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A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants.

Related Articles

A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants.

Mol Cell. 2017 Oct 19;68(2):456-470.e10

Authors: Gabrielsen M, Buetow L, Nakasone MA, Ahmed SF, Sibbet GJ, Smith BO, Zhang W, Sidhu SS, Huang DT

Abstract
RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

PMID: 29053960 [PubMed - in process]



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Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or Protein Abundance Using Fluorescent Proteins in Saccharomyces cerevisiae.

Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or Protein Abundance Using Fluorescent Proteins in Saccharomyces cerevisiae.

Methods Mol Biol. 2018;1672:613-629

Authors: Göttert H, Mattiazzi Usaj M, Rosebrock AP, Andrews BJ

Abstract
Fluorescent reporter genes have long been used to quantify various cell features such as transcript and protein abundance. Here, we describe a method, reporter synthetic genetic array (R-SGA) analysis, which allows for the simultaneous quantification of any fluorescent protein readout in thousands of yeast strains using an automated pipeline. R-SGA combines a fluorescent reporter system with standard SGA analysis and can be used to examine any array-based strain collection available to the yeast community. This protocol describes the R-SGA methodology for screening different arrays of yeast mutants including the deletion collection, a collection of temperature-sensitive strains for the assessment of essential yeast genes and a collection of inducible overexpression strains. We also present an alternative pipeline for the analysis of R-SGA output strains using flow cytometry of cells in liquid culture. Data normalization for both pipelines is discussed.

PMID: 29043651 [PubMed - in process]



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DNA Replication Profiling Using Deep Sequencing.

DNA Replication Profiling Using Deep Sequencing.

Methods Mol Biol. 2018;1672:195-207

Authors: Saayman X, Ramos-Pérez C, Brown GW

Abstract
Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

PMID: 29043626 [PubMed - in process]



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Abstracts and Workshops 7th National Spinal Cord Injury Conference November 9 - 11, 2017 Fallsview Casino Resort Niagara Falls, Ontario, Canada.

Abstracts and Workshops 7th National Spinal Cord Injury Conference November 9 - 11, 2017 Fallsview Casino Resort Niagara Falls, Ontario, Canada.

J Spinal Cord Med. 2017 Nov;40(6):813-869

Authors: Shojaei MH, Alavinia M, Craven BC, Cheng CL, Plashkes T, Shen T, Fallah N, Humphreys S, O'Connell C, Linassi AG, Ho C, Short C, Ethans K, Charbonneau R, Paquet J, Noonan VK, Furlan JC, Fehlings MG, Craven BC, Likitlersuang J, Sumitro E, Kalsi-Ryan S, Zariffa J, Wolfe D, Cornell S, Gagliardi J, Marrocco S, Rivers CS, Fallah NN, Noonan VK, Whitehurst D, Schwartz C, Finkelstein J, Craven BC, Ethans K, O'Connell C, Truchon C, Ho C, Linassi AG, Short C, Tsai E, Drew B, Ahn H, Dvorak MF, Paquet J, Fehlings MG, Noreau L, Lenz K, Bailey KA, Allison D, Ditor D, Baron J, Tomasone J, Curran D, Miller T, Grimshaw J, Moineau B, Alizadeh-Meghrazi M, Stefan G, Masani K, Popovic MR, Sumitro E, Likitlersuang J, Kalsi-Ryan S, Zariffa J, Garcia-Garcia MG, Marquez-Chin C, Popovic MR, Furlan JC, Gulasingam S, Craven BC, Furlan JC, Gulasingam S, Craven BC, Khan A, Pujol C, Laylor M, Unic N, Pakosh M, Musselman K, Brisbois LM, Catharine Craven B, Verrier MC, Jones MK, O'Shea R, Valika S, Holtz K, Szefer E, Noonan V, Kwon B, Mills P, Morin C, Harris A, Cheng C, Aspinall A, Plashkes T, Noonan VK, Chan K, Verrier MC, Craven BC, Alappat C, Flett HM, Furlan JC, Musselman KE, Milligan J, Hillier LM, Bauman C, Donaldson L, Lee J, Milligan J, Lee J, Hillier LM, Slonim K, Wolfe D, Sleeth L, Jeske S, Kras-Dupuis A, Marrocco S, McRae S, Flett H, Mokry J, Zee J, Bayley M, Lemay JF, Roy A, Gagnon HD, Jones MK, O'Shea R, Theiss R, Flett H, Guy K, Johnston G, Kokotow M, Mills S, Mokry J, Bain P, Scovil C, Houghton P, Lala D, Orr L, Holyoke P, Wolfe D, Orr L, Brooke J, Holyoke P, Lala D, Houghton P, Martin Ginis KA, Shaw RB, Stork MJ, McBride CB, Furlan JC, Craven BC, Giangregorio L, Hitzig S, Kapadia N, Popovic MR, Zivanovic V, Valiante T, Popovic MR, Patsakos E, Brisbois L, Farahani F, Kaiser A, Craven BC, Patsakos E, Kaiser A, Brisbois L, Farahani F, Craven BC, Mortenson B, MacGillivray M, Mahsa S, Adams J, Sawatzky B, Mills P, Arbour-Nicitopoulos K, Bassett-Gunter R, Leo J, Sharma R, Latimer-Cheung A, Olds T, Martin Ginis K, Graco M, Cross S, Thiyagarajan C, Shafazand S, Ayas N, Schembri R, Booker L, Nicholls C, Burns P, Nash M, Green S, Berlowitz DJ, Taran S, Rocchi M, Martin Ginis KA, Sweet SN, Caron JG, Sweet SN, Rocchi MA, Zelaya W, Sweet SN, Bergquist AJ, Del Castillo-Valenzuela MF, Popovic MR, Masani K, Ethans K, Casey A, Namaka M, Krassiokov-Enns D, Marquez-Chin C, Marquis A, Desai N, Zivanovic V, Hebert D, Popovic MR, Furlan JC, Craven BC, McLeod J, Hicks A, Gauthier C, Arel J, Brosseau R, Hicks AL, Gagnon DH, Nejatbakhsh N, Kaiser A, Hitzig SL, Cappe S, McGillivray C, Singh H, Sam J, Flett H, Craven BC, Verrier M, Musselman K, Koh RGL, Garai P, Zariffa J, Unger J, Oates AR, Arora T, Musselman K, Moshe B, Anthony B, Gulasingam S, Craven BC, Michalovic E, Gainforth HL, Baron J, Graham ID, Sweet SN, Chan B, Craven BC, Wodchis W, Cadarette S, Krahn M, Mittmann N, Chemtob K, Rocchi MA, Arbour-Nicitopoulos K, Kairy D, Sweet SN, Sabetian P, Koh RGL, Zariffa J, Yoo P, Iwasa SN, Babona-Pilipos R, Schneider P, Velayudhan P, Ahmed U, Popovic MR, Morshead CM, Yoo J, Shinya M, Milosevic M, Masani K, Gabison S, Mathur S, Nussbaum E, Popovic M, Verrier MC, Musselman K, Lemay JF, McCullum S, Guy K, Walden K, Zariffa J, Kalsi-Ryan S, Alizadeh-Meghrazi M, Lee J, Milligan J, Smith M, Athanasopoulos P, Jeji T, Howcroft J, Howcroft J, Townson A, Willms R, Plashkes T, Mills S, Flett H, Scovil C, Mazzella F, Morris H, Ventre A, Loh E, Guy S, Kramer J, Jeji T, Xia N, Mehta S, Martin Ginis KA, McBride CB, Shaw RB, West C, Ethans K, O'Connell C, Charlifue S, Gagnon DH, Escalona Castillo MJ, Vermette M, Carvalho LP, Karelis A, Kairy D, Aubertin-Leheudre M, Duclos C, Houghton PE, Orr L, Holyoke P, Kras-Dupuis A, Wolfe D, Munro B, Sweeny M, Craven BC, Flett H, Hitzig S, Farahani F, Alavinia SM, Omidvar M, Bayley M, Sweet SN, Gassaway J, Shaw R, Hong M, Everhart-Skeels S, Houlihan B, Burns A, Bilsky G, Lanig I, Graco M, Cross S, Thiyagarajan C, Shafazand S, Ayas N, Schembri R, Booker L, Nicholls C, Burns P, Nash M, Green S, Berlowitz D, Furlan JC, Kalsi-Ryan S

PMID: 29034821 [PubMed - in process]



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Emerging and evolving concepts in gene essentiality.

Related Articles

Emerging and evolving concepts in gene essentiality.

Nat Rev Genet. 2017 Oct 16;:

Authors: Rancati G, Moffat J, Typas A, Pavelka N

Abstract
Gene essentiality is a founding concept of genetics with important implications in both fundamental and applied research. Multiple screens have been performed over the years in bacteria, yeasts, animals and more recently in human cells to identify essential genes. A mounting body of evidence suggests that gene essentiality, rather than being a static and binary property, is both context dependent and evolvable in all kingdoms of life. This concept of a non-absolute nature of gene essentiality changes our fundamental understanding of essential biological processes and could directly affect future treatment strategies for cancer and infectious diseases.

PMID: 29033457 [PubMed - as supplied by publisher]



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Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition.

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition.

Elife. 2017 Oct 13;6:

Authors: Mahadevan V, Khademullah CS, Dargaei Z, Chevrier J, Uvarov P, Kwan J, Bagshaw RD, Pawson T, Emili A, De Koninck Y, Anggono V, Airaksinen M, Woodin MA

Abstract
KCC2 is a neuron-specific K(+)-Cl(-) cotransporter essential for establishing the Cl(-) gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with diverse proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons increases KCC2 expression and hyperpolarizes the reversal potential for Cl(-). Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2.

PMID: 29028184 [PubMed - in process]



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The Anti-CRISPR Story: A Battle for Survival.

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The Anti-CRISPR Story: A Battle for Survival.

Mol Cell. 2017 Oct 05;68(1):8-14

Authors: Maxwell KL

Abstract
The last decade has seen the fields of molecular biology and genetics transformed by the development of CRISPR-based gene editing technologies. These technologies were derived from bacterial defense systems that protect against viral invasion. Elegant studies focused on the evolutionary battle between CRISPR-encoding bacteria and the viruses that infect and kill them revealed the next step in this arms race, the anti-CRISPR proteins. Investigation of these proteins has provided important new insight into how CRISPR-Cas systems work and how bacterial genomes evolve. They have also led to the development of important biotechnological tools that can be used for genetic engineering, including off switches for CRISPR-Cas9 genome editing in human cells.

PMID: 28985512 [PubMed - indexed for MEDLINE]



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A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family.

A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family.

Protein Eng Des Sel. 2017 Aug 30;:1-8

Authors: Miersch S, Kuruganti S, Walter MR, Sidhu SS

Abstract
The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease. Using phage-displayed synthetic antibody libraries, we have conducted selections against 12 human IFNα subtypes to explore our ability to obtain fine-specificity antibodies that recognize and antagonize the biological signals induced by a single IFNα subtype. For the first time, we have isolated antibodies that specifically recognize individual IFNα subtypes (IFNα2a/b, IFNα6, IFNα8b and IFNα16) with high affinity that antagonize signaling. Our results show that highly specific antibodies capable of distinguishing between closely related cytokines can be isolated from synthetic libraries and can be used to characterize cytokine abundance and function.

PMID: 28981904 [PubMed - as supplied by publisher]



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A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis.

A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis.

Nat Nanotechnol. 2017 Oct 02;:

Authors: Ghosh E, Srivastava A, Baidya M, Kumari P, Dwivedi H, Nidhi K, Ranjan R, Dogra S, Koide A, Yadav PN, Sidhu SS, Koide S, Shukla AK

Abstract
Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.

PMID: 28967893 [PubMed - as supplied by publisher]



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Pathway-based discovery of genetic interactions in breast cancer.

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Pathway-based discovery of genetic interactions in breast cancer.

PLoS Genet. 2017 Sep;13(9):e1006973

Authors: Wang W, Xu ZZ, Costanzo M, Boone C, Lange CA, Myers CL

Abstract
Breast cancer is the second largest cause of cancer death among U.S. women and the leading cause of cancer death among women worldwide. Genome-wide association studies (GWAS) have identified several genetic variants associated with susceptibility to breast cancer, but these still explain less than half of the estimated genetic contribution to the disease. Combinations of variants (i.e. genetic interactions) may play an important role in breast cancer susceptibility. However, due to a lack of statistical power, the current tests for genetic interactions from GWAS data mainly leverage prior knowledge to focus on small sets of genes or SNPs that are known to have an association with breast cancer. Thus, many genetic interactions, particularly among novel variants, remain understudied. Reverse-genetic interaction screens in model organisms have shown that genetic interactions frequently cluster into highly structured motifs, where members of the same pathway share similar patterns of genetic interactions. Based on this key observation, we recently developed a method called BridGE to search for such structured motifs in genetic networks derived from GWAS studies and identify pathway-level genetic interactions in human populations. We applied BridGE to six independent breast cancer cohorts and identified significant pathway-level interactions in five cohorts. Joint analysis across all five cohorts revealed a high confidence consensus set of genetic interactions with support in multiple cohorts. The discovered interactions implicated the glutathione conjugation, vitamin D receptor, purine metabolism, mitotic prometaphase, and steroid hormone biosynthesis pathways as major modifiers of breast cancer risk. Notably, while many of the pathways identified by BridGE show clear relevance to breast cancer, variants in these pathways had not been previously discovered by traditional single variant association tests, or single pathway enrichment analysis that does not consider SNP-SNP interactions.

PMID: 28957314 [PubMed - indexed for MEDLINE]



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