An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Hematopoietic stem and progenitor cells confer cross-protective trained immunity in mouse models.

Recent studies suggest that infection reprograms hematopoietic stem and progenitor cells (HSPCs) to enhance innate immune responses upon secondary infectious challenge, a process called "trained immunity." However, the specificity and cell types responsible for this response remain poorly defined. We established a model of trained immunity in mice in response to Mycobacterium avium infection. scRNA-seq analysis revealed that HSPCs activate interferon gamma-response genes heterogeneously upon primary challenge, while rare cell populations expand. Macrophages derived from trained HSPCs demonstrated enhanced bacterial killing and metabolism, and a single dose of recombinant interferon gamma exposure was sufficient to induce similar training. Mice transplanted with influenza-trained HSPCs displayed enhanced immunity against M. avium challenge and vice versa, demonstrating cross protection against antigenically distinct pathogens. Together, these results indicate that heterogeneous responses to infection by HSPCs can lead to long-term production of bone marrow derived macrophages with enhanced function and confer cross-protection against alternative pathogens.

Asp263 missense variants perturb the active site of human phosphoglucomutase 1.

The enzyme phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis. Clinical studies have identified mutations in human PGM1 as the cause of PGM1 deficiency, an inherited metabolic disease. One residue, Asp263, has two known variants associated with disease: D263G and D263Y. Biochemical studies have shown that these mutants are soluble and well folded, but have significant catalytic impairment. To better understand this catalytic defect, we determined crystal structures of these two missense variants, both of which reveal a similar and indirect structural change due to the loss of a conserved salt bridge between Asp263 and Arg293. The arginine reorients into the active site, making interactions with residues responsible for substrate binding. Biochemical studies also show that the catalytic phosphoserine of the missense variants is more stable to hydrolysis relative to wild-type enzyme. The structural perturbation resulting from mutation of this single amino acid reveals the molecular mechanism underlying PGM1 deficiency in these missense variants. DATABASE: Structural data are available in the PDB under the accession numbers 5JN5 and 5TR2.

Induced Structural Disorder as a Molecular Mechanism for Enzyme Dysfunction in Phosphoglucomutase 1 Deficiency.

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, mediating the switch between glycolysis and gluconeogenesis through the conversion of glucose 1-phosphate and glucose 6-phosphate. Recent clinical studies have identified mutations in this enzyme as the cause of PGM1 deficiency, an inborn error of metabolism classified as both a glycogen storage disease and a congenital disorder of glycosylation. Reported here are the first crystal structures of two disease-related missense variants of PGM1, along with the structure of the wild-type enzyme. Two independent glycine-to-arginine substitutions (G121R and G291R), both affecting key active site loops of PGM1, are found to induce regions of structural disorder, as evidenced by a nearly complete loss of electron density for as many as 23 aa. The disordered regions are not contiguous in sequence to the site of mutation, and even cross domain boundaries. Other structural rearrangements include changes in the conformations of loops and side chains, some of which occur nearly 20 Šaway from the site of mutation. The induced structural disorder is correlated with increased sensitivity to proteolysis and lower-resolution diffraction, particularly for the G291R variant. Examination of the multi-domain effects of these G➔R mutations establishes a correlation between interdomain interfaces of the enzyme and missense variants of PGM1 associated with disease. These crystal structures provide the first insights into the structural basis of enzyme dysfunction in PGM1 deficiency and highlight a growing role for biophysical characterization of proteins in the field of precision medicine.

Compromised catalysis and potential folding defects in in vitro studies of missense mutants associated with hereditary phosphoglucomutase 1 deficiency.

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. Affected patients show multiple disease phenotypes, including dilated cardiomyopathy, exercise intolerance, and hepatopathy, reflecting the central role of the enzyme in glucose metabolism. We present here the first in vitro biochemical characterization of 13 missense mutations involved in PGM1 deficiency. The biochemical phenotypes of the PGM1 mutants cluster into two groups: those with compromised catalysis and those with possible folding defects. Relative to the recombinant wild-type enzyme, certain missense mutants show greatly decreased expression of soluble protein and/or increased aggregation. In contrast, other missense variants are well behaved in solution, but show dramatic reductions in enzyme activity, with kcat/Km often <1.5% of wild-type. Modest changes in protein conformation and flexibility are also apparent in some of the catalytically impaired variants. In the case of the G291R mutant, severely compromised activity is linked to the inability of a key active site serine to be phosphorylated, a prerequisite for catalysis. Our results complement previous in vivo studies, which suggest that both protein misfolding and catalytic impairment may play a role in PGM1 deficiency.