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High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.

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High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.

Mol Cell. 2018 Feb 01;69(3):517-532.e11

Authors: Youn JY, Dunham WH, Hong SJ, Knight JDR, Bashkurov M, Chen GI, Bagci H, Rathod B, MacLeod G, Eng SWM, Angers S, Morris Q, Fabian M, Côté JF, Gingras AC

Abstract
mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.

PMID: 29395067 [PubMed - in process]



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Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PDB.

Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PDB.

Proc Natl Acad Sci U S A. 2018 Jan 29;:

Authors: Garton M, Nim S, Stone TA, Wang KE, Deber CM, Kim PM

Abstract
Biologics are a rapidly growing class of therapeutics with many advantages over traditional small molecule drugs. A major obstacle to their development is that proteins and peptides are easily destroyed by proteases and, thus, typically have prohibitively short half-lives in human gut, plasma, and cells. One of the most effective ways to prevent degradation is to engineer analogs from dextrorotary (D)-amino acids, with up to 105-fold improvements in potency reported. We here propose a general peptide-engineering platform that overcomes limitations of previous methods. By creating a mirror image of every structure in the Protein Data Bank (PDB), we generate a database of ∼2.8 million D-peptides. To obtain a D-analog of a given peptide, we search the (D)-PDB for similar configurations of its critical-"hotspot"-residues. As a proof of concept, we apply our method to two peptides that are Food and Drug Administration approved as therapeutics for diabetes and osteoporosis, respectively. We obtain D-analogs that activate the GLP1 and PTH1 receptors with the same efficacy as their natural counterparts and show greatly increased half-life.

PMID: 29378946 [PubMed - as supplied by publisher]



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Modeling signaling-dependent pluripotency with Boolean logic to predict cell fate transitions.

Modeling signaling-dependent pluripotency with Boolean logic to predict cell fate transitions.

Mol Syst Biol. 2018 Jan 29;14(1):e7952

Authors: Yachie-Kinoshita A, Onishi K, Ostblom J, Langley MA, Posfai E, Rossant J, Zandstra PW

Abstract
Pluripotent stem cells (PSCs) exist in multiple stable states, each with specific cellular properties and molecular signatures. The mechanisms that maintain pluripotency, or that cause its destabilization to initiate development, are complex and incompletely understood. We have developed a model to predict stabilized PSC gene regulatory network (GRN) states in response to input signals. Our strategy used random asynchronous Boolean simulations (R-ABS) to simulate single-cell fate transitions and strongly connected components (SCCs) strategy to represent population heterogeneity. This framework was applied to a reverse-engineered and curated core GRN for mouse embryonic stem cells (mESCs) and used to simulate cellular responses to combinations of five signaling pathways. Our simulations predicted experimentally verified cell population compositions and input signal combinations controlling specific cell fate transitions. Extending the model to PSC differentiation, we predicted a combination of signaling activators and inhibitors that efficiently and robustly generated a Cdx2+Oct4- cells from naïve mESCs. Overall, this platform provides new strategies to simulate cell fate transitions and the heterogeneity that typically occurs during development and differentiation.

PMID: 29378814 [PubMed - in process]



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Unification of Protein Abundance Datasets Yields a Quantitative Saccharomyces cerevisiae Proteome.

Unification of Protein Abundance Datasets Yields a Quantitative Saccharomyces cerevisiae Proteome.

Cell Syst. 2018 Jan 17;:

Authors: Ho B, Baryshnikova A, Brown GW

Abstract
Protein activity is the ultimate arbiter of function in most cellular pathways, and protein concentration is fundamentally connected to protein action. While the proteome of yeast has been subjected to the most comprehensive analysis of any eukaryote, existing datasets are difficult to compare, and there is no consensus abundance value for each protein. We evaluated 21 quantitative analyses of the S. cerevisiae proteome, normalizing and converting all measurements of protein abundance into the intuitive measurement of absolute molecules per cell. We estimate the cellular abundance of 92% of the proteins in the yeast proteome and assess the variation in each abundance measurement. Using our protein abundance dataset, we find that a global response to diverse environmental stresses is not detected at the level of protein abundance, we find that protein tags have only a modest effect on protein abundance, and we identify proteins that are differentially regulated at the mRNA abundance, mRNA translation, and protein abundance levels.

PMID: 29361465 [PubMed - as supplied by publisher]



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The Chemical Fluctuation Theorem governing gene expression.

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The Chemical Fluctuation Theorem governing gene expression.

Nat Commun. 2018 Jan 19;9(1):297

Authors: Park SJ, Song S, Yang GS, Kim PM, Yoon S, Kim JH, Sung J

Abstract
Gene expression is a complex stochastic process composed of numerous enzymatic reactions with rates coupled to hidden cell-state variables. Despite advances in single-cell technologies, the lack of a theory accurately describing the gene expression process has restricted a robust, quantitative understanding of gene expression variability among cells. Here we present the Chemical Fluctuation Theorem (CFT), providing an accurate relationship between the environment-coupled chemical dynamics of gene expression and gene expression variability. Combined with a general, accurate model of environment-coupled transcription processes, the CFT provides a unified explanation of mRNA variability for various experimental systems. From this analysis, we construct a quantitative model of transcription dynamics enabling analytic predictions for the dependence of mRNA noise on the mRNA lifetime distribution, confirmed against stochastic simulation. This work suggests promising new directions for quantitative investigation into cellular control over biological functions by making complex dynamics of intracellular reactions accessible to rigorous mathematical deductions.

PMID: 29352116 [PubMed - in process]



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Cardiolipin synthesizing enzymes form a complex that interacts with cardiolipin-dependent membrane organizing proteins.

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Cardiolipin synthesizing enzymes form a complex that interacts with cardiolipin-dependent membrane organizing proteins.

Biochim Biophys Acta. 2018 Jan 14;:

Authors: Serricchio M, Vissa A, Kim PK, Yip CM, McQuibban GA

Abstract
The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes.

PMID: 29343430 [PubMed - as supplied by publisher]



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A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

Nature. 2018 Jan 17;:

Authors: Bahr C, von Paleske L, Uslu VV, Remeseiro S, Takayama N, Ng SW, Murison A, Langenfeld K, Petretich M, Scognamiglio R, Zeisberger P, Benk AS, Amit I, Zandstra PW, Lupien M, Dick JE, Trumpp A, Spitz F

Abstract
The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a 'super-enhancer' is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a 'blood enhancer cluster' (BENC). We show that BENC is also essential for the maintenance of MLL-AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.

PMID: 29342133 [PubMed - as supplied by publisher]



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Drug development: Allosteric inhibitors hit USP7 hard.

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Drug development: Allosteric inhibitors hit USP7 hard.

Nat Chem Biol. 2018 Jan 16;14(2):110-111

Authors: Zhang W, Sidhu SS

PMID: 29337966 [PubMed - in process]



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Cellular Biomechanics in Skeletal Muscle Regeneration.

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Cellular Biomechanics in Skeletal Muscle Regeneration.

Curr Top Dev Biol. 2018;126:125-176

Authors: Li EW, McKee-Muir OC, Gilbert PM

Abstract
Satellite cells, adult stem cells in skeletal muscle tissue, reside within a mechanically dynamic three-dimensional microenvironment. With each contraction-relaxation cycle, a satellite cell is expected to experience tensile, shear, and compressive stresses, and through cell-extracellular matrix interactions, also gauge the stiffness of the niche. Via mechanoreceptors, cells can sense these biophysical parameters of the niche, which serve to physically induce conformational changes that impact biomolecule activity, and thereby alter downstream signal transduction pathways and ultimately cell fate. An emerging body of literature supports the notion that myogenic cells, too, integrate biochemical factors together with biomechanical stresses and that this may serve to provide spatio-temporal control of cell fate in the complicated three-dimensional niche. Further, skeletal muscle regenerative medicine therapies are being improved by applying this fresh insight. In this focused chapter, the progression of skeletal muscle regeneration is dissected into a dynamic conversation between muscle progenitor cells and the mechanical properties of the extracellular matrix. The significance of biophysical regulation to myogenic repair is reinforced by the exaggerative influences of extrinsic mechanical stresses and the pathological implications of ECM dysregulation. Additional fundamental studies that further define the satellite cell biophysical environment in health, regeneration, aging, and disease may serve to close knowledge gaps and bolster skeletal muscle regenerative medicine.

PMID: 29304997 [PubMed - in process]



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Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling.

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Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling.

Cell Rep. 2017 Dec 26;21(13):3970-3986

Authors: Yuzwa SA, Borrett MJ, Innes BT, Voronova A, Ketela T, Kaplan DR, Bader GD, Miller FD

Abstract
Adult neural stem cells (NSCs) derive from embryonic precursors, but little is known about how or when this occurs. We have addressed this issue using single-cell RNA sequencing at multiple developmental time points to analyze the embryonic murine cortex, one source of adult forebrain NSCs. We computationally identify all major cortical cell types, including the embryonic radial precursors (RPs) that generate adult NSCs. We define the initial emergence of RPs from neuroepithelial stem cells at E11.5. We show that, by E13.5, RPs express a transcriptional identity that is maintained and reinforced throughout their transition to a non-proliferative state between E15.5 and E17.5. These slowly proliferating late embryonic RPs share a core transcriptional phenotype with quiescent adult forebrain NSCs. Together, these findings support a model wherein cortical RPs maintain a core transcriptional identity from embryogenesis through to adulthood and wherein the transition to a quiescent adult NSC occurs during late neurogenesis.

PMID: 29281841 [PubMed - in process]



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