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A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases.

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A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases.

Structure. 2018 Jun 26;:

Authors: Gorelik M, Manczyk N, Pavlenco A, Kurinov I, Sidhu SS, Sicheri F

Abstract
Skp1-Cul1-F-box (SCF) E3 ligases constitute the largest and best-characterized family of the multisubunit E3 ligases with important cellular functions and numerous disease links. The specificity of an SCF E3 ligase is established by one of the 69 human F-box proteins that are recruited to Cul1 through the Skp1 adaptor. We previously reported generation of ubiquitin variants (UbVs) targeting Fbw7 and Fbw11, which inhibit ligase activity by binding at the F-box-Skp1 interface to competitively displace Cul1. In the present study, we employed an optimized engineering strategy to generate specific binding UbVs against 17 additional Skp1-F-box complexes. We validated our design strategy and uncovered the structural basis of binding specificity by crystallographic analyses of representative UbVs bound to Skp1-Fbl10 and Skp1-Fbl11. Our study highlights the power of combining phage display with structure-based design to develop UbVs targeting specific protein surfaces.

PMID: 30033217 [PubMed - as supplied by publisher]



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Single-Cell RNA Sequencing: A New Window into Cell Scale Dynamics.

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Single-Cell RNA Sequencing: A New Window into Cell Scale Dynamics.

Biophys J. 2018 Jul 11;:

Authors: Dasgupta S, Bader GD, Goyal S

Abstract
Single-cell genomics has recently emerged as a powerful tool for observing multicellular systems at a much higher level of resolution and depth than previously possible. High-throughput single-cell RNA sequencing techniques are able to simultaneously quantify expression levels of several thousands of genes within individual cells for tens of thousands of cells within a complex tissue. This has led to development of novel computational methods to analyze this high-dimensional data, investigating longstanding and fundamental questions regarding the granularity of cell types, the definition of cell states, and transitions from one cell type to another along developmental trajectories. In this perspective, we outline this emerging field starting from the "input data" (e.g., quantifying transcription levels in single cells), which are analyzed to define "identities" (e.g., cell types, states, and key genes) and to build "interactions" using models that can infer relations and transitions between cells.

PMID: 30033145 [PubMed - as supplied by publisher]



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The shieldin complex mediates 53BP1-dependent DNA repair.

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The shieldin complex mediates 53BP1-dependent DNA repair.

Nature. 2018 08;560(7716):117-121

Authors: Noordermeer SM, Adam S, Setiaputra D, Barazas M, Pettitt SJ, Ling AK, Olivieri M, Álvarez-Quilón A, Moatti N, Zimmermann M, Annunziato S, Krastev DB, Song F, Brandsma I, Frankum J, Brough R, Sherker A, Landry S, Szilard RK, Munro MM, McEwan A, Goullet de Rugy T, Lin ZY, Hart T, Moffat J, Gingras AC, Martin A, van Attikum H, Jonkers J, Lord CJ, Rottenberg S, Durocher D

Abstract
53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

PMID: 30022168 [PubMed - indexed for MEDLINE]



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Two protein/protein interaction assays in one go.

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Two protein/protein interaction assays in one go.

Mol Syst Biol. 2018 07 18;14(7):e8485

Authors: Taipale M

PMID: 30021847 [PubMed - indexed for MEDLINE]



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New and Prospective Roles for lncRNAs in Organelle Formation and Function.

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New and Prospective Roles for lncRNAs in Organelle Formation and Function.

Trends Genet. 2018 Jul 14;:

Authors: Krause HM

Abstract
The observation that long noncoding RNAs (lncRNAs) represent the majority of transcripts in humans has led to a rapid increase in interest and study. Most of this interest has focused on their roles in the nucleus. However, increasing evidence is beginning to reveal even more functions outside the nucleus, and even outside cells. Many of these roles are mediated by newly discovered properties, including the ability of lncRNAs to interact with lipids, membranes, and disordered protein domains, and to form differentially soluble RNA-protein sub-organelles. This review explores the possibilities enabled by these new properties and abilities, such as likely roles in exosome formation and function.

PMID: 30017312 [PubMed - as supplied by publisher]



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A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A.

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A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A.

Genetics. 2018 09;210(1):203-218

Authors: Ciftci-Yilmaz S, Au WC, Mishra PK, Eisenstatt JR, Chang J, Dawson AR, Zhu I, Rahman M, Bilke S, Costanzo M, Baryshnikova A, Myers CL, Meltzer PS, Landsman D, Baker RE, Boone C, Basrai MA

Abstract
Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A lead to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide synthetic genetic array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to noncentromeric regions for genome stability. Hir2 interacts with Cse4 in vivo, and hir2∆ strains exhibit defects in Cse4 proteolysis and stabilization of chromatin-bound Cse4 Mislocalization of Cse4 to noncentromeric regions with a preferential enrichment at promoter regions was observed in hir2∆ strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1, and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2∆ strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4, thereby promoting genome stability.

PMID: 30012561 [PubMed - indexed for MEDLINE]



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Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress.

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Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress.

Diseases. 2018 Jul 13;6(3):

Authors: Han Z, Schwoerer MP, Hicks P, Liang J, Ruthel G, Berry CT, Freedman BD, Sagum CA, Bedford MT, Sidhu SS, Sudol M, Harty RN

Abstract
Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins.

PMID: 30011814 [PubMed]



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The impact of oncogenic EGFRvIII on the proteome of extracellular vesicles released from glioblastoma cells.

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The impact of oncogenic EGFRvIII on the proteome of extracellular vesicles released from glioblastoma cells.

Mol Cell Proteomics. 2018 Jul 13;:

Authors: Choi D, Montermini L, Kim DK, Meehan B, Roth FP, Rak J

Abstract
Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and down-regulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins. Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also co-localized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.

PMID: 30006486 [PubMed - as supplied by publisher]



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Functional genetic discovery of enzymes using full-scan mass spectrometry metabolomics.

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Functional genetic discovery of enzymes using full-scan mass spectrometry metabolomics.

Biochem Cell Biol. 2018 Jul 12;:

Authors: Caudy AA, Hanchard JA, Hsieh A, Shaan S, Rosebrock AP

Abstract
Our understanding of metabolic networks is incomplete, and new enzymatic activities await discovery in well studied organisms. Mass spectrometric methods for measuring cellular metabolism reveal compounds inside cells that are unexplained by existing maps of metabolic reactions. Current computational models are unable to account for all activities and contents observed within cells. Additional large-scale genetic and biochemical approaches are required to elucidate metabolic gene function. We have used full-scan mass spectrometry metabolomics to examine deletions of candidate enzymes in the model budding yeast Saccharomyces cerevisiae and report the identification of twenty-five candidates that alter metabolite levels. Triumphs and pitfalls of metabolic phenotyping screens are discussed, including estimates of the frequency of uncharacterized eukaryotic genes affecting metabolism and key issues to consider when searching for new enzymatic functions in other organisms.

PMID: 30001498 [PubMed - as supplied by publisher]



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Author Correction: Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

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Author Correction: Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Nature. 2018 Jul 11;:

Authors: Parikshak NN, Swarup V, Belgard TG, Irimia M, Ramaswami G, Gandal MJ, Hartl C, Leppa V, de la Torre Ubieta L, Huang J, Lowe JK, Blencowe BJ, Horvath S, Geschwind DH

Abstract
Change history: In this Letter, the labels for splicing events A3SS and A5SS were swapped in column D of Supplementary Table 3a and b. This has been corrected online.

PMID: 29995847 [PubMed - as supplied by publisher]



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