LIVIVO - The Search Portal for Life Sciences

zur deutschen Oberfläche wechseln
Advanced search

Search results

Result 1 - 3 of total 3

Search options

  1. Article ; Online: Isolating and Analyzing Protein Containing Granules from Cells.

    Victor, Rachel A / Thompson, Valery F / Schwartz, Jacob C

    Current protocols

    2021  Volume 1, Issue 3, Page(s) e35

    Abstract: Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to ...

    Abstract Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to separate condensates from cells using chemical cross-linking followed by size-exclusion chromatography (SEC). After SEC fractionation, the samples can be characterized by a variety of approaches including enzyme-linked immunosorbent assay, dynamic light scattering, electron microscopy, and mass spectrometry. The protocol described here has been optimized for cultured mammalian cells and E. coli expressing recombinant proteins. Since the lysates are fractionated by size, this protocol can be modified to study other large protein assemblies, including the nuclear pore complex, and for other tissues or organisms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: SEC separation of cross-linked mammalian cell lysates Alternate Protocol: Preparation of non-crosslinked mammalian cells Basic Protocol 2: SEC separation of E. coli lysate Support Protocol 1: Detecting protein of interest by ELISA Support Protocol 2: TCA precipitation of SEC fractions.
    MeSH term(s) Animals ; Chromatography, Gel ; Dynamic Light Scattering ; Escherichia coli ; Mass Spectrometry ; Proteins
    Chemical Substances Proteins
    Language English
    Publishing date 2021-03-18
    Publishing country United States
    Document type Journal Article
    ISSN 2691-1299
    ISSN (online) 2691-1299
    DOI 10.1002/cpz1.35
    Database MEDical Literature Analysis and Retrieval System OnLINE

    More links

    Kategorien

    Order via subito

    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

    Inter-library loan at ZB MED

    Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

  2. Article ; Online: Fused in sarcoma silences HIV gene transcription and maintains viral latency through suppressing AFF4 gene activation.

    Krasnopolsky, Simona / Marom, Lital / Victor, Rachel A / Kuzmina, Alona / Schwartz, Jacob C / Fujinaga, Koh / Taube, Ran

    Retrovirology

    2019  Volume 16, Issue 1, Page(s) 16

    Abstract: Background: The human immunodeficiency virus (HIV) cell reservoir is currently a main obstacle towards complete eradication of the virus. This infected pool is refractory to anti-viral therapy and harbors integrated proviruses that are transcriptionally ...

    Abstract Background: The human immunodeficiency virus (HIV) cell reservoir is currently a main obstacle towards complete eradication of the virus. This infected pool is refractory to anti-viral therapy and harbors integrated proviruses that are transcriptionally repressed but replication competent. As transcription silencing is key for establishing the HIV reservoir, significant efforts have been made to understand the mechanism that regulate HIV gene transcription, and the role of the elongation machinery in promoting this step. However, while the role of the super elongation complex (SEC) in enhancing transcription activation of HIV is well established, the function of SEC in modulating viral latency is less defined and its cell partners are yet to be identified.
    Results: In this study we identify fused in sarcoma (FUS) as a partner of AFF4 in cells. FUS inhibits the activation of HIV transcription by AFF4 and ELL2, and silences overall HIV gene transcription. Concordantly, depletion of FUS elevates the occupancy of AFF4 and Cdk9 on the viral promoter and activates HIV gene transcription. Live cell imaging demonstrates that FUS co-localizes with AFF4 within nuclear punctuated condensates, which are disrupted upon treating cells with aliphatic alcohol. In HIV infected cells, knockout of FUS delays the gradual entry of HIV into latency, and similarly promotes viral activation in a T cell latency model that is treated with JQ1. Finally, effects of FUS on HIV gene transcription are also exhibited genome wide, where FUS mainly occupies gene promoters at transcription starting sites, while its knockdown leads to an increase in AFF4 and Cdk9 occupancy on gene promoters of FUS affected genes.
    Conclusions: Towards eliminating the HIV infected reservoir, understanding the mechanisms by which the virus persists in the face of therapy is important. Our observations show that FUS regulates both HIV and global gene transcription and modulates viral latency, thus can potentially serve as a target for future therapy that sets to reactivate HIV from its latent state.
    MeSH term(s) Cyclin-Dependent Kinase 9 ; Disease Reservoirs/virology ; Gene Silencing ; HEK293 Cells ; HIV Infections/virology ; HIV-1/genetics ; HIV-1/physiology ; Humans ; Jurkat Cells ; Promoter Regions, Genetic ; Proviruses/genetics ; RNA-Binding Protein FUS/metabolism ; T-Lymphocytes/virology ; Transcription, Genetic ; Transcriptional Elongation Factors/genetics ; Virus Activation ; Virus Latency/genetics
    Chemical Substances AFF4 protein, human ; ELL2 protein, human ; FUS protein, human ; RNA-Binding Protein FUS ; Transcriptional Elongation Factors ; CDK9 protein, human (EC 2.7.11.22) ; Cyclin-Dependent Kinase 9 (EC 2.7.11.22)
    Language English
    Publishing date 2019-06-25
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2142602-8
    ISSN 1742-4690 ; 1742-4690
    ISSN (online) 1742-4690
    ISSN 1742-4690
    DOI 10.1186/s12977-019-0478-x
    Database MEDical Literature Analysis and Retrieval System OnLINE

    More links

    Kategorien

    Order via subito

    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

  3. Article ; Online: Transcription-Dependent Formation of Nuclear Granules Containing FUS and RNA Pol II.

    Thompson, Valery F / Victor, Rachel A / Morera, Andres A / Moinpour, Mahta / Liu, Meilani N / Kisiel, Conner C / Pickrel, Kaitlyn / Springhower, Charis E / Schwartz, Jacob C

    Biochemistry

    2018  Volume 57, Issue 51, Page(s) 7021–7032

    Abstract: Purified recombinant FUsed in Sarcoma (FUS) assembles into an oligomeric state in an RNA-dependent manner to form large condensates. FUS condensates bind and concentrate the C-terminal domain of RNA polymerase II (RNA Pol II). We asked whether a granule ... ...

    Abstract Purified recombinant FUsed in Sarcoma (FUS) assembles into an oligomeric state in an RNA-dependent manner to form large condensates. FUS condensates bind and concentrate the C-terminal domain of RNA polymerase II (RNA Pol II). We asked whether a granule in cells contained FUS and RNA Pol II as suggested by the binding of FUS condensates to the polymerase. We developed cross-linking protocols to recover protein particles containing FUS from cells and separated them by size exclusion chromatography. We found a significant fraction of RNA Pol II in large granules containing FUS with diameters of >50 nm or twice that of the RNA Pol II holoenzyme. Inhibition of transcription prevented the polymerase from associating with the granules. Altogether, we found physical evidence of granules containing FUS and RNA Pol II in cells that possess properties comparable to those of in vitro FUS condensates.
    MeSH term(s) Cell Nucleus/metabolism ; Cell Nucleus/ultrastructure ; Cross-Linking Reagents ; HEK293 Cells ; Humans ; Microscopy, Electron, Transmission ; Models, Biological ; Particle Size ; Protein Interaction Domains and Motifs ; RNA Polymerase II/chemistry ; RNA Polymerase II/genetics ; RNA Polymerase II/metabolism ; RNA-Binding Protein FUS/chemistry ; RNA-Binding Protein FUS/genetics ; RNA-Binding Protein FUS/metabolism ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Transcription, Genetic
    Chemical Substances Cross-Linking Reagents ; FUS protein, human ; RNA-Binding Protein FUS ; Recombinant Proteins ; RNA Polymerase II (EC 2.7.7.-)
    Language English
    Publishing date 2018-12-11
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/acs.biochem.8b01097
    Database MEDical Literature Analysis and Retrieval System OnLINE

    More links

    Kategorien

    In stock of ZB MED Cologne/Königswinter

    Zs.A 217: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
    ab Jg. 2022: Lesesaal (EG)

    Order via subito

    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

To top