ABSTRACT
Genomic DNA sequencing technologies have been one of the great advances of the 21st century, having decreased in cost by seven orders of magnitude and opening up new fields of investigation throughout research and clinical medicine. Genomics coupled with biochemical investigation has allowed the molecular definition of a growing number of new genetic diseases that reveal new concepts of immune regulation. Also, defining the genetic pathogenesis of these diseases has led to improved diagnosis, prognosis, genetic counseling, and, most importantly, new therapies. We highlight the investigational journey from patient phenotype to treatment using the newly defined XMEN disease, caused by the genetic loss of the MAGT1 magnesium transporter, as an example. This disease illustrates how genomics yields new fundamental immunoregulatory insights as well as how research genomics is integrated into clinical immunology. At the end, we discuss two other recently described diseases, CHAI/LATAIE (CTLA-4 deficiency) and PASLI (PI3K dysregulation), as additional examples of the journey from unknown immunological diseases to new precision medicine treatments using genomics.
Subject(s)
CTLA-4 Antigen/genetics , Cation Transport Proteins/genetics , Genomics , Immune System Diseases/genetics , Mutation/genetics , Phosphatidylinositol 3-Kinases/genetics , X-Linked Combined Immunodeficiency Diseases/genetics , Animals , High-Throughput Nucleotide Sequencing , Humans , Immune System Diseases/therapy , Male , Molecular Targeted Therapy , X-Linked Combined Immunodeficiency Diseases/therapyABSTRACT
Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.
Subject(s)
Adaptive Immunity/physiology , Immunity, Innate/physiology , Ion Channels/metabolism , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Humans , Hypersensitivity/genetics , Hypersensitivity/immunology , Hypersensitivity/metabolism , Immunologic Deficiency Syndromes/drug therapy , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Immunologic Deficiency Syndromes/metabolism , Immunotherapy/methods , Ion Channels/genetics , Lymphocytes/cytology , Lymphocytes/immunology , Lymphocytes/metabolism , Mast Cells/immunology , Mast Cells/metabolism , Molecular Targeted Therapy , Mutation , Signal TransductionABSTRACT
The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.
Subject(s)
Carcinoma, Hepatocellular/immunology , Cation Transport Proteins/genetics , Inflammation/immunology , Liver Neoplasms/immunology , Membrane Glycoproteins/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Communication/genetics , Cell Communication/immunology , Cell Lineage/genetics , Cell Lineage/immunology , Dendritic Cells/immunology , Dendritic Cells/pathology , Gene Expression Regulation, Neoplastic , Humans , Inflammation/genetics , Inflammation/pathology , Leukocyte Common Antigens/immunology , Liver/immunology , Liver/pathology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocytes/immunology , Lymphocytes/pathology , Lysosomal Membrane Proteins/genetics , Macrophages/immunology , Macrophages/pathology , Myeloid Cells/immunology , Myeloid Cells/pathology , Neoplasm Proteins/genetics , Sequence Analysis, RNA , Single-Cell Analysis , Transcriptome/genetics , Transcriptome/immunology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.
Subject(s)
Agammaglobulinemia/immunology , B-Lymphocytes/immunology , Cation Transport Proteins/immunology , Zinc/immunology , Agammaglobulinemia/genetics , Agammaglobulinemia/metabolism , Animals , B-Lymphocytes/metabolism , Cation Transport Proteins/deficiency , Cation Transport Proteins/genetics , Child, Preschool , Cytosol/immunology , Cytosol/metabolism , Disease Models, Animal , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Female , Gene Expression Profiling , Humans , Infant , Male , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Pedigree , Zinc/metabolismABSTRACT
CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD â¼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (â¼ 10-25 Å) by hinge-like motions as large as â¼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.
Subject(s)
Bacterial Proteins/ultrastructure , Cation Transport Proteins/ultrastructure , Magnesium/metabolism , Thermotoga maritima/chemistry , Bacterial Proteins/chemistry , Cation Transport Proteins/chemistry , Cryoelectron Microscopy , Models, Molecular , Molecular Dynamics SimulationABSTRACT
Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.
Subject(s)
Cation Transport Proteins/chemistry , Multiprotein Complexes/chemistry , Sorting Nexins/chemistry , Vesicular Transport Proteins/chemistry , Amino Acid Sequence , Cation Transport Proteins/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Scattering, Small Angle , Sorting Nexins/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
Ligand binding usually moves the target protein from an ensemble of inactive states to a well-defined active conformation. Matthies et al. flip this scheme around, finding that, for the magnesium channel CorA, loss of ligand binding induces an ensemble of conformations that turn the channel on.
Subject(s)
Bacterial Proteins/ultrastructure , Cation Transport Proteins/ultrastructure , Magnesium/metabolism , Thermotoga maritima/chemistryABSTRACT
Calcium (Ca2+) is an essential nutrient for plants and a cellular signal, but excessive levels can be toxic and inhibit growth1,2. To thrive in dynamic environments, plants must monitor and maintain cytosolic Ca2+ homeostasis by regulating numerous Ca2+ transporters3. Here we report two signalling pathways in Arabidopsis thaliana that converge on the activation of vacuolar Ca2+/H+ exchangers (CAXs) to scavenge excess cytosolic Ca2+ in plants. One mechanism, activated in response to an elevated external Ca2+ level, entails calcineurin B-like (CBL) Ca2+ sensors and CBL-interacting protein kinases (CIPKs), which activate CAXs by phosphorylating a serine (S) cluster in the auto-inhibitory domain. The second pathway, triggered by molecular patterns associated with microorganisms, engages the immune receptor complex FLS2-BAK1 and the associated cytoplasmic kinases BIK1 and PBL1, which phosphorylate the same S-cluster in CAXs to modulate Ca2+ signals in immunity. These Ca2+-dependent (CBL-CIPK) and Ca2+-independent (FLS2-BAK1-BIK1/PBL1) mechanisms combine to balance plant growth and immunity by regulating cytosolic Ca2+ homeostasis.
Subject(s)
Arabidopsis , Calcium , Homeostasis , Plant Immunity , Arabidopsis/cytology , Arabidopsis/growth & development , Arabidopsis/immunology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Calcium/metabolism , Calcium-Binding Proteins/metabolism , Cytosol/metabolism , Phosphorylation , Phosphoserine/metabolism , Protein Serine-Threonine Kinases/metabolism , Cation Transport Proteins/metabolism , Antiporters/metabolismABSTRACT
Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.
Subject(s)
Calcium-Binding Proteins/metabolism , Calcium , Cation Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Adenosine Triphosphate , Animals , Calcium/metabolism , Calcium Channels , Calcium-Binding Proteins/genetics , Disulfides/metabolism , Humans , Mice , Mitochondrial Membrane Transport Proteins/genetics , Reactive Oxygen Species/metabolismABSTRACT
Osteoarthritis (OA), primarily characterized by cartilage degeneration, is caused by an imbalance between anabolic and catabolic factors. Here, we investigated the role of zinc (Zn2+) homeostasis, Zn2+ transporters, and Zn(2+)-dependent transcription factors in OA pathogenesis. Among Zn2+ transporters, the Zn2+ importer ZIP8 was specifically upregulated in OA cartilage of humans and mice, resulting in increased levels of intracellular Zn2+ in chondrocytes. ZIP8-mediated Zn2+ influx upregulated the expression of matrix-degrading enzymes (MMP3, MMP9, MMP12, MMP13, and ADAMTS5) in chondrocytes. Ectopic expression of ZIP8 in mouse cartilage tissue caused OA cartilage destruction, whereas Zip8 knockout suppressed surgically induced OA pathogenesis, with concomitant modulation of Zn2+ influx and matrix-degrading enzymes. Furthermore, MTF1 was identified as an essential transcription factor in mediating Zn2+/ZIP8-induced catabolic factor expression, and genetic modulation of Mtf1 in mice altered OA pathogenesis. We propose that the zinc-ZIP8-MTF1 axis is an essential catabolic regulator of OA pathogenesis.
Subject(s)
Osteoarthritis/metabolism , Osteoarthritis/pathology , Signal Transduction , ADAM Proteins/metabolism , Aged , Animals , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Chondrocytes/metabolism , Chondrocytes/pathology , Humans , Kruppel-Like Transcription Factors/metabolism , Male , Matrix Metalloproteinases/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Up-Regulation , Zinc/metabolismABSTRACT
Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S. cerevisiae HOL1 mRNA is under translational control by polyamines, and we reveal that the encoded membrane transporter Hol1 is a high-affinity polyamine transporter and is required for yeast growth under limiting polyamine conditions. Moreover, we show that polyamine inhibition of the translation factor eIF5A impairs translation termination at a Pro-Ser-stop motif in a conserved upstream open reading frame on the HOL1 mRNA to repress Hol1 synthesis under conditions of elevated polyamines. Our findings reveal that polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels.
Subject(s)
Cation Transport Proteins/metabolism , Membrane Transport Proteins/metabolism , Polyamines/metabolism , Protein Biosynthesis , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Biological Transport , Cation Transport Proteins/genetics , Gene Expression Regulation, Fungal , Membrane Transport Proteins/genetics , Open Reading Frames , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Eukaryotic Translation Initiation Factor 5AABSTRACT
Several intracellular pathogens, including Salmonella enterica and Mycobacterium tuberculosis, require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm, whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein.
Subject(s)
Bacterial Proteins/metabolism , Cation Transport Proteins/metabolism , Proton-Translocating ATPases/antagonists & inhibitors , Salmonella Infections/microbiology , Salmonella typhimurium/cytology , Salmonella typhimurium/pathogenicity , Virulence Factors/metabolism , Adenosine Triphosphate/metabolism , Animals , Female , Hydrogen-Ion Concentration , Macrophages/microbiology , Membrane Potentials , Mice , Mice, Inbred C3H , Protein Subunits/antagonists & inhibitors , Salmonella typhimurium/enzymology , VirulenceABSTRACT
Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.
Subject(s)
Mitochondria , Mitochondrial Proteins , Cation Transport Proteins , Cell Cycle Proteins , Energy Metabolism , Humans , Mass Spectrometry , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Transcription Factors , rab5 GTP-Binding ProteinsABSTRACT
Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.
Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Cation Transport Proteins/metabolism , Cell Survival , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Apoptosis , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membranes/metabolismABSTRACT
Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.
Subject(s)
Cation Transport Proteins/metabolism , GTP Phosphohydrolases/metabolism , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Dynamics , Mitochondrial Proteins/metabolism , Mitophagy , Adenosine Triphosphate/metabolism , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Cation Transport Proteins/genetics , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Dynamins , Energy Metabolism , GTP Phosphohydrolases/genetics , HEK293 Cells , HeLa Cells , Humans , Membrane Potential, Mitochondrial , Microtubule-Associated Proteins/genetics , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mutation , Neurons/metabolism , Neurons/pathology , Protein Binding , Protein Interaction Domains and Motifs , Rats, Sprague-Dawley , Signal Transduction , Time Factors , Zinc/metabolismABSTRACT
Transcription regulation underlies stem cell function and development. Here, we elucidate an unexpected role of an essential ribogenesis factor, WDR43, as a chromatin-associated RNA-binding protein (RBP) and release factor in modulating the polymerase (Pol) II activity for pluripotency regulation. WDR43 binds prominently to promoter-associated noncoding/nascent RNAs, occupies thousands of gene promoters and enhancers, and interacts with the Pol II machinery in embryonic stem cells (ESCs). Nascent transcripts and transcription recruit WDR43 to active promoters, where WDR43 facilitates releases of the elongation factor P-TEFb and paused Pol II. Knockdown of WDR43 causes genome-wide defects in Pol II release and pluripotency-associated gene expression. Importantly, auxin-mediated rapid degradation of WDR43 drastically reduces Pol II activity, precluding indirect consequences. These results reveal an RNA-mediated recruitment and feedforward regulation on transcription and demonstrate an unforeseen role of an RBP in promoting Pol II elongation and coordinating high-level transcription and translation in ESC pluripotency.
Subject(s)
Cation Transport Proteins/genetics , Chromatin/chemistry , Gene Expression Regulation, Developmental , Mouse Embryonic Stem Cells/metabolism , RNA Polymerase II/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Transcription, Genetic , Zebrafish Proteins/genetics , Animals , Binding Sites , Cation Transport Proteins/metabolism , Cell Differentiation , Cell Line , Chromatin/metabolism , Embryo, Mammalian , Enhancer Elements, Genetic , Gene Deletion , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Positive Transcriptional Elongation Factor B/genetics , Positive Transcriptional Elongation Factor B/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Biosynthesis , Proteolysis , RNA Polymerase II/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Signal Transduction , Zebrafish Proteins/metabolismABSTRACT
Intracellular sensors detect changes in levels of essential metals to initiate homeostatic responses. But, a mammalian manganese (Mn) sensor is unknown, representing a major gap in understanding of Mn homeostasis. Using human-relevant models, we recently reported that: 1) the primary homeostatic response to elevated Mn is upregulation of hypoxia-inducible factors (HIFs), which increases expression of the Mn efflux transporter SLC30A10; and 2) elevated Mn blocks the prolyl hydroxylation of HIFs by prolyl hydroxylase domain (PHD) enzymes, which otherwise targets HIFs for degradation. Thus, the mammalian mechanism for sensing elevated Mn likely relates to PHD inhibition. Moreover, 1) Mn substitutes for a catalytic iron (Fe) in PHD structures; and 2) exchangeable cellular levels of Fe and Mn are comparable. Therefore, we hypothesized that elevated Mn directly inhibits PHD by replacing its catalytic Fe. In vitro assays using catalytically active PHD2, the primary PHD isoform, revealed that Mn inhibited, and Fe supplementation rescued, PHD2 activity. However, a mutation in PHD2 (D315E) that selectively reduced Mn binding without substantially impacting Fe binding or enzymatic activity resulted in complete insensitivity of PHD2 to Mn in vitro. Additionally, hepatic cells expressing full-length PHD2D315E were less sensitive to Mn-induced HIF activation and SLC30A10 upregulation than PHD2wild-type. These results: 1) define a fundamental Mn sensing mechanism for controlling Mn homeostasis-elevated Mn inhibits PHD2, which functions as a Mn sensor, by outcompeting its catalytic Fe, and PHD2 inhibition activates HIF signaling to up-regulate SLC30A10; and 2) identify a unique mode of metal sensing that may have wide applicability.
Subject(s)
Homeostasis , Hypoxia-Inducible Factor-Proline Dioxygenases , Manganese , Humans , Manganese/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Cation Transport Proteins/metabolism , Cation Transport Proteins/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , HEK293 Cells , Iron/metabolismSubject(s)
Cation Transport Proteins , Zinc , B-Lymphocytes , Endoplasmic Reticulum , Lymphocyte ActivationABSTRACT
ABSTRACT: Iron-mediated induction of bone morphogenetic protein (BMP)6 expression by liver endothelial cells is essential for iron homeostasis regulation. We used multiple dietary and genetic mouse cohorts to demonstrate a minor functional role for the metal-ion transporter ZIP8 in regulating BMP6 expression under high-iron conditions.
Subject(s)
Bone Morphogenetic Protein 6 , Cation Transport Proteins , Iron , Animals , Bone Morphogenetic Protein 6/metabolism , Bone Morphogenetic Protein 6/genetics , Mice , Cation Transport Proteins/metabolism , Cation Transport Proteins/genetics , Iron/metabolism , Endothelial Cells/metabolism , Mice, Knockout , Gene Expression Regulation , Liver/metabolism , Mice, Inbred C57BL , HomeostasisABSTRACT
Ferroportin (Fpn) is the only known iron exporter in humans and is essential for maintaining iron homeostasis. Fpn activity is suppressed by hepcidin, an endogenous peptide hormone, which inhibits iron export and promotes endocytosis of Fpn. Hepcidin deficiency leads to hemochromatosis and iron-loading anemia. Previous studies have shown that small peptides that mimic the first few residues of hepcidin, i.e., minihepcidins, are more potent than hepcidin. However, the mechanism of enhanced inhibition by minihepcidins remains unclear. Here, we report the structure of human ferroportin in complex with a minihepcidin, PR73 that mimics the first 9 residues of hepcidin, at 2.7 Å overall resolution. The structure reveals novel interactions that were not present between Fpn and hepcidin. We validate PR73-Fpn interactions through binding and transport assays. These results provide insights into how minihepcidins increase inhibition potency and will guide future development of Fpn inhibitors.