The importance of the role of iron regulatory proteins (IRPs) in mitochondrial iron homeostasis and function has been raised. To understand how an IRP affects mitochondrial function, we used globally Irp2-depleted mouse embryonic fibroblasts (MEFs) and found that Irp2 ablation significantly induced the expression of both hypoxia-inducible factor subunits, Hif1α and Hif2α. The increase of Hif1α up-regulated its targeted genes, enhancing glycolysis, and the increase of Hif2α down-regulated the expression of iron–sulfur cluster (Fe–S) biogenesis-related and electron transport chain (ETC)-related genes, weakening mitochondrial respiration. Inhibition of Hif1α by genetic knockdown or a specific inhibitor prevented Hif1α-targeted gene expression, leading to decreased aerobic glycolysis. Inhibition of Hif2α by genetic knockdown or selective disruption of the heterodimerization of Hif2α and Hif1β restored the mitochondrial ETC and coupled oxidative phosphorylation (OXPHOS) by enhancing Fe–S biogenesis and increasing ETC-related gene expression. Our results indicate that Irp2 modulates the metabolic switch from aerobic glycolysis to OXPHOS that is mediated by Hif1α and Hif2α in MEFs.

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Signal regulatory proteins (SIRPs) comprise a family of cell surface signaling receptors differentially expressed in leukocytes and the central nervous system. Although the extracellular domains of SIRPs are highly similar, classical motifs in the cytoplasmic or transmembrane domains distinguish them as either activating (␤) or inhibitory (␣) isoforms. We reported previously that human neutrophils (polymorphonuclear leukocytes (PMN)) express multiple SIRP isoforms and that SIRP␣ binding to its ligand CD47 regulates PMN transmigration. Here we further characterized the expression of PMN SIRPs, and we reported that the major SIRP␣ and SIRP␤ isoforms expressed in PMN include Bit/PTPNS-1 and SIRP␤1, respectively. Furthermore, although SIRP␣ (Bit/PTPNS-1) is expressed as a monomer, we showed that SIRP␤1 is expressed on the cell surface as a disulfide-linked homodimer with bond formation mediated by Cys-320 in the membrane-proximal Ig loop. Subcellular fractionation studies revealed a major pool of SIRP␤1 within the plasma membrane fractions of PMN. In contrast, the majority of SIRP␣ (Bit/PTPNS-1) is present in fractions enriched in secondary granules and is translocated to the cell surface after chemoattractant (formylmethionylleucylphenylalanine) stimulation. Functional studies revealed that antibody-mediated ligation of SIRP␤1 enhanced formylmethionylleucylphenylalanine-driven PMN transepithelial migration. Co-immunoprecipitation experiments to identify associated adaptor proteins revealed a 10 -12-kDa protein associated with SIRP␤1 that was tyrosine-phosphorylated after PMN stimulation and is not DAP10/12 or Fc receptor ␥ chain. These results provide new insights into the structure and function of SIRPs in leukocytes and their potential role(s) in fine-tuning responses to inflammatory stimuli. Signal regulatory proteins (SIRPs)3 are a family of transmembrane receptor-like signaling proteins that are abundantly expressed in hematopoietic cells, including granulocytes, monocytes, dendritic cells, and lymphocytes (1-3). In addition, SIRPs are expressed in neuronal cells (4 -6) and certain types of cancer cells (7-10). SIRPs can be divided into two subfamilies, SIRP␣ and SIRP␤, based on the putative structures of their C-terminal intracellular domains (11). SIRPs share typical immunoglobulin superfamily structures with an N-terminal extracellular domain containing three cysteine-bound Ig-like loops, a single membrane-spanning transmembrane domain, and a C-terminal intracellular domain (11). The C-terminal intracellular domains of the SIRP␣ subfamily contain a relatively long amino acid sequence (110 amino acids for SIRP␣1) that includes four tyrosine residues to form two immunoreceptor tyrosine-based inhibition motifs (ITIM). Conversely, SIRP␤ subfamily members have a short intracellular domain containing only a few amino acids (4 amino acids for SIRP␤1). Despite a short cytoplasmic tail, SIRP␤1 contains a positively charged lysine in the transmembrane domain that can mediate interactions with an immunoreceptor tyrosi...

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.

Human mast cell tryptases vary strikingly in secretion, catalytic competence, and inheritance. To explore the basis of variation, we compared genes from a range of primates, including humans, great apes (chimpanzee, gorilla, orangutan), Old- and New-World monkeys (macaque and marmoset), and a prosimian (galago), tracking key changes. Our analysis reveals that extant soluble tryptase-like proteins, including α- and β-like tryptases, mastins, and implantation serine proteases, likely evolved from membrane-anchored ancestors because their more deeply rooted relatives (γ tryptases, pancreasins, prostasins) are type I transmembrane peptidases. Function-altering mutations appeared at widely separated times during primate speciation, with tryptases evolving by duplication, gene conversion, and point mutation. The α-tryptase Gly216Asp catalytic domain mutation, which diminishes activity, is present in macaque tryptases, and thus arose before great apes and Old World monkeys shared an ancestor, and before the αβ split. However, the Arg−3Gln processing mutation appeared recently, affecting only human α. By comparison, the transmembrane γ-tryptase gene, which anchors the telomeric end of the multigene tryptase locus, changed little during primate evolution. Related transmembrane peptidase genes were found in reptiles, amphibians, and fish. We identified soluble tryptase-like genes in the full spectrum of mammals, including marsupial (opossum) and monotreme (platypus), but not in nonmammalian vertebrates. Overall, our analysis suggests that soluble tryptases evolved rapidly from membrane-anchored, two-chain peptidases in ancestral vertebrates into soluble, single-chain, self-compartmentalizing, inhibitor-resistant oligomers expressed primarily by mast cells, and that much of present numerical, behavioral, and genetic diversity of α- and β-like tryptases was acquired during primate evolution.