14 research outputs found

    Inhibition of HIV-1 Vif by Pokeweed Antiviral Protein and its Impact on Cellular Immune Defense

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    Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G (APOBEC3G) is a DNA editing enzyme. APOBEC3G hypermutates human immunodeficiency virus type 1 (HIV-1) DNA, resulting in the inhibition of viral propagation. The HIV-1 accessory protein viral infectivity factor (Vif) targets APOBEC3G for degradation. The objective of this work was to evaluate whether the inhibitory effect of Vif on A3G can be diminished by pokeweed antiviral protein (PAP). PAP is an N-glycosidase synthesized by the pokeweed plant, Phytolacca americana. In this study, I show that PAP reduced Vif protein accumulation by depurinating Vif open reading frame (ORF). Decreased Vif protein levels in the presence of PAP were correlated with increased A3G levels. The antiviral enzyme reduced viral particle release by approximately 100-fold and the virions released from PAP expressing cells were 11-fold less infectious. The expression of PAP also reduced the levels of integrated HIV-1 DNA

    Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells.

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    In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution

    miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells

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    SummaryTo investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance

    TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate

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    It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.ISSN:1934-5909ISSN:1875-977

    Dichotomous regulation of lysosomes by MYC and TFEB controls hematopoietic stem cell fate

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    It is critical to understand how quiescent long-term hematopoietic stem cells (LT-HSC) sense demand from daily and stress-mediated cues and transition into bioenergetically active progeny to differentiate and meet these cellular needs. Here, we show that lysosomes, which are sophisticated nutrient sensing and signaling centers, are dichotomously regulated by the Transcription Factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSC and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, which promotes quiescence, self-renewal and governs erythroid-myeloid commitment. By contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism to drive LT-HSC activation. Collectively, our study identifies lysosomes as a central regulatory hub for proper and coordinated stem cell fate determination
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