Arginine-rich peptides can penetrate cells and consequently be used as delivery agents in various cellular applications. The activity of these reagents is often context-dependent, and the parameters that impact cell entry are not fully understood, giving rise to variability and limiting progress toward their usage. Herein, we report that the cytosolic penetration of linear polyarginine peptides is dependent on the oxidation state of the cell. In particular, we find that hypoxia and cellular antioxidants inhibit cell penetration. In contrast, oxidants promote cytosolic cell entry with an efficiency proportional to the level of reactive oxygen species generated within membranes. Moreover, an antibody that binds to oxidized lipids inhibits cell penetration, whereas extracellularly administered pure oxidized lipids enhance peptide transport into cells. Overall, these data indicate that oxidized lipids are capable of mediating the transport of polyarginine peptides across membranes. These data may also explain variability in cell-penetrating peptide performance in different experimental conditions. These new findings therefore provide new opportunities for the rational design of future cell-permeable compounds and for the optimization of delivery protocols.

Poster Sessions 4TTHA0252 is a conserved hypothetical protein that belongs to the β-CASP family, within the metallo-β-lactamase superfamily. Archetypal metallo β-lactamases degrade β-lactam antibiotics, whereas the β-CASP family proteins degrade nucleic acids. Recently, it was reported that two β-CASP family proteins from Bacillus subtilis are functional homologues of E. coli RNase E. RNase E is a key enzyme for mRNA degradation in E. coli but no homolog is found in most bacteria. To reveal the biological role of this novel RNase family with a β-CASP fold in RNA metabolism, we currently address the structure and function of TTHA0252 from Thermus thermophilus HB8. We have determined the crystal structure of TTHA0252, which represents the first report of the tertiary structure of a β-CASP family protein (1). TTHA0252 comprises two separate domains: a metallo-β-lactamase domain and a clamp domain. The active site of the enzyme is located in a cleft between the two domains. The width of the cleft (10 Å) suggests that TTHA0252 can recognize a single-stranded region, but not a double-stranded region (diameter of 20 Å), of RNA as substrate. The active site of TTHA0252 comprises two zinc ions and seven conserved residues which are similar to those of other β-lactamases. A sulfate ion was also observed near the active site. Since the position of the sulfate ion, appears to mimic the 5'-terminal phosphate group of the substrate, we predicted TTHA0252 to have 5' to 3' exonuclease activity. TTHA0252 actually showed single-strand-specific 5' to 3' exonuclease activity to various oligonucleotides. The effects of mutations of active site residues are also discussed. (1) and AMP, and with Mg 2+ and a nonhydrolyzable ADPR analogue 2) . Although Ndx2 recognizes the AMP moiety in a manner similar to other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg 2+ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms 3) but different mechanisms of substrate recognition. 1) Yoshiba, S. et al. (2004) (atToc33) is a GTPase and a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein import into chloroplasts. By inference from the crystal structure of psToc34, a homologue in pea, the arginine at residue 130 (Arg130) has been implicated in formation of the atToc33 dimer an...

Acute lymphoblastic leukemia is the most common pediatric cancer and leading cause of cancer related mortality in pediatric populations. A key challenge to bridge better therapies to patients that fail conventional therapy are to understand their tumor landscape and aberrations in cell signaling, particularly in relation to normal hematopoietic development. To address this gap, we produced a unified reference map of pediatric T, B, and myeloid cell development from the HSPC using single cell RNA-seq and single-cell ATAC-seq on healthy pediatric bone marrow and thymus. We employed 6 different FACS sorting strategies in order to capture rare, but informative, progenitor cell states, including those of the CCR9+ CD34+ CD1A- CD4- CD8- early-T-cell precursor, CD34+ CD1A- CD4- CD8- pro-T cell, and CD34+ CD1A+ CD4- CD8- pre-T cell and Lin-CD34+CD38- multipotent, lymphoid, and myeloid progenitors from the bone marrow. We mapped leukemic cells from patients from 4 different subtypes of pediatric leukemia (T-ALL, ETP-ALL, MPAL, AML) to our healthy reference and found that our reference map can distinguish between subtle differences in transcriptome and epigenome that were undetectable using surface marker or canonical gene expression. Notably, using trajectories inferred from our healthy reference map, we discovered a large amount of inter-tumoral and intra-tumoral heterogeneity, with leukemic blasts from different patients and different populations within any one patient projecting to different cell states along normal development. Finally, we mapped engrafted leukemic cells from patient derived xenografts (PDX) back to our healthy reference. While we observed patient-specific transcriptomic shifts in engrafted versus primary leukemic blasts, we found that the overall transcriptomic hierarchy is maintained in the most PDX, with engrafted cells projecting to near-identical stages of arrest along our healthy hematopoietic trajectory. Interestingly, for PDX that projected to different areas in development compared to primary sample, we discovered alterations in expression of key transcription factors that regulate hematopoietic development. Our single cell multi-omic reference map of pediatric hematopoiesis serves as a valuable reference for mapping RNA-seq and ATAC-seq data back to nearest healthy precursors in normal hematopoietic development. On-going analysis is utilizing single cell transcriptomic, chromatin accessibility data from additional leukemic patients, including patients with B-ALL, to determined key genes and regulators that are altered in comparison to nearest healthy cell-types. In addition, population level signatures learned from healthy reference are being tested in bulk-transcriptomic ALL datasets. We are eager to present the results of these analyses at ASH. *CC and JX, as well as, DTT and KT contributed equally to this work Figure 1 Figure 1. Disclosures Teachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.