RESUMO
A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand ß-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Fatores de Transcrição/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Citocromos/metabolismo , Transporte de Elétrons/fisiologia , Elétrons , Heme/metabolismo , Complexos Multiproteicos/ultraestrutura , Oxirredução , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Long-range (>10 µm) transport of electrons along networks of Geobacter sulfurreducens protein filaments, known as microbial nanowires, has been invoked to explain a wide range of globally important redox phenomena. These nanowires were previously thought to be type IV pili composed of PilA protein. Here, we report a 3.7 Å resolution cryoelectron microscopy structure, which surprisingly reveals that, rather than PilA, G. sulfurreducens nanowires are assembled by micrometer-long polymerization of the hexaheme cytochrome OmcS, with hemes packed within â¼3.5-6 Å of each other. The inter-subunit interfaces show unique structural elements such as inter-subunit parallel-stacked hemes and axial coordination of heme by histidines from neighboring subunits. Wild-type OmcS filaments show 100-fold greater conductivity than other filaments from a ΔomcS strain, highlighting the importance of OmcS to conductivity in these nanowires. This structure explains the remarkable capacity of soil bacteria to transport electrons to remote electron acceptors for respiration and energy sharing.
Assuntos
Transporte de Elétrons/fisiologia , Geobacter/metabolismo , Heme/metabolismo , Biofilmes , Condutividade Elétrica , Elétrons , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Nanofios , OxirreduçãoRESUMO
For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress.
Assuntos
Antioxidantes/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Glicólise/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Animais , Antioxidantes/administração & dosagem , Fatores de Transcrição de Zíper de Leucina Básica/genética , Movimento Celular/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Heme/metabolismo , Hexoquinase/antagonistas & inibidores , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fator 2 Relacionado a NF-E2/metabolismo , Metástase Neoplásica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80hiCD115hiC3aRhiCD88hi), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80+HO-1+ bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80+HO-1+ TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1+ myeloid subgroup as a new antimetastatic target and prognostic blood marker.
Assuntos
Biomarcadores Tumorais/metabolismo , Heme Oxigenase-1/metabolismo , Neoplasias Pulmonares/imunologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Linhagem Celular Tumoral/transplante , Quimioterapia Adjuvante/métodos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/imunologia , Feminino , Heme/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/sangue , Heme Oxigenase-1/genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Masculino , Melanoma/mortalidade , Melanoma/secundário , Melanoma/terapia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/metabolismo , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/metabolismoRESUMO
Iron is essential for the survival of most bacteria but presents a significant challenge given its limited bioavailability. Furthermore, the toxicity of iron combined with the need to maintain physiological iron levels within a narrow concentration range requires sophisticated systems to sense, regulate, and transport iron. Most bacteria have evolved mechanisms to chelate and transport ferric iron (Fe3+) via siderophore receptor systems, and pathogenic bacteria have further lowered this barrier by employing mechanisms to utilize the host's hemoproteins. Once internalized, heme is cleaved by both oxidative and nonoxidative mechanisms to release iron. Heme, itself a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such, pathogenic bacteria have evolved sophisticated cell surface signaling and transport systems to obtain heme from the host. In this review, we summarize the structure and function of the heme-sensing and transport systems of pathogenic bacteria and the potential of these systems as antimicrobial targets.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Heme/antagonistas & inibidores , Ferro/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Expressão Gênica , Heme/metabolismo , Metaloporfirinas/síntese química , Metaloporfirinas/farmacologia , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sideróforos/antagonistas & inibidores , Sideróforos/biossíntese , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismoRESUMO
Red blood cell rupture (hemolysis) activates innate immunity and inflammation by releasing heme. Sundaram et al.1 implicate the immune sensor NLRP12 in hemolytic disease, showing that it controls necrotic cell death induction in response to heme combined with pathogen-associated molecules.
Assuntos
Heme , Moléculas com Motivos Associados a Patógenos , Humanos , Heme/metabolismo , Inflamação/metabolismo , Imunidade Inata , Hemólise , Necrose , Morte Celular , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.
Assuntos
Ferro , eIF-2 Quinase , Ferro/metabolismo , eIF-2 Quinase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Células Eritroides/metabolismo , Heme/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.
Assuntos
NADPH Oxidase 2 , Fagócitos , Humanos , Elétrons , Ativação Enzimática , Flavina-Adenina Dinucleotídeo/metabolismo , Heme/química , Heme/metabolismo , NADP/metabolismo , NADPH Oxidase 2/química , NADPH Oxidase 2/metabolismo , Fagócitos/enzimologia , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Superóxidos/metabolismo , Ligação ProteicaRESUMO
Iron is the most abundant transition metal essential for numerous cellular processes. Although most mammalian cells acquire iron through transferrin receptors, molecular players of iron utilization under iron restriction are incompletely understood. To address this, we performed metabolism-focused CRISPRa gain-of-function screens, which revealed metabolic limitations under stress conditions. Iron restriction screens identified not only expected members of iron utilization pathways but also SLCO2B1, a poorly characterized membrane carrier. SLCO2B1 expression is sufficient to increase intracellular iron, bypass the essentiality of the transferrin receptor, and enable proliferation under iron restriction. Mechanistically, SLCO2B1 mediates heme analog import in cellular assays. Heme uptake by SLCO2B1 provides sufficient iron for proliferation through heme oxygenases. Notably, SLCO2B1 is predominantly expressed in microglia in the brain, and primary Slco2b1-/- mouse microglia exhibit strong defects in heme analog import. Altogether, our work identifies SLCO2B1 as a microglia-enriched plasma membrane heme importer and provides a genetic platform to identify metabolic limitations under stress conditions.
Assuntos
Heme , Ferro , Transportadores de Ânions Orgânicos/metabolismo , Animais , Transporte Biológico , Heme/genética , Heme/metabolismo , Ferro/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Ativação TranscricionalRESUMO
Splenic red pulp macrophages (RPMs) contribute to erythrocyte homeostasis and are required for iron recycling. Heme induces the expression of SPIC transcription factor in monocyte-derived macrophages and promotes their differentiation into RPM precursors, pre-RPMs. However, the requirements for differentiation into mature RPMs remain unknown. Here, we have demonstrated that interleukin (IL)-33 associated with erythrocytes and co-cooperated with heme to promote the generation of mature RPMs through activation of the MyD88 adaptor protein and ERK1/2 kinases downstream of the IL-33 receptor, IL1RL1. IL-33- and IL1RL1-deficient mice showed defective iron recycling and increased splenic iron deposition. Gene expression and chromatin accessibility studies revealed a role for GATA transcription factors downstream of IL-33 signaling during the development of pre-RPMs that retained full potential to differentiate into RPMs. Thus, IL-33 instructs the development of RPMs as a response to physiological erythrocyte damage with important implications to iron recycling and iron homeostasis.
Assuntos
Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-33/imunologia , Ferro/metabolismo , Macrófagos/imunologia , Transdução de Sinais/imunologia , Baço/metabolismo , Animais , Eritrócitos/imunologia , Eritrócitos/metabolismo , Heme/imunologia , Heme/metabolismo , Homeostase/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Macrófagos/metabolismo , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Baço/citologiaRESUMO
Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80(+)VCAM1(+) bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Heme/metabolismo , Ferro/metabolismo , Monócitos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Baço/citologia , Baço/metabolismoRESUMO
The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/ultraestrutura , Regulação Alostérica , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Heme/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Simulação de Dinâmica Molecular , Palmitoil Coenzima A/metabolismo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Methylamine dehydrogenase (MADH) catalyzes the oxidative deamination of methylamine to formaldehyde and ammonia. Tryptophan tryptophylquinone (TTQ) is the protein-derived cofactor of MADH required for this catalytic activity. TTQ is biosynthesized through the posttranslational modification of two tryptophan residues within MADH, during which the indole rings of two tryptophan side chains are cross-linked and two oxygen atoms are inserted into one of the indole rings. MauG is a c-type diheme enzyme that catalyzes the final three reactions in TTQ formation. In total, this is a six-electron oxidation process requiring three cycles of MauG-dependent two-electron oxidation events using either H2O2 or O2. The MauG redox form responsible for the catalytic activity is an unprecedented bis-Fe(IV) species. The amino acids of MADH that are modified are ≈ 40 Å from the site where MauG binds oxygen, and the reaction proceeds by a hole hopping electron transfer mechanism. This review addresses these highly unusual aspects of the long-range catalytic reaction mediated by MauG.
Assuntos
Heme/metabolismo , Indolquinonas/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Paracoccus denitrificans/enzimologia , Processamento de Proteína Pós-Traducional/fisiologia , Triptofano/análogos & derivados , Triptofano/metabolismo , Catálise , Transporte de Elétrons , Oxirredução , Paracoccus denitrificans/genética , Paracoccus denitrificans/metabolismo , Triptofano/biossínteseRESUMO
Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.
Assuntos
Infecções por Bactérias Gram-Negativas/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Heme/metabolismo , Hemólise/imunologia , Macrófagos/imunologia , Fagocitose , Sepse/imunologia , Animais , Antibacterianos/uso terapêutico , Citoesqueleto/metabolismo , Feminino , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/genética , Heme Oxigenase-1/genética , Hemólise/efeitos dos fármacos , Humanos , Evasão da Resposta Imune , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Quinina/uso terapêutico , Células RAW 264.7 , Sepse/tratamento farmacológico , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Haem is an iron-containing tetrapyrrole that is critical for a variety of cellular and physiological processes1-3. Haem binding proteins are present in almost all cellular compartments, but the molecular mechanisms that regulate the transport and use of haem within the cell remain poorly understood2,3. Here we show that haem-responsive gene 9 (HRG-9) (also known as transport and Golgi organization 2 (TANGO2)) is an evolutionarily conserved haem chaperone with a crucial role in trafficking haem out of haem storage or synthesis sites in eukaryotic cells. Loss of Caenorhabditis elegans hrg-9 and its paralogue hrg-10 results in the accumulation of haem in lysosome-related organelles, the haem storage site in worms. Similarly, deletion of the hrg-9 homologue TANGO2 in yeast and mammalian cells induces haem overload in mitochondria, the site of haem synthesis. We demonstrate that TANGO2 binds haem and transfers it from cellular membranes to apo-haemoproteins. Notably, homozygous tango2-/- zebrafish larvae develop pleiotropic symptoms including encephalopathy, cardiac arrhythmia and myopathy, and die during early development. These defects partially resemble the symptoms of human TANGO2-related metabolic encephalopathy and arrhythmias, a hereditary disease caused by mutations in TANGO24-8. Thus, the identification of HRG-9 as an intracellular haem chaperone provides a biological basis for exploring the aetiology and treatment of TANGO2-related disorders.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Heme , Animais , Humanos , Arritmias Cardíacas/metabolismo , Encefalopatias/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Heme/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro, or taken from humans in vivo, and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.
Assuntos
Eritrócitos , Hemoglobinas , Humanos , Camundongos , Animais , Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Oxirredução , Heme/metabolismo , Ritmo CircadianoRESUMO
In response to an immune challenge, naive T cells undergo a transition from a quiescent to an activated state acquiring the effector function. Concurrently, these T cells reprogram cellular metabolism, which is regulated by iron. We and others have shown that iron homeostasis controls proliferation and mitochondrial function, but the underlying mechanisms are poorly understood. Given that iron derived from heme makes up a large portion of the cellular iron pool, we investigated iron homeostasis in T cells using mice with a T cell-specific deletion of the heme exporter, FLVCR1 [referred to as knockout (KO)]. Our finding revealed that maintaining heme and iron homeostasis is essential to keep naive T cells in a quiescent state. KO naive CD4 T cells exhibited an iron-overloaded phenotype, with increased spontaneous proliferation and hyperactive mitochondria. This was evidenced by reduced IL-7R and IL-15R levels but increased CD5 and Nur77 expression. Upon activation, however, KO CD4 T cells have defects in proliferation, IL-2 production, and mitochondrial functions. Iron-overloaded CD4 T cells failed to induce mitochondrial iron and exhibited more fragmented mitochondria after activation, making them susceptible to ferroptosis. Iron overload also led to inefficient glycolysis and glutaminolysis but heightened activity in the hexosamine biosynthetic pathway. Overall, these findings highlight the essential role of iron in controlling mitochondrial function and cellular metabolism in naive CD4 T cells, critical for maintaining their quiescent state.
Assuntos
Linfócitos T CD4-Positivos , Ferro , Camundongos , Animais , Ferro/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Heme/metabolismoRESUMO
ABSTRACT: Chronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with the progressive deterioration of renal health, resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vaso-occlusive crisis associated with acute intravascular hemolysis. However, the mechanisms of hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of the vascular endothelial protein C receptor (EPCR) in the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR), indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of patients with SCD, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, the restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD.
Assuntos
Anemia Falciforme , Receptor de Proteína C Endotelial , Heme , Camundongos Transgênicos , Insuficiência Renal Crônica , Animais , Anemia Falciforme/complicações , Anemia Falciforme/patologia , Anemia Falciforme/metabolismo , Anemia Falciforme/sangue , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/etiologia , Receptor de Proteína C Endotelial/metabolismo , Receptor de Proteína C Endotelial/genética , Camundongos , Heme/metabolismo , Humanos , Masculino , Feminino , Hemólise , Rim/metabolismo , Rim/patologiaRESUMO
Extracellular free heme can intercalate into membranes and promote damage to cellular macromolecules. Thus it is likely that specific intercellular pathways exist for the directed transport, trafficking, and delivery of heme to cellular destinations, although none have been found to date. Here we show that Caenorhabditis elegans HRG-3 is required for the delivery of maternal heme to developing embryos. HRG-3 binds heme and is exclusively secreted by maternal intestinal cells into the interstitial fluid for transport of heme to extraintestinal cells, including oocytes. HRG-3 deficiency results either in death during embryogenesis or in developmental arrest immediately post-hatching-phenotypes that are fully suppressed by maternal but not zygotic hrg-3 expression. Our results establish a role for HRG-3 as an intercellular heme-trafficking protein.
Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Heme/deficiência , Hemeproteínas/química , Hemeproteínas/genética , Mucosa Intestinal/metabolismo , Mutação , Fenótipo , Transporte Proteico , Via SecretóriaRESUMO
Most proteins associate into multimeric complexes with specific architectures1,2, which often have functional properties such as cooperative ligand binding or allosteric regulation3. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and ß-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical 'missing link' through which the modern tetramer evolved-a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and ß-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein's structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.