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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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DNA Sequence Recognition of Human CXXC Domains and Their Structural Determinants.

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DNA Sequence Recognition of Human CXXC Domains and Their Structural Determinants.

Structure. 2017 Dec 16;:

Authors: Xu C, Liu K, Lei M, Yang A, Li Y, Hughes TR, Min J

Abstract
The CXXC domain, first identified as the reader of unmodified CpG dinucleotide, plays important roles in epigenetic regulation by targeting various activities to CpG islands. Here we systematically measured and compared the DNA-binding selectivities of all known human CXXC domains by different binding assays, and complemented the existing function-based classification of human CXXC domains with a classification based on their DNA selectivities. Through a series of crystal structures of CXXC domains with DNA ligands, we unravel the molecular mechanisms of how these CXXC domains, including single CXXC domains and tandem CXXC-PHD domains, recognize distinct DNA ligands, which further supports our classification of human CXXC domains and also provides insights into selective recruitment of chromatin modifiers to their respective targets via CXXC domains recognizing different genomic DNA sequences. Our study facilitates the understanding of the relationship between the DNA-binding specificities of the CXXC proteins and their biological functions.

PMID: 29276034 [PubMed - as supplied by publisher]



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A framework for exhaustively mapping functional missense variants.

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A framework for exhaustively mapping functional missense variants.

Mol Syst Biol. 2017 Dec 21;13(12):957

Authors: Weile J, Sun S, Cote AG, Knapp J, Verby M, Mellor JC, Wu Y, Pons C, Wong C, van Lieshout N, Yang F, Tasan M, Tan G, Yang S, Fowler DM, Nussbaum R, Bloom JD, Vidal M, Hill DE, Aloy P, Roth FP

Abstract
Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

PMID: 29269382 [PubMed - in process]



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Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFα signaling.

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Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFα signaling.

Sci Transl Med. 2017 Dec 20;9(421):

Authors: Wang W, Fujii H, Kim HJ, Hermans K, Usenko T, Xie S, Luo ZJ, Ma J, Celso CL, Dick JE, Schroeder T, Krueger J, Wall D, Egeler RM, Zandstra PW

Abstract
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell-derived inflammatory signals. Donor T cell-derived tumor necrosis factor-α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell-derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell-derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.

PMID: 29263228 [PubMed - in process]



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FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors.

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FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors.

Stem Cell Reports. 2017 Dec 13;:

Authors: Yoon C, Song H, Yin T, Bausch-Fluck D, Frei AP, Kattman S, Dubois N, Witty AD, Hewel JA, Guo H, Emili A, Wollscheid B, Keller G, Zandstra PW

Abstract
The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs) generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm. Additionally, we utilized FZD4, in conjunction with FLK1 and PDGFRA, to further purify CPCs and increase cardiomyocyte (CM) enrichment in both mouse and human systems. Moreover, we have shown that NORRIN presented to FZD4 further increases CM output via proliferation through the canonical WNT pathway. Taken together, these findings demonstrate a role for FZD4 in mammalian cardiac development.

PMID: 29249665 [PubMed - as supplied by publisher]



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Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

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Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

MBio. 2017 Dec 12;8(6):

Authors: Pawluk A, Shah M, Mejdani M, Calmettes C, Moraes TF, Davidson AR, Maxwell KL

Abstract
CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system.

PMID: 29233895 [PubMed - in process]



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