RESUMO
Redox signaling, a mode of signal transduction that involves the transfer of electrons from a nucleophilic to electrophilic molecule, has emerged as an essential regulator of inflammatory macrophages. Redox reactions are driven by reactive oxygen/nitrogen species (ROS and RNS) and redox-sensitive metabolites such as fumarate and itaconate, which can post-translationally modify specific cysteine residues in target proteins. In the past decade our understanding of how ROS, RNS, and redox-sensitive metabolites control macrophage function has expanded dramatically. In this review, we discuss the latest evidence of how ROS, RNS, and metabolites regulate macrophage function and how this is dysregulated with disease. We highlight the key tools to assess redox signaling and important questions that remain.
Assuntos
Macrófagos , Oxirredução , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio , Transdução de Sinais , Succinatos , Macrófagos/metabolismo , Humanos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , AnimaisRESUMO
Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.
Assuntos
Creatina Quinase , Creatina , Creatina Quinase/química , Creatina Quinase/metabolismo , Creatina/farmacologia , Cisteína , Fosfotransferases , Isoformas de ProteínasRESUMO
Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.
Assuntos
Tecido Adiposo Marrom , Proteoma , Humanos , Camundongos , Animais , Tecido Adiposo Marrom/metabolismo , Proteoma/metabolismo , Termogênese/fisiologia , Adiposidade , Obesidade/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Obesity is one of the leading preventable causes of cancer; however, little is known about the effects of obesity on anti-tumor immunity. Here, we investigated the effects of obesity on CD8 T cells in mouse models and patients with endometrial cancer. Our findings revealed that CD8 T cell infiltration is suppressed in obesity, which was associated with a decrease in chemokine production. Tumor-resident CD8 T cells were also functionally suppressed in obese mice, which was associated with a suppression of amino acid metabolism. Similarly, we found that a high BMI negatively correlated with CD8 infiltration in human endometrial cancer and that weight loss was associated with a complete pathological response in six of nine patients. Moreover, immunotherapy using anti-PD-1 led to tumor rejection in lean and obese mice and partially restored CD8 metabolism and anti-tumor immunity. These findings highlight the suppressive effects of obesity on CD8 T cell anti-tumor immunity, which can partially be reversed by weight loss and/or immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/etiologia , Neoplasias/metabolismo , Obesidade/metabolismo , Microambiente Tumoral/imunologia , Aminoácidos/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Imunoterapia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Obesos , Neoplasias/patologia , Neoplasias/terapia , Obesidade/etiologiaRESUMO
Uncoupling protein 1 (UCP1) is a major regulator of brown and beige adipocyte energy expenditure and metabolic homeostasis. However, the widely employed UCP1 loss-of-function model has recently been shown to have a severe deficiency in the entire electron transport chain of thermogenic fat. As such, the role of UCP1 in metabolic regulation in vivo remains unclear. We recently identified cysteine-253 as a regulatory site on UCP1 that elevates protein activity upon covalent modification. Here, we examine the physiological importance of this site through the generation of a UCP1 cysteine-253-null (UCP1 C253A) mouse, a precise genetic model for selective disruption of UCP1 in vivo. UCP1 C253A mice exhibit significantly compromised thermogenic responses in both males and females but display no measurable effect on fat accumulation in an obesogenic environment. Unexpectedly, we find that a lack of C253 results in adipose tissue redox stress, which drives substantial immune cell infiltration and systemic inflammatory pathology in adipose tissues and liver of male, but not female, mice. Elevation of systemic estrogen reverses this male-specific pathology, providing a basis for protection from inflammation due to loss of UCP1 C253 in females. Together, our results establish the UCP1 C253 activation site as a regulator of acute thermogenesis and sex-dependent tissue inflammation.
Assuntos
Tecido Adiposo Marrom , Cisteína , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Cisteína/metabolismo , Metabolismo Energético , Feminino , Inflamação/metabolismo , Masculino , Camundongos , Termogênese/fisiologia , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Activated macrophages undergo metabolic reprogramming, which not only supports their energetic demands but also allows for the production of specific metabolites that function as signaling molecules. Several Krebs cycles, or Krebs-cycle-derived metabolites, including succinate, α-ketoglutarate, and itaconate, have recently been shown to modulate macrophage function. The accumulation of 2-hydroxyglutarate (2HG) has also been well documented in transformed cells and more recently shown to play a role in T cell and dendritic cell function. Here we have found that the abundance of both enantiomers of 2HG is increased in LPS-activated macrophages. We show that L-2HG, but not D-2HG, can promote the expression of the proinflammatory cytokine IL-1ß and the adoption of an inflammatory, highly glycolytic metabolic state. These changes are likely mediated through activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) by L-2HG, a known inhibitor of the HIF prolyl hydroxylases. Expression of the enzyme responsible for L-2HG degradation, L-2HG dehydrogenase (L-2HGDH), was also found to be decreased in LPS-stimulated macrophages and may therefore also contribute to L-2HG accumulation. Finally, overexpression of L-2HGDH in HEK293 TLR4/MD2/CD14 cells inhibited HIF-1α activation by LPS, while knockdown of L-2HGDH in macrophages boosted the induction of HIF-1α-dependent genes, as well as increasing LPS-induced HIF-1α activity. Taken together, this study therefore identifies L-2HG as a metabolite that can regulate HIF-1α in macrophages.
Assuntos
Glutaratos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Lipopolissacarídeos , Macrófagos , Glutaratos/metabolismo , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismoRESUMO
Age and sex are major risk factors in Alzheimer's disease (AD) with a higher incidence of the disease in females. Neuroinflammation, which is a hallmark of AD, contributes to disease pathogenesis and is inexorably linked with inappropriate microglial activation and neurodegeneration. We investigated sex-related differences in microglia in APP/PS1 mice and in post-mortem tissue from AD patients. Changes in genes that are indicative of microglial activation were preferentially increased in cells from female APP/PS1 mice and cells from males and females were morphological, metabolically and functionally distinct. Microglia from female APP/PS1 mice were glycolytic and less phagocytic and associated with increased amyloidosis whereas microglia from males were amoeboid and this was also the case in post-mortem tissue from male AD patients, where plaque load was reduced. We propose that the sex-related differences in microglia are likely to explain, at least in part, the sexual dimorphism in AD.
Assuntos
Doença de Alzheimer/metabolismo , Microglia/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Feminino , Regulação da Expressão Gênica , Glicólise , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microglia/patologia , Fatores SexuaisRESUMO
Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.
Assuntos
Envelhecimento/genética , Cisteína/genética , Proteínas/genética , Proteoma/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Cisteína/metabolismo , Humanos , Camundongos , Especificidade de Órgãos/genética , Oxirredução , Estresse Oxidativo/genética , Proteômica/métodos , Espécies Reativas de Oxigênio , Transdução de Sinais/genéticaRESUMO
Metabolic reprogramming has become a key focus for both immunologists and cancer biologists, with exciting advances providing new insights into underlying mechanisms of disease. Metabolites traditionally associated with bioenergetics or biosynthesis have been implicated in immunity and malignancy in transformed cells, with a particular focus on intermediates of the mitochondrial pathway known as the Krebs cycle. Among these, the intermediates succinate, fumarate, itaconate, 2-hydroxyglutarate isomers (D-2-hydroxyglutarate and L-2-hydroxyglutarate) and acetyl-CoA now have extensive evidence for "non-metabolic" signalling functions in both physiological immune contexts and in disease contexts, such as the initiation of carcinogenesis. This review will describe how metabolic reprogramming, with emphasis placed on these metabolites, leads to altered immune cell and transformed cell function. The latest findings are informative for new therapeutic approaches which could be transformative for a range of diseases.
Assuntos
Ciclo do Ácido Cítrico , Imunidade Inata , Neoplasias/metabolismo , Transdução de Sinais , Humanos , Macrófagos/metabolismo , Succinato Desidrogenase/antagonistas & inibidores , Succinatos/metabolismo , Ácido Succínico/metabolismoRESUMO
The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.
Assuntos
Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Succinatos/metabolismo , Alquilação , Animais , Carboxiliases , Bovinos , Cisteína/química , Cisteína/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Hidroliases/biossíntese , Interferon beta/imunologia , Interferon beta/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas/metabolismo , Ratos , Ratos Wistar , Succinatos/químicaRESUMO
Recent evidence indicates that mitochondria lie at the heart of immunity. Mitochondrial DNA acts as a danger-associated molecular pattern (DAMP), and the mitochondrial outer membrane is a platform for signaling molecules such as MAVS in RIG-I signaling, and for the NLRP3 inflammasome. Mitochondrial biogenesis, fusion and fission have roles in aspects of immune-cell activation. Most important, Krebs cycle intermediates such as succinate, fumarate and citrate engage in processes related to immunity and inflammation, in both innate and adaptive immune cells. These discoveries are revealing mitochondrial targets that could potentially be exploited for therapeutic gain in inflammation and cancer.
Assuntos
Imunidade Adaptativa , Ciclo do Ácido Cítrico/imunologia , Imunidade Inata , Mitocôndrias/imunologia , Membranas Mitocondriais/imunologia , Animais , Proteína DEAD-box 58/metabolismo , Metabolismo Energético , Humanos , Imunomodulação , Inflamassomos/metabolismo , Ativação Linfocitária , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores Imunológicos , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de SinaisRESUMO
Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.
Assuntos
Inflamação/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Mitocôndrias/enzimologia , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Ciclo do Ácido Cítrico , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Malonatos/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Succinato Desidrogenase/genética , TranscriptomaRESUMO
Mitochondria are master regulators of metabolism. Mitochondria generate ATP by oxidative phosphorylation using pyruvate (derived from glucose and glycolysis) and fatty acids (FAs), both of which are oxidized in the Krebs cycle, as fuel sources. Mitochondria are also an important source of reactive oxygen species (ROS), creating oxidative stress in various contexts, including in the response to bacterial infection. Recently, complex changes in mitochondrial metabolism have been characterized in mouse macrophages in response to varying stimuli in vitro. In LPS and IFN-γ-activated macrophages (M1 macrophages), there is decreased respiration and a broken Krebs cycle, leading to accumulation of succinate and citrate, which act as signals to alter immune function. In IL-4-activated macrophages (M2 macrophages), the Krebs cycle and oxidative phosphorylation are intact and fatty acid oxidation (FAO) is also utilized. These metabolic alterations in response to the nature of the stimulus are proving to be determinants of the effector functions of M1 and M2 macrophages. Furthermore, reprogramming of macrophages from M1 to M2 can be achieved by targeting metabolic events. Here, we describe the role that metabolism plays in macrophage function in infection and immunity, and propose that reprogramming with metabolic inhibitors might be a novel therapeutic approach for the treatment of inflammatory diseases.