RESUMEN
Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.
RESUMEN
Streptococcus pneumoniae (Spn) strains cause pneumonia that kills millions every year worldwide. Spn produces Ply, a hemolysin that lyses erythrocytes releasing hemoglobin, and also produces the pro-oxidant hydrogen peroxide (Spn-H2O2) during growth. The hallmark of the pathophysiology of hemolytic diseases is the oxidation of hemoglobin, but oxidative reactions catalyzed by Spn-H2O2 have been poorly studied. We characterized the oxidation of hemoglobin by Spn-H2O2. We prepared a series of single-mutant (ΔspxB or ΔlctO), double-mutant (ΔspxB ΔlctO), and complemented strains in TIGR4, D39, and EF3030. We then utilized an in vitro model with oxyhemoglobin to demonstrate that oxyhemoglobin was oxidized rapidly, within 30 min of incubation, by Spn-H2O2 to methemoglobin and that the main source of Spn-H2O2 was pyruvate oxidase (SpxB). Moreover, extended incubation caused the release and the degradation of heme. We then assessed oxidation of hemoglobin and heme degradation by other bacterial inhabitants of the respiratory tract. All hydrogen peroxide-producing streptococci tested caused the oxidation of hemoglobin and heme degradation, whereas bacterial species that produce <1 µM H2O2 neither oxidized hemoglobin nor degraded heme. An ex vivo bacteremia model confirmed that oxidation of hemoglobin and heme degradation occurred concurrently with hemoglobin that was released from erythrocytes by Ply. Finally, gene expression studies demonstrated that heme, but not red blood cells or hemoglobin, induced upregulated transcription of the spxB gene. Oxidation of hemoglobin may be important for pathogenesis and for the symbiosis of hydrogen peroxide-producing bacteria with other species by providing nutrients such as iron.
