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1.
Clin Cancer Res ; 24(23): 6028-6039, 2018 12 01.
Article in English | MEDLINE | ID: mdl-30131386

ABSTRACT

PURPOSE: Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES. EXPERIMENTAL DESIGN: Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy. RESULTS: Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed. CONCLUSIONS: Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.


Subject(s)
Aminopyridines/pharmacology , Benzimidazoles/pharmacology , Cell Cycle , DNA Methylation , Interferons/metabolism , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Signal Transduction/drug effects , Animals , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/genetics , Humans , Mice , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/pathology , Xenograft Model Antitumor Assays
2.
Neurogenesis (Austin) ; 4(1): e1316888, 2017.
Article in English | MEDLINE | ID: mdl-28596979

ABSTRACT

Epigenetic regulators play a crucial role in neurodevelopment. One such epigenetic complex, Ehmt1/2 (G9a/GLP), is essential for repressing gene transcription by methylating H3K9 in a highly tissue- and temporal-specific manner. Recently, data has emerged suggesting that this complex plays additional roles in regulating the activity of numerous other non-histone proteins. While much is known about the downstream effects of Ehmt1/2 function, evidence is only beginning to come to light suggesting the control of Ehmt1/2 function may be, at least in part, due to context-dependent binding partners. Here we review emerging roles for the Ehmt1/2 complex suggesting that it may play a much larger role than previously recognized, and discuss binding partners that we and others have recently characterized which act to coordinate its activity during early neurodevelopment.

3.
Stem Cell Reports ; 7(3): 454-470, 2016 09 13.
Article in English | MEDLINE | ID: mdl-27546533

ABSTRACT

Proliferating progenitor cells undergo changes in competence to give rise to post-mitotic progeny of specialized function. These cell-fate transitions typically involve dynamic regulation of gene expression by histone methyltransferase (HMT) complexes. However, the composition, roles, and regulation of these assemblies in regulating cell-fate decisions in vivo are poorly understood. Using unbiased affinity purification and mass spectrometry, we identified the uncharacterized C2H2-like zinc finger protein ZNF644 as a G9a/GLP-interacting protein and co-regulator of histone methylation. In zebrafish, functional characterization of ZNF644 orthologs, znf644a and znf644b, revealed complementary roles in regulating G9a/H3K9me2-mediated gene silencing during neurogenesis. The non-overlapping requirements for znf644a and znf644b during retinal differentiation demarcate critical aspects of retinal differentiation programs regulated by differential G9a-ZNF644 associations, such as transitioning proliferating progenitor cells toward differentiation. Collectively, our data point to ZNF644 as a critical co-regulator of G9a/H3K9me2-mediated gene silencing during neuronal differentiation.


Subject(s)
Gene Expression Regulation, Developmental , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurogenesis/genetics , Transcription Factors/metabolism , Animals , Binding Sites , Biomarkers , Cell Differentiation , Cell Proliferation , Cell Survival/genetics , Gene Silencing , Histocompatibility Antigens/genetics , Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Humans , Methylation , Neurons/cytology , Neurons/metabolism , Phenotype , Protein Binding , Protein Interaction Mapping , Protein Interaction Maps , Retina/metabolism , Transcription Factors/genetics , Zebrafish
4.
Mol Cell Proteomics ; 15(3): 892-905, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26750096

ABSTRACT

The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation.


Subject(s)
Carcinoma, Squamous Cell/metabolism , Cell Culture Techniques/methods , Esophageal Neoplasms/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Mass Spectrometry/methods , Proteome/isolation & purification , Proteomics/methods , Amino Acid Motifs , Benzamides/pharmacology , Cell Line, Tumor , Esophageal Squamous Cell Carcinoma , Gene Expression Regulation, Neoplastic/drug effects , Humans , Isotope Labeling , Lysine/metabolism , Methylation , Proteome/chemistry , Pyrrolidines/pharmacology , Substrate Specificity
5.
EMBO Rep ; 16(10): 1334-57, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26265008

ABSTRACT

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.


Subject(s)
Embryonic Stem Cells/physiology , Gene Regulatory Networks/genetics , Nerve Tissue Proteins/genetics , Ribonucleoproteins/genetics , Animals , Cytoplasm/metabolism , Genomics , Mice , Nerve Tissue Proteins/chemistry , Proteomics , RNA/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/chemistry , Ubiquitin-Protein Ligases/metabolism
6.
Nat Methods ; 12(8): 725-31, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26121405

ABSTRACT

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibody Specificity , Chromatin/chemistry , Immunoprecipitation/methods , Proteomics/methods , Cloning, Molecular , Computational Biology/methods , Escherichia coli/metabolism , HEK293 Cells , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin G/chemistry , Mass Spectrometry/methods , Peptide Library , Proteins/chemistry , Proteome , Reproducibility of Results
7.
J Biol Chem ; 290(22): 13641-53, 2015 May 29.
Article in English | MEDLINE | ID: mdl-25825497

ABSTRACT

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.


Subject(s)
Antineoplastic Agents/chemistry , Benzamides/chemistry , Enzyme Inhibitors/chemistry , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Neoplasms/enzymology , Pyrrolidines/chemistry , Cell Line, Tumor , Cell Proliferation , Chromatin/chemistry , Computational Biology , Crystallization , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Epigenesis, Genetic , Histones/chemistry , Humans , Mass Spectrometry , Neoplasms/drug therapy , Peptides/chemistry , Protein Denaturation , Proteomics , Tumor Suppressor Protein p53/metabolism
8.
Cell Rep ; 8(1): 297-310, 2014 Jul 10.
Article in English | MEDLINE | ID: mdl-24981860

ABSTRACT

Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.


Subject(s)
Carrier Proteins/metabolism , Chromatin/metabolism , Chromosome Segregation , Histone Demethylases/metabolism , Membrane Proteins/metabolism , Carrier Proteins/genetics , HEK293 Cells , Humans , Membrane Proteins/genetics , Protein Binding
9.
Nat Struct Mol Biol ; 21(8): 686-695, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24997600

ABSTRACT

The RNA polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeats (1-YSPTSPS-7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD-interaction domains (CIDs) with RNAPII CTD repeats phosphorylated at S2 and S7. Crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with RNAPII CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.


Subject(s)
Cell Cycle Proteins/chemistry , Neoplasm Proteins/chemistry , Protein Processing, Post-Translational , RNA Polymerase II/chemistry , Repressor Proteins/chemistry , Amino Acid Sequence , Amino Acid Substitution , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Crystallography, X-Ray , HEK293 Cells , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Secondary , RNA Polymerase II/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Serine/chemistry
10.
Dev Cell ; 30(2): 177-91, 2014 Jul 28.
Article in English | MEDLINE | ID: mdl-25073155

ABSTRACT

Intergenic transcription within repetitive loci such as the ribosomal DNA (rDNA) repeats of yeast commonly triggers aberrant recombination. Major mechanisms suppressing aberrant rDNA recombination rely on chromatin silencing or RNAPII repression at intergenic spacers within the repeats. We find ancient processes operating at rDNA intergenic spacers and other loci to maintain genome stability via repression of RNA-DNA hybrids. The yeast Ataxin-2 protein Pbp1 binds noncoding RNA, suppresses RNA-DNA hybrids, and prevents aberrant rDNA recombination. Repression of RNA-DNA hybrids in Pbp1-deficient cells through RNaseH overexpression, deletion of the G4DNA-stabilizing Stm1, or caloric restriction operating via RNaseH/Pif1 restores rDNA stability. Pbp1 also limits hybrids at non-rDNA G4DNA loci including telomeres. Moreover, cells lacking Pbp1 have a short replicative lifespan that is extended upon hybrid suppression. Thus, we find roles for Pbp1 in genome maintenance and reveal that caloric restriction counteracts Pbp1 deficiencies by engaging RNaseH and Pif1.


Subject(s)
Carrier Proteins/metabolism , DNA, Fungal/genetics , Genome, Fungal , Genomic Instability , RNA, Fungal/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Carrier Proteins/genetics , DNA Helicases/genetics , DNA Helicases/metabolism , DNA, Ribosomal/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , G-Quadruplexes , Protein Binding , RNA, Untranslated/genetics , Recombination, Genetic , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcription, Genetic
11.
ACS Nano ; 8(6): 5515-26, 2014 Jun 24.
Article in English | MEDLINE | ID: mdl-24797313

ABSTRACT

A nanoparticle's physical and chemical properties at the time of cell contact will determine the ensuing cellular response. Aggregation and the formation of a protein corona in the extracellular environment will alter nanoparticle size, shape, and surface properties, giving it a "biological identity" that is distinct from its initial "synthetic identity". The biological identity of a nanoparticle depends on the composition of the surrounding biological environment and determines subsequent cellular interactions. When studying nanoparticle-cell interactions, previous studies have ignored the dynamic composition of the extracellular environment as cells deplete and secrete biomolecules in a process known as "conditioning". Here, we show that cell conditioning induces gold nanoparticle aggregation and changes the protein corona composition in a manner that depends on nanoparticle diameter, surface chemistry, and cell phenotype. The evolution of the biological identity in conditioned media enhances the cell membrane affinity, uptake, and retention of nanoparticles. These results show that dynamic extracellular environments can alter nanoparticle-cell interactions by modulating the biological identity. The effect of the dynamic nature of biological environments on the biological identity of nanoparticles must be considered to fully understand nano-bio interactions and prevent data misinterpretation.


Subject(s)
Metal Nanoparticles/chemistry , Nanotechnology/methods , Proteins/chemistry , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Culture Media, Conditioned/chemistry , Electrophoresis, Polyacrylamide Gel , Gold/chemistry , HeLa Cells , Humans , Kinetics , Mass Spectrometry , Particle Size , Phenotype , Protein Binding , Surface Properties
12.
ACS Nano ; 8(3): 2439-55, 2014 Mar 25.
Article in English | MEDLINE | ID: mdl-24517450

ABSTRACT

Using quantitative models to predict the biological interactions of nanoparticles will accelerate the translation of nanotechnology. Here, we characterized the serum protein corona 'fingerprint' formed around a library of 105 surface-modified gold nanoparticles. Applying a bioinformatics-inspired approach, we developed a multivariate model that uses the protein corona fingerprint to predict cell association 50% more accurately than a model that uses parameters describing nanoparticle size, aggregation state, and surface charge. Our model implicates a set of hyaluronan-binding proteins as mediators of nanoparticle-cell interactions. This study establishes a framework for developing a comprehensive database of protein corona fingerprints and biological responses for multiple nanoparticle types. Such a database can be used to develop quantitative relationships that predict the biological responses to nanoparticles and will aid in uncovering the fundamental mechanisms of nano-bio interactions.


Subject(s)
Blood Proteins/metabolism , Gold/chemistry , Gold/metabolism , Metal Nanoparticles , Silver/chemistry , Silver/metabolism , Cell Line , Humans , Nanotechnology , Particle Size , Protein Binding
13.
Dev Cell ; 28(3): 282-94, 2014 Feb 10.
Article in English | MEDLINE | ID: mdl-24462187

ABSTRACT

During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora B kinase activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologs are highly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest that BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes.


Subject(s)
Cell Cycle Proteins/chemistry , Chromosomes, Human/genetics , Glioblastoma/pathology , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Spindle Apparatus/physiology , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Aurora Kinase B/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Fluorescent Antibody Technique , Glioblastoma/genetics , Glioblastoma/metabolism , Histones/metabolism , Humans , Immunoblotting , Mice , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Mitosis/physiology , Molecular Sequence Data , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Phosphorylation , Phylogeny , Poly-ADP-Ribose Binding Proteins , Protein Stability , RNA, Small Interfering/genetics , Sequence Homology, Amino Acid
14.
Mol Cell Proteomics ; 13(3): 679-700, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24361864

ABSTRACT

Multiple sclerosis is a chronic demyelinating disorder characterized by the infiltration of auto-reactive immune cells from the periphery into the central nervous system resulting in axonal injury and neuronal cell death. Experimental autoimmune encephalomyelitis represents the best characterized animal model as common clinical, histological, and immunological features are recapitulated. A label-free mass spectrometric proteomics approach was used to detect differences in protein abundance within specific fractions of disease-affected tissues including the soluble lysate derived from the spinal cord and membrane protein-enriched peripheral blood mononuclear cells. Tissues were harvested from actively induced experimental autoimmune encephalomyelitis mice and sham-induced ("vehicle" control) counterparts at the disease peak followed by subsequent analysis by nanoflow liquid chromatography tandem mass spectrometry. Relative protein quantitation was performed using both intensity- and fragmentation-based approaches. After statistical evaluation of the data, over 500 and 250 differentially abundant proteins were identified in the spinal cord and peripheral blood mononuclear cell data sets, respectively. More than half of these observations have not previously been linked to the disease. The biological significance of all candidate disease markers has been elucidated through rigorous literature searches, pathway analysis, and validation studies. Results from comprehensive targeted mass spectrometry analyses have confirmed the differential abundance of ∼ 200 candidate markers (≥ twofold dysregulated expression) at a 70% success rate. This study is, to our knowledge, the first to examine the cell-surface proteome of peripheral blood mononuclear cells in experimental autoimmune encephalomyelitis. These data provide a unique mechanistic insight into the dynamics of peripheral immune cell infiltration into CNS-privileged sites at a molecular level and has identified several candidate markers, which represent promising targets for future multiple sclerosis therapies. The mass spectrometry proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000255.


Subject(s)
Cell Membrane/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Membrane Proteins/metabolism , Multiple Sclerosis/metabolism , Proteomics/methods , Animals , Biomarkers/cerebrospinal fluid , Biomarkers/metabolism , Blotting, Western , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/cerebrospinal fluid , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Leukocytes, Mononuclear/metabolism , Mass Spectrometry , Membrane Proteins/cerebrospinal fluid , Mice , Mice, Inbred C57BL , Multiple Sclerosis/cerebrospinal fluid , Multiple Sclerosis/pathology , Peptides/metabolism , Reproducibility of Results , Signal Transduction , Spinal Cord/metabolism , Spinal Cord/pathology , Staining and Labeling
15.
Mol Cell Proteomics ; 11(7): M111.016642, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22357554

ABSTRACT

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Antifungal Agents/metabolism , Fungal Proteins/metabolism , GTP-Binding Proteins/metabolism , Methyltransferases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Adaptor Proteins, Signal Transducing/analysis , Cell-Free System , Chromatography, Liquid , Dopamine Agonists/metabolism , Escherichia coli , Fungal Proteins/analysis , GTP-Binding Proteins/analysis , HeLa Cells , Humans , Hydroxamic Acids/metabolism , Indenes/metabolism , Macrolides/metabolism , Mass Spectrometry , Methotrexate/metabolism , Methyltransferases/analysis , Molecular Targeted Therapy , Phosphotransferases (Alcohol Group Acceptor)/analysis , Protein Binding , Proteomics , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/analysis
16.
J Am Chem Soc ; 134(4): 2139-47, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-22191645

ABSTRACT

Delivery and toxicity are critical issues facing nanomedicine research. Currently, there is limited understanding and connection between the physicochemical properties of a nanomaterial and its interactions with a physiological system. As a result, it remains unclear how to optimally synthesize and chemically modify nanomaterials for in vivo applications. It has been suggested that the physicochemical properties of a nanomaterial after synthesis, known as its "synthetic identity", are not what a cell encounters in vivo. Adsorption of blood components and interactions with phagocytes can modify the size, aggregation state, and interfacial composition of a nanomaterial, giving it a distinct "biological identity". Here, we investigate the role of size and surface chemistry in mediating serum protein adsorption to gold nanoparticles and their subsequent uptake by macrophages. Using label-free liquid chromatography tandem mass spectrometry, we find that over 70 different serum proteins are heterogeneously adsorbed to the surface of gold nanoparticles. The relative density of each of these adsorbed proteins depends on nanoparticle size and poly(ethylene glycol) grafting density. Variations in serum protein adsorption correlate with differences in the mechanism and efficiency of nanoparticle uptake by a macrophage cell line. Macrophages contribute to the poor efficiency of nanomaterial delivery into diseased tissues, redistribution of nanomaterials within the body, and potential toxicity. This study establishes principles for the rational design of clinically useful nanomaterials.


Subject(s)
Blood Proteins/chemistry , Gold/chemistry , Macrophages/chemistry , Metal Nanoparticles/chemistry , Adsorption , Gold/pharmacokinetics , Humans , Particle Size , Polyethylene Glycols/chemistry , Surface Properties , Tissue Distribution
17.
Methods Mol Biol ; 781: 31-45, 2011.
Article in English | MEDLINE | ID: mdl-21877275

ABSTRACT

Protein complexes and protein-protein interactions (PPIs) are fundamental for most biological functions. Deciphering the extensive protein interaction networks that occur within cellular contexts has become a logical extension to the human genome project. Proteome-scale interactome analysis of mammalian systems requires efficient methods for accurately detecting PPIs with specific considerations for the intrinsic technical challenges of mammalian genome manipulation. In this chapter, we outline in detail an innovative lentiviral-based functional proteomic approach that can be used to rapidly characterize protein complexes from a broad range of mammalian cell lines. This method integrates the following key features: (1) lentiviral elements for efficient delivery of tagged constructs into mammalian cell lines; (2) site-specific Gateway™ recombination sites for easy cloning; (3) versatile epitope-tagging system for flexible affinity purification strategies; and (4) LC-MS-based protein identification using tandem mass spectrometry.


Subject(s)
Lentivirus/genetics , Protein Interaction Mapping/methods , Protein Interaction Maps , Proteins/analysis , Proteomics/methods , Animals , Cell Line , Chromatography, Affinity , Cloning, Molecular , HEK293 Cells , Humans , Protein Binding , Proteins/metabolism , Proteome/analysis , Tandem Mass Spectrometry
18.
Dev Cell ; 20(6): 867-79, 2011 Jun 14.
Article in English | MEDLINE | ID: mdl-21664583

ABSTRACT

Heterochromatin, or silent chromatin, preferentially resides at the nuclear envelope. Telomeres and rDNA repeats are the two major perinuclear silent chromatin domains of Saccharomyces cerevisiae. The Cohibin protein complex maintains rDNA repeat stability in part through silent chromatin assembly and perinuclear rDNA anchoring. We report here a role for Cohibin at telomeres and show that functions of the complex at chromosome ends and rDNA maintain replicative life span. Cohibin binds LEM/SUN domain-containing nuclear envelope proteins and telomere-associated factors. Disruption of Cohibin or the envelope proteins abrogates telomere localization and silent chromatin assembly within subtelomeres. Loss of Cohibin limits Sir2 histone deacetylase localization to chromosome ends, disrupts subtelomeric DNA stability, and shortens life span even when rDNA repeats are stabilized. Restoring telomeric Sir2 concentration abolishes chromatin and life span defects linked to the loss of telomeric Cohibin. Our work uncovers roles for Cohibin complexes and reveals relationships between nuclear compartmentalization, chromosome stability, and aging.


Subject(s)
Heterochromatin/genetics , Longevity , Nuclear Envelope/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/genetics , Telomere/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA, Fungal/genetics , DNA, Ribosomal/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polymerase Chain Reaction , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/genetics , Sirtuin 2/metabolism
19.
Transcription ; 2(5): 237-42, 2011.
Article in English | MEDLINE | ID: mdl-22231121

ABSTRACT

RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation is important for various transcription-related processes. Here, we identify by affinity purification and mass spectrometry three previously uncharacterized human CTD-interaction domain (CID)-containing proteins, RPRD1A, RPRD1B and RPRD2, which co-purify with RNAP II and three other RNAP II-associated proteins, RPAP2, GRINL1A and RECQL5, but not with the Mediator complex. RPRD1A and RPRD1B can accompany RNAP II from promoter regions to 3'-untranslated regions during transcription in vivo, predominantly interact with phosphorylated RNAP II, and can reduce CTD S5- and S7-phosphorylated RNAP II at target gene promoters. Thus, the RPRD proteins are likely to have multiple important roles in transcription.


Subject(s)
Cell Cycle Proteins/metabolism , Neoplasm Proteins/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Repressor Proteins/metabolism , 3' Untranslated Regions , Carrier Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , HEK293 Cells , Humans , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Phosphorylation , Protein Interaction Domains and Motifs , RNA Polymerase II/chemistry , RNA Polymerase II/genetics , RecQ Helicases/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Transcription, Genetic
20.
Mol Cell Proteomics ; 9(11): 2460-73, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20467045

ABSTRACT

Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including "label-free" quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations.


Subject(s)
Chromatography, Liquid/methods , Peptides/chemistry , Protein Array Analysis/methods , Proteins/analysis , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Animals , Mice , Peptides/chemical synthesis , Peptides/genetics , Proteomics/methods
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