Abemaciclib Is Active in Preclinical Models of Ewing Sarcoma via Multipronged Regulation of Cell Cycle, DNA Methylation, and Interferon Pathway Signaling.

Purpose: Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The 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.

The expanding role of the Ehmt2/G9a complex in neurodevelopment.

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.

G9a and ZNF644 Physically Associate to Suppress Progenitor Gene Expression during Neurogenesis.

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.

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics.

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.

Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.

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.

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

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.

LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2.

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.

Roles for Pbp1 and caloric restriction in genome and lifespan maintenance via suppression of RNA-DNA hybrids.

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.

RPRD1A and RPRD1B are human RNA polymerase II C-terminal domain scaffolds for Ser5 dephosphorylation.

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.

Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

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.

Secreted biomolecules alter the biological identity and cellular interactions of nanoparticles.

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.

Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles.

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.

BuGZ is required for Bub3 stability, Bub1 kinetochore function, and chromosome alignment.

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.

Discovery of novel disease-specific and membrane-associated candidate markers in a mouse model of multiple sclerosis.

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.

Target identification by chromatographic co-elution: monitoring of drug-protein interactions without immobilization or chemical derivatization.

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.

Control of the RNA polymerase II phosphorylation state in promoter regions by CTD interaction domain-containing proteins RPRD1A and RPRD1B.

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.

Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake.

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.

Identification of mammalian protein complexes by lentiviral-based affinity purification and mass spectrometry.

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.

Perinuclear cohibin complexes maintain replicative life span via roles at distinct silent chromatin domains.

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.

Synthetic peptide arrays for pathway-level protein monitoring by liquid chromatography-tandem mass spectrometry.

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.

A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.