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  1. Article ; Online: ACE2 protein expression within isogenic cell lines is heterogeneous and associated with distinct transcriptomes

    Emily J. Sherman / Brian T. Emmer

    Scientific Reports, Vol 11, Iss 1, Pp 1-

    2021  Volume 13

    Abstract: Abstract The membrane protein angiotensin-converting enzyme 2 (ACE2) is a physiologic regulator of the renin-angiotensin system and the cellular receptor for the SARS-CoV-2 virus. Prior studies of ACE2 expression have primarily focused on mRNA abundance, ...

    Abstract Abstract The membrane protein angiotensin-converting enzyme 2 (ACE2) is a physiologic regulator of the renin-angiotensin system and the cellular receptor for the SARS-CoV-2 virus. Prior studies of ACE2 expression have primarily focused on mRNA abundance, with investigation at the protein level limited by uncertain specificity of commercial ACE2 antibodies. Here, we report our development of a sensitive and specific flow cytometry-based assay for cellular ACE2 protein abundance. Application of this approach to multiple cell lines revealed an unexpected degree of cellular heterogeneity, with detectable ACE2 protein in only a subset of cells in each isogenic population. This heterogeneity was mediated at the mRNA level by transcripts predominantly initiated from the ACE2 proximal promoter. ACE2 expression was heritable but not fixed over multiple generations of daughter cells, with gradual drift toward the original heterogeneous background. RNA-seq profiling identified distinct transcriptomes of ACE2-expressing relative cells to non-expressing cells, with enrichment in functionally related genes and transcription factor target sets. Our findings provide a validated approach for the specific detection of ACE2 protein at the surface of single cells, support an epigenetic mechanism of ACE2 gene regulation, and identify specific pathways associated with ACE2 expression in HuH7 cells.
    Keywords Medicine ; R ; Science ; Q
    Subject code 612
    Language English
    Publishing date 2021-08-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Murine Surf4 is essential for early embryonic development.

    Brian T Emmer / Paul J Lascuna / Vi T Tang / Emilee N Kotnik / Thomas L Saunders / Rami Khoriaty / David Ginsburg

    PLoS ONE, Vol 15, Iss 1, p e

    2020  Volume 0227450

    Abstract: Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor ...

    Abstract Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4 in vivo, we used CRISPR/Cas9-mediated gene editing to generate mice with germline loss-of-function mutations in Surf4. Heterozygous Surf4+/- mice exhibit grossly normal appearance, behavior, body weight, fecundity, and organ development, with no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol, and a detectable accumulation of intrahepatic apoliprotein B. Homozygous Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. In contrast to the milder murine phenotypes associated with deficiency of known SURF4 cargoes, the embryonic lethality of Surf4-/- mice implies the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570 ; 572
    Language English
    Publishing date 2020-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Identification of cell type specific ACE2 modifiers by CRISPR screening.

    Emily J Sherman / Carmen Mirabelli / Vi T Tang / Taslima G Khan / Kyle Leix / Andrew A Kennedy / Sarah E Graham / Cristen J Willer / Andrew W Tai / Jonathan Z Sexton / Christiane E Wobus / Brian T Emmer

    PLoS Pathogens, Vol 18, Iss 3, p e

    2022  Volume 1010377

    Abstract: SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 ... ...

    Abstract SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.
    Keywords Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2022-03-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: IgV somatic mutation of human anti–SARS-CoV-2 monoclonal antibodies governs neutralization and breadth of reactivity

    Mayara Garcia de Mattos Barbosa / Hui Liu / Daniel Huynh / Greg Shelley / Evan T. Keller / Brian T. Emmer / Emily Sherman / David Ginsburg / Andrew A. Kennedy / Andrew W. Tai / Christiane Wobus / Carmen Mirabeli / Thomas M. Lanigan / Milagros Samaniego / Wenzhao Meng / Aaron M. Rosenfeld / Eline T. Luning Prak / Jeffrey L. Platt / Marilia Cascalho

    JCI Insight, Vol 6, Iss

    2021  Volume 9

    Abstract: Abs that neutralize SARS-CoV-2 are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing Abs are especially sought. Here, we ...

    Abstract Abs that neutralize SARS-CoV-2 are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing Abs are especially sought. Here, we report a possibly novel approach to rapid generation of potent broadly neutralizing human anti–SARS-CoV-2 Abs. We isolated SARS-CoV-2 spike protein–specific memory B cells by panning from the blood of convalescent subjects after infection with SARS-CoV-2 and sequenced and expressed Ig genes from individual B cells as human mAbs. All of 43 human mAbs generated in this way neutralized SARS-CoV-2. Eighteen of the forty-three human mAbs exhibited half-maximal inhibitory concentrations (IC50) of 6.7 × 10–12 M to 6.7 × 10–15 M for spike-pseudotyped virus. Seven of the human mAbs also neutralized (with IC50 < 6.7 × 10–12 M) viruses pseudotyped with mutant spike proteins (including receptor-binding domain mutants and the S1 C-terminal D614G mutant). Neutralization of the Wuhan Hu-1 founder strain and of some variants decreased when coding sequences were reverted to germline, suggesting that potency of neutralization was acquired by somatic hypermutation and selection of B cells. These results indicate that infection with SARS-CoV-2 evokes high-affinity B cell responses, some products of which are broadly neutralizing and others highly strain specific. We also identify variants that would potentially resist immunity evoked by infection with the Wuhan Hu-1 founder strain or by vaccines developed with products of that strain, suggesting evolutionary courses that SARS-CoV-2 could take.
    Keywords COVID-19 ; Immunology ; Medicine ; R
    Subject code 572
    Language English
    Publishing date 2021-05-01T00:00:00Z
    Publisher American Society for Clinical investigation
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

    Brian T Emmer / Geoffrey G Hesketh / Emilee Kotnik / Vi T Tang / Paul J Lascuna / Jie Xiang / Anne-Claude Gingras / Xiao-Wei Chen / David Ginsburg

    eLife, Vol

    2018  Volume 7

    Abstract: PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, ...

    Abstract PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extracellular secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient cellular secretion of PCSK9.
    Keywords secretion ; cholesterol ; PCSK9 ; SURF4 ; cargo receptor ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 616
    Language English
    Publishing date 2018-09-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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