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Structural and Functional Analysis of Ubiquitin-based inhibitors that Target the Backsides of E2 Enzymes.

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Structural and Functional Analysis of Ubiquitin-based inhibitors that Target the Backsides of E2 Enzymes.

J Mol Biol. 2019 Oct 18;:

Authors: Garg P, Ceccarelli DF, Keszei AF, Kourinov I, Sicheri F, Sidhu SS

Abstract
Ubiquitin-conjugating E2 enzymes are central to the ubiquitination cascade and have been implicated in cancer and other diseases. Despite strong interest in developing specific E2 inhibitors, the shallow and exposed active site has proven recalcitrant to targeting with reversible small-molecule inhibitors. Here, we used phage display to generate highly potent and selective ubiquitin variants (UbVs) that target the E2 backside, which is located opposite to the active site. A UbV targeting Ube2D1 did not affect charging but greatly attenuated chain elongation. Likewise, a UbV targeting the E2 variant Ube2V1 did not interfere with the charging of its partner E2 enzyme but inhibited formation of di-ubiquitin. In contrast, a UbV that bound to the backside of Ube2G1 impeded the generation of thioester-linked ubiquitin to the active site cysteine of Ube2G1 by the E1 enzyme. Crystal structures of UbVs in complex with three E2 proteins revealed distinctive molecular interactions in each case, but they also highlighted a common backside pocket that the UbVs utilized for enhanced affinity and specificity. These findings validate the E2 backside as a target for inhibition and provide structural insights to aid inhibitor design and screening efforts.

PMID: 31634471 [PubMed - as supplied by publisher]



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High-throughput micropatterning platform reveals Nodal-dependent bisection of peri-gastrulation-associated versus preneurulation-associated fate patterning.

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High-throughput micropatterning platform reveals Nodal-dependent bisection of peri-gastrulation-associated versus preneurulation-associated fate patterning.

PLoS Biol. 2019 Oct 21;17(10):e3000081

Authors: Tewary M, Dziedzicka D, Ostblom J, Prochazka L, Shakiba N, Heydari T, Aguilar-Hidalgo D, Woodford C, Piccinini E, Becerra-Alonso D, Vickers A, Louis B, Rahman N, Danovi D, Geens M, Watt FM, Zandstra PW

Abstract
In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous nodal expression, with up-regulation of nodal correlated with expression of a gastrulation-associated gene profile, and nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.

PMID: 31634368 [PubMed - as supplied by publisher]



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Sphingolipid Modulation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal.

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Sphingolipid Modulation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal.

Cell Stem Cell. 2019 Oct 11;:

Authors: Xie SZ, Garcia-Prat L, Voisin V, Ferrari R, Gan OI, Wagenblast E, Kaufmann KB, Zeng AGX, Takayanagi SI, Patel I, Lee EK, Jargstorf J, Holmes G, Romm G, Pan K, Shoong M, Vedi A, Luberto C, Minden MD, Bader GD, Laurenti E, Dick JE

Abstract
Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.

PMID: 31631013 [PubMed - as supplied by publisher]



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Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution.

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Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution.

Mol Cell. 2019 Oct 17;76(2):329-345

Authors: Ule J, Blencowe BJ

Abstract
High-throughput sequencing-based methods and their applications in the study of transcriptomes have revolutionized our understanding of alternative splicing. Networks of functionally coordinated and biologically important alternative splicing events continue to be discovered in an ever-increasing diversity of cell types in the context of physiologically normal and disease states. These studies have been complemented by efforts directed at defining sequence codes governing splicing and their cognate trans-acting factors, which have illuminated important combinatorial principles of regulation. Additional studies have revealed critical roles of position-dependent, multivalent protein-RNA interactions that direct splicing outcomes. Investigations of evolutionary changes in RNA binding proteins, splice variants, and associated cis elements have further shed light on the emergence, mechanisms, and functions of splicing networks. Progress in these areas has emphasized the need for a coordinated, community-based effort to systematically address the functions of individual splice variants associated with normal and disease biology.

PMID: 31626751 [PubMed - in process]



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Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.

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Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.

Elife. 2019 10 15;8:

Authors: Laflamme C, McKeever PM, Kumar R, Schwartz J, Kolahdouzan M, Chen CX, You Z, Benaliouad F, Gileadi O, McBride HM, Durcan TM, Edwards AM, Healy LM, Robertson J, McPherson PS

Abstract
Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.

PMID: 31612854 [PubMed - indexed for MEDLINE]



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Protein-Protein Interaction Profiling in Candida albicans Revealed by Biochemical Purification-Mass Spectrometry (BP/MS).

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Protein-Protein Interaction Profiling in Candida albicans Revealed by Biochemical Purification-Mass Spectrometry (BP/MS).

Methods Mol Biol. 2019;2049:203-211

Authors: Pourhaghighi R, O'Meara TR, Cowen LE, Emili A

Abstract
BP/MS is a new experimental proteomic platform for systematic study of native protein complexes and global protein-protein interaction networks based on deep biochemical purification of soluble protein extracts and mass spectrometry identification of coeluting proteins. Herein, we describe the application of this methodology to draft a high-confidence protein interaction network for Candida albicans.

PMID: 31602613 [PubMed - in process]



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Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Am J Physiol Renal Physiol. 2019 Sep 30;:

Authors: Cina DP, Ketela T, Brown KR, Chandrashekhar M, Mero P, Li C, Onay T, Fu Y, Han Z, Saleem MA, Moffat J, Quaggin SE

Abstract
Background: Podocyte function is tightly linked to the complex organization of its cytoskeleton, and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. Methods: To identify novel genes that are important for podocyte function, we designed an in vitrogenetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble FLT1/Fc. Results: A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against each of DPH1, DPH2, DPH3, and DPH4were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2,or DPH3which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Last, we showed that Drosophila nephrocyte-specific knock-down of Dph1, Dph2, and Dph4results in altered nephrocyte function. Conclusions: In summary, we report a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.

PMID: 31566424 [PubMed - as supplied by publisher]



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Neuronal-specific microexon splicing of TAF1 mRNA is directly regulated by SRRM4/nSR100.

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Neuronal-specific microexon splicing of TAF1 mRNA is directly regulated by SRRM4/nSR100.

RNA Biol. 2019 Sep 27;:1-13

Authors: Capponi S, Stöffler N, Irimia M, Van Schaik FMA, Ondik MM, Biniossek ML, Lehmann L, Mitschke J, Vermunt MW, Creyghton MP, Graybiel AM, Reinheckel T, Schilling O, Blencowe BJ, Crittenden JR, Timmers HTM

Abstract
Neuronal microexons represent the most highly conserved class of alternative splicing events and their timed expression shapes neuronal biology, including neuronal commitment and differentiation. The six-nt microexon 34' is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this study, we investigate the tissue distribution of TAF1-34' mRNA and protein and the mechanism responsible for its neuronal-specific splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34' have different distributions in the brain, which distinguish proliferating from post-mitotic neurons. Knockdown and ectopic expression experiments demonstrate that the neuronal-specific splicing factor SRRM4/nSR100 promotes the inclusion of microexon 34' into TAF1 mRNA, through the recognition of UGC sequences in the poly-pyrimidine tract upstream of the regulated microexon. These results show that SRRM4 regulates temporal and spatial expression of alternative TAF1 mRNAs to generate a neuronal-specific TFIID complex.

PMID: 31559909 [PubMed - as supplied by publisher]



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Single-cell transcriptomic profiling of the aging mouse brain.

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Single-cell transcriptomic profiling of the aging mouse brain.

Nat Neurosci. 2019 10;22(10):1696-1708

Authors: Ximerakis M, Lipnick SL, Innes BT, Simmons SK, Adiconis X, Dionne D, Mayweather BA, Nguyen L, Niziolek Z, Ozek C, Butty VL, Isserlin R, Buchanan SM, Levine SS, Regev A, Bader GD, Levin JZ, Rubin LL

Abstract
The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.

PMID: 31551601 [PubMed - indexed for MEDLINE]



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High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting.

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High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting.

Nat Biomed Eng. 2019 Sep 23;:

Authors: Mair B, Aldridge PM, Atwal RS, Philpott D, Zhang M, Masud SN, Labib M, Tong AHY, Sargent EH, Angers S, Moffat J, Kelley SO

Abstract
Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 108 cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.

PMID: 31548591 [PubMed - as supplied by publisher]



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