Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions.

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation

Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains

Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified

Meprinα transactivates the epidermal growth factor receptor (EGFR) via ligand shedding, thereby enhancing colorectal cancer cell proliferation and migration

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression

CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications

Correction: Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses.

[This corrects the article DOI: 10.1371/journal.ppat.1006892.]

Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na⁺/K⁺ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

<div><p>Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na⁺/K⁺ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.</p></div

Effect of knock-down of genes identified LC-MS/MS analysis on LCMV multiplication.

<p><b>(A)</b> Effect of siRNA-mediated knockdown expression of genes identified by proteomics analysis. A549 cells (1,000 cells/well) in a 384-well plate were reverse transfected with siRNA pools targeting each indicated gene. At 72 h post-transfection, cells were infected (MOI = 0.05) with rLCMV/ZsG. At 48 h pi, cells were fixed and stained with DAPI. ZsGreen and DAPI signals were measured by a fluorescence plate reader. ZsGreen signal was normalized to DAPI signal. Normalized values from cells transfected with the control (NC, black bar) non-specific siRNA was set to 100%. A panel of 154 siRNAs including NC siRNA was evaluated three independent times. Results from three independent experiments were sorted by mean values and assigned to three graphs with SD. Each graph includes identical NC results (black bar) for reference. <b>(B)</b> Reduction of protein expression of ATP1A1 and PHB by siRNA-mediated gene knockdown. A549 cells (3.0 x 10⁴ cells/well) were reverse transfected in a 24-well plate with siRNA pools against either ATP1A1 or PHB or with NC siRNA. At 72 h post-transfection, total cell lysate was prepared, and expression of ATP1A1 and PHB in cell lysates were determined by western blots. <b>(C)</b> Effect of siRNA-mediated kd of ATP1A1 on production of infectious LCMV progeny. A549 cells (1.5 x 10⁴ cells/well; 48-well plate) were reverse-transfected with siRNAs against ATP1A1 or with NC siRNA. 72 h later, cells were infected (MOI = 0.01) with rLCMV/ZsG. At 24 h and 48 h pi, TCSs were collected. At 48 h pi, cells were fixed and ZsGreen expression examined by fluorescence microscopy (i). Bar, 200 μm. Virus titers in TCSs were determined by IFFA (ii). Data represent means ± SD of results from three independent experiments. LoD, limit of detection.</p

Synergistic antiviral effect of ouabain and rocaglamide on rLCMV/eGFP multiplication.

<p>A549 cells seeded (2.0 x 10⁴ cells/well) in 96-well plates and cultured overnight were treated with combinations of ouabain and Roc-A at indicated concentrations for 2 h and then infected (MOI = 0.01) with rLCMV/eGFP. Compounds were present throughout the end of experiment. At 48 h pi, cells were fixed and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader. eGFP signal was normalized to DAPI signal, and the normalized data were used to analyze synergistic effect by MacSynergy II software. Data represent % synergy (% inhibition over the expected [additive effect]) at the 95% confidence interval from five independent experiments.</p

LC-MS/MS analysis of NP-binding proteins.

<p><b>(A)</b> Flow chart of the experimental approach to identify NP-interacting host-cell proteins in LCMV-infected cells. A549 cells prepared in six 15-cm dishes (total of 1.0 x 10⁸ cells) were infected (MOI = 0.1) with either rLCMV/Strep-NP or r3LCMV/eGFP. At 48 h pi, total cell lysates were prepared, and NP- or eGFP-interacting proteins were pulled down (PD) using Streptactin-coated sepharose resin. Protein complexes bound to the resin were eluted using 2.5 mM of desthiobiotin. Eluates were precipitated using TCA followed by trypsin digestion. Tryptic peptides were subjected to LC-MS/MS analysis. <b>(B)</b> Detection of proteins present in PD samples. Protein complexes present in PD samples were separated by SDS-PAGE and visualized by SYPRO staining. Some protein bands present only in the Strep-NP PD sample are indicated by asterisks. <b>(C)</b> Venn diagram of the NP- and eGFP-interacting proteins identified by LC-MS/MS analysis. <b>(D, E)</b> Gene Onthology (GO) analysis of the NP-interacting proteins identified by LC-MS/MS. Bioinformatic analysis by PANTHER was performed showing the number of genes of identified NP-interacting proteins classified by biological process <b>(D)</b> and protein class <b>(E)</b>.</p

ATP1A1 and PHB are involved in different steps of the mammarenavirus life cycle.

<p><b>(A)</b> Influence of time of addition of ouabain (OUA) or rocaglamide (Roc-A) on virus multiplication. A549 cells seeded (2.5 x 10⁵ cells/well) in a 12-well plate and cultured overnight were infected (moi = 0.1) with rLCMV/eGFP or remained uninfected (mock). OUA (10 nM), Roc-A (100 nM), or DMSO (0.01%) was added to the culture media at the indicated time points and remained present throughout the end of the experiment. Ammonium chloride (20 mM) was added to culture medium at 4 h pi to prevent multiple rounds of virus infection. At 24 h pi, eGFP expression in infected cells was examined by flow cytometry. Data represent mean ± SD of the results of three independent experiments. <b>(B)</b> Effect of ouabain and Roc-A on LCMV replication. A549 cells seeded (1.25 x 10⁵ cells/well) in 24-well plates and cultured overnight were infected (MOI = 1) with rLCMVΔGPC/eGFP, followed by addition of the indicated concentrations of ouabain or Roc-A. At 72 h pi, total cell lysates were prepared, and eGFP expression levels were measured using a fluorescent plate reader. Data represent mean ± SD of three independent experiments. <b>(C-E)</b> Effect of ouabain and Roc-A on Z-mediated budding. Cells (HEK 293T) seeded (3.5 x 10⁵ cells/well) in a 12-well plate and cultured overnight were transfected with 0.5 μg of either pC-Empty or pC-LCMV-Z-Strep (LCMV-Z-Strep) <b>(C)</b> or pC-LASV-Z-FLAG (LASV-Z-FLAG) <b>(D, E)</b>. At 24 h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag <b>(C)</b> and FLAG-tag <b>(D)</b>. Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel <b>C</b> correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel <b>E</b> correspond to the average and SD from four independent experiments including the one shown in panel <b>D</b>. The mean budding efficiency of DMSO treated-samples was set to 100%. Data represent mean ± SD of four independent experiments. <b>(F)</b> Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded (4.0 x 10⁵ cells/well) in a 12-well plate and cultured overnight were infected (MOI = 0.1) with scrLCMV/ZsG (1^st infection) for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM (OUA). At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells (2^nd infection) seeded (4.0 x 10⁵ cells/well) in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate (2^nd) by that in 293T cell lysate (1^st). The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. <b>(G)</b> Effect of ouabain on the late stage of LCMV infection. A549 cells seeded (1.25 x 10⁵ cells/well) and cultured overnight were infected (MOI = 0.1) with rLCMV/eGFP. At 48 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh medium in the presence of ouabain (OUA) at indicated concentrations. At 72 h pi, TCS was collected and virus titers were determined by IFFA. Data represent means ± SD from three independent experiments.</p