progression to regulate distinct cellular events. Moreover, loss of PLK-2 activity partially restores tripartite SC structure in a mutant background, suggesting that the defect in pairwise SC assembly in mutants reflects inappropriate PLK activity. Together, our data support a model in which the nucleoplasmic HAL-2/HAL-3 protein complex constrains both localization and activity of meiotic Polo-like kinases, thereby preventing premature interaction with stage-inappropriate targets.
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http://dx.doi.org/10.1534/genetics.119.302479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727811PMC
September 2019

Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin.

Mol Cell 2018 11 1;72(4):739-752.e9. Epub 2018 Nov 1.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany; MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN, UK; Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. Electronic address:

The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside" of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.
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http://dx.doi.org/10.1016/j.molcel.2018.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242706PMC
November 2018

Temporal dynamics of Wnt-dependent transcriptome reveal an oncogenic Wnt/MYC/ribosome axis.

J Clin Invest 2018 12 12;128(12):5620-5633. Epub 2018 Nov 12.

Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.

Activating mutations in the Wnt pathway drive a variety of cancers, but the specific targets and pathways activated by Wnt ligands are not fully understood. To bridge this knowledge gap, we performed a comprehensive time-course analysis of Wnt-dependent signaling pathways in an orthotopic model of Wnt-addicted pancreatic cancer, using a porcupine (PORCN) inhibitor currently in clinical trials, and validated key results in additional Wnt-addicted models. The temporal analysis of the drug-perturbed transcriptome demonstrated direct and indirect regulation of more than 3,500 Wnt-activated genes (23% of the transcriptome). Regulation was both via Wnt/β-catenin and through the modulation of protein abundance of important transcription factors, including MYC, via Wnt-dependent stabilization of proteins (Wnt/STOP). Our study identifies a central role of Wnt/β-catenin and Wnt/STOP signaling in controlling ribosome biogenesis, a key driver of cancer proliferation.
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http://dx.doi.org/10.1172/JCI122383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264740PMC
December 2018

Neuronatin regulates pancreatic β cell insulin content and secretion.

J Clin Invest 2018 08 9;128(8):3369-3381. Epub 2018 Jul 9.

MRC London Institute of Medical Sciences, London, United Kingdom.

Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone- and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in β cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat-null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in β cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of β cell function and whole-animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.
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http://dx.doi.org/10.1172/JCI120115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063487PMC
August 2018

Transient and Partial Nuclear Lamina Disruption Promotes Chromosome Movement in Early Meiotic Prophase.

Dev Cell 2018 04;45(2):212-225.e7

Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, 1030 Vienna, Austria. Electronic address:

Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.
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http://dx.doi.org/10.1016/j.devcel.2018.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920155PMC
April 2018

The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis.

Elife 2016 Feb 27;5:e12039. Epub 2016 Feb 27.

Department of Genetics, Harvard Medical School, Boston, United States.

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.
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http://dx.doi.org/10.7554/eLife.12039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805554PMC
February 2016

mTOR regulates MAPKAPK2 translation to control the senescence-associated secretory phenotype.

Nat Cell Biol 2015 Sep 17;17(9):1205-17. Epub 2015 Aug 17.

Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Campus, London W12 0NN, UK.

Senescent cells secrete a combination of factors collectively known as the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence and activates an immune surveillance response, but it can also show pro-tumorigenic properties and contribute to age-related pathologies. In a drug screen to find new SASP regulators, we uncovered the mTOR inhibitor rapamycin as a potent SASP suppressor. Here we report a mechanism by which mTOR controls the SASP by differentially regulating the translation of the MK2 (also known as MAPKAPK2) kinase through 4EBP1. In turn, MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1 during senescence, inhibiting its ability to degrade the transcripts of numerous SASP components. Consequently, mTOR inhibition or constitutive activation of ZFP36L1 impairs the non-cell-autonomous effects of senescent cells in both tumour-suppressive and tumour-promoting contexts. Altogether, our results place regulation of the SASP as a key mechanism by which mTOR could influence cancer, age-related diseases and immune responses.
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http://dx.doi.org/10.1038/ncb3225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589897PMC
September 2015

Macrophage epoxygenase determines a profibrotic transcriptome signature.

J Immunol 2015 May 3;194(10):4705-4716. Epub 2015 Apr 3.

Physiological Genomics and Medicine, MRC Clinical Sciences Centre, Imperial College London, W12 0NN, UK.

Epoxygenases belong to the cytochrome P450 family. They generate epoxyeicosatrienoic acids, which are known to have anti-inflammatory effects, but little is known about their role in macrophage function. By high-throughput sequencing of RNA in primary macrophages derived from rodents and humans, we establish the relative expression of epoxygenases in these cells. Zinc-finger nuclease-mediated targeted gene deletion of the major rat macrophage epoxygenase Cyp2j4 (ortholog of human CYP2J2) resulted in reduced epoxyeicosatrienoic acid synthesis. Cyp2j4(-/-) macrophages have relatively increased peroxisome proliferator-activated receptor-γ levels and show a profibrotic transcriptome, displaying overexpression of a specific subset of genes (260 transcripts) primarily involved in extracellular matrix, with fibronectin being the most abundantly expressed transcript. Fibronectin expression is under the control of epoxygenase activity in human and rat primary macrophages. In keeping with the in vitro findings, Cyp2j4(-/-) rats show upregulation of type I collagen following unilateral ureter obstruction of the kidney, and quantitative proteomics analysis (liquid chromatography-tandem mass spectrometry) showed increased renal type I collagen and fibronectin protein abundance resulting from experimentally induced crescentic glomerulonephritis in these rats. Taken together, these results identify the rat epoxygenase Cyp2j4 as a determinant of a profibrotic macrophage transcriptome that could have implications in various inflammatory conditions, depending on macrophage function.
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http://dx.doi.org/10.4049/jimmunol.1402979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417646PMC
May 2015

Integrating phosphoproteome and transcriptome reveals new determinants of macrophage multinucleation.

Mol Cell Proteomics 2015 Mar 22;14(3):484-98. Epub 2014 Dec 22.

§Centre for Complement and Inflammation Research (CCIR), Imperial College London, UK;

Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages.
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http://dx.doi.org/10.1074/mcp.M114.043836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349971PMC
March 2015

Characterization of a TiO₂ enrichment method for label-free quantitative phosphoproteomics.

Methods 2011 Aug 18;54(4):370-8. Epub 2011 Feb 18.

Analytical Signalling Laboratory, Centre for Cell Signalling, Barts Cancer Institute, Bart's and the London School of Medicine, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK.

Phosphorylation is a protein post-translational modification with key roles in the regulation of cell biochemistry and signaling. In-depth analysis of phosphorylation using mass spectrometry is permitting the investigation of processes controlled by phosphorylation at the system level. A critical step of these phosphoproteomics methods involves the isolation of phosphorylated peptides from the more abundant unmodified peptides produced by the digestion of cell lysates. Although different techniques to enrich for phosphopeptides have been reported, there are limited data on their suitability for direct quantitative analysis by MS. Here we report a TiO(2) based enrichment method compatible with large-scale and label-free quantitative analysis by LC-MS/MS. Starting with just 500 μg of protein, the technique reproducibly isolated hundreds of peptides, >85% of which were phosphorylated. These results were obtained by using relatively short LC-MS/MS gradient runs (45 min) and without any previous separation step. In order to characterize the performance of the method for quantitative analyses, we employed label-free LC-MS/MS using extracted ion chromatograms as the quantitative readout. After normalization, phosphopeptides were quantified with good precision (coefficient of variation was 20% on average, n=900 phosphopeptides), linearity (correlation coefficients >0.98) and accuracy (deviations <20%). Thus, phosphopeptide ion signals correlated with the concentration of the respective phosphopeptide in samples, making the approach suitable for in-depth relative quantification of phosphorylation by label-free LC-MS/MS.
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http://dx.doi.org/10.1016/j.ymeth.2011.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158853PMC
August 2011