RESUMO
SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 focus retention and increases NHEJ and radioresistance. Collectively, our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.
Assuntos
Cisteína Endopeptidases/metabolismo , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Recombinação Homóloga/genética , Sumoilação/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Cisteína Endopeptidases/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Células HeLa , Humanos , Raios Infravermelhos , Proteínas Nucleares/metabolismo , Tolerância a Radiação/genética , Transdução de Sinais/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteína com Valosina/metabolismoRESUMO
The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.
Assuntos
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Replicação do DNA , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Linhagem Celular Tumoral , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Isomerismo , Mutação , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismoRESUMO
AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis that functions to restore the energy balance by phosphorylating its substrates during altered metabolic conditions. AMPK activity is tightly controlled by diverse regulators including its upstream kinases LKB1 and CaMKK2. Recent studies have also identified the localization of AMPK at different intracellular compartments as another key mechanism for regulating AMPK signaling in response to specific stimuli. This review discusses the AMPK signaling associated with different subcellular compartments, including lysosomes, endoplasmic reticulum, mitochondria, Golgi apparatus, nucleus, and cell junctions. Because altered AMPK signaling is associated with various pathologic conditions including cancer, targeting AMPK signaling in different subcellular compartments may present attractive therapeutic approaches for treatment of disease.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Organelas/enzimologia , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Humanos , Neoplasias/patologia , Organelas/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
AMPK is a crucial regulator of energy homeostasis that acts downstream of its upstream kinase liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase 2 (CaMKK2). LKB1 primarily phosphorylates AMPK after energy stress, whereas calcium-mediated activation of AMPK requires CaMKK2, although the regulatory mechanisms of calcium-mediated AMPK activation remain unclear. Using biochemical, microscopic, and genetic approaches, we demonstrate that the stromal interaction molecule (STIM)2, a calcium sensor, acts as a novel regulator of CaMKK2-AMPK signaling. We reveal that STIM2 interacts with AMPK and CaMKK2 and that the increase in intracellular calcium levels promotes AMPK colocalization and interaction with STIM2. We further show that STIM2 deficiency attenuates calcium-induced but not energy stress-induced AMPK activation, possibly by regulating the CaMKK2-AMPK interaction. Together, our results identify a previously unappreciated mechanism that modulates calcium-mediated AMPK activation.-Chauhan, A. S., Liu, X., Jing, J., Lee, H., Yadav, R. K., Liu, J., Zhou, Y., Gan B. STIM2 interacts with AMPK and regulates calcium-induced AMPK activation.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Molécula 2 de Interação Estromal/genéticaRESUMO
During physiologic stresses, like micronutrient starvation, infection, and cancer, the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is trafficked to the plasma membrane (PM) and extracellular milieu (ECM). Our work demonstrates that GAPDH mobilized to the PM, and the ECM does not utilize the classic endoplasmic reticulum-Golgi route of secretion; instead, it is first selectively translocated into early and late endosomes from the cytosol via microautophagy. GAPDH recruited to this common entry point is subsequently delivered into multivesicular bodies, leading to its membrane trafficking through secretion via exosomes and secretory lysosomes. We present evidence that both pathways of GAPDH membrane trafficking are up-regulated upon iron starvation, potentially by mobilization of intracellular calcium. These pathways also play a role in clearance of misfolded intracellular polypeptide aggregates. Our findings suggest that cells build in redundancy for vital cellular pathways to maintain micronutrient homeostasis and prevent buildup of toxic intracellular misfolded protein refuse.-Chauhan, A. S., Kumar, M., Chaudhary, S., Dhiman, A., Patidar, A., Jakhar, P., Jaswal, P., Sharma, K., Sheokand, N., Malhotra, H., Raje, C. I., Raje. M. Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways.
Assuntos
Endossomos/metabolismo , Microautofagia/fisiologia , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Animais , Autofagia/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Exossomos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Camundongos , Corpos Multivesiculares/metabolismo , Regulação para Cima/fisiologiaRESUMO
FoxO transcription factors serve as the central regulator of cellular homeostasis and are tumor suppressors in human cancers. Recent studies have revealed that, besides their classic functions in promoting cell death and inducing cell cycle arrest, FoxOs also regulate cancer metabolism, an emerging hallmark of cancer. In this review, we summarize the regulatory mechanisms employed to control FoxO activities in the context of cancer biology, and discuss FoxO function in metabolism reprogramming in cancer and interaction with other key cancer metabolism pathways. A deeper understanding of FoxOs in cancer metabolism may reveal novel therapeutic opportunities in cancer treatment.
Assuntos
Fatores de Transcrição Forkhead/genética , Redes e Vias Metabólicas/genética , Neoplasias/metabolismo , Apoptose/genética , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Humanos , Neoplasias/genética , Neoplasias/patologiaRESUMO
BACKGROUND/AIMS: Hypoxia triggers a rapid increase in iron demand to meet the requirements of enhanced erythropoiesis. The mobilization of iron stores from macrophage to plasma as holo-transferrin (Tf) from where it is accessible to erythroid precursor cells impacts iron homeostasis. Despite the immediate need for enhanced iron uptake by bone marrow cells, numerous studies have shown that transferrin receptor levels do not rise until more than 24 hours after the onset of hypoxia, suggesting the existence of heretofore unknown rapid response cellular machinery for iron acquisition in the early stages of cellular hypoxia. METHODS: We performed flow cytometry to measure cell surface levels of TfR1, GAPDH, and Tf binding after hypoxia treatment. We utilized FRET analysis and co-immunoprecipitation methods to establish the interaction between Tf and GAPDH. RESULTS: In the current study, we demonstrated that hypoxia induces K562 cells to translocate the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) onto cell surfaces and into the extracellular milieu to acquire transferrin-bound iron, even while levels of the classical transferrin receptor TfR1 (CD71) remain suppressed. GAPDH knockdown confirmed this protein's role in transferrin acquisition. Interestingly, macrophages did not show enhanced levels of extracellular GAPDH under hypoxia. CONCLUSION: Our results suggest the role of GAPDH-mediated Tf uptake as a rapid response mechanism by which cells acquire iron during the early stages of hypoxia. This is a tissue-specific phenomenon for the distinct requirements of cells that are consumers of iron versus cells that play a role in iron storage and recycling. This rapid deployment of an abundantly available multipurpose molecule allows hypoxic cells to internalize more Tf and maintain enhanced iron supplies in the early stages of hypoxia before specialized receptors can be synthesized and deployed to the cell membrane.
Assuntos
Hipóxia Celular , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ferro/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Células K562 , Macrófagos/citologia , Macrófagos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transferrina/metabolismoRESUMO
Iron (Fe), a vital micronutrient for all organisms, must be managed judiciously because both deficiency or excess can trigger severe pathology. Although cellular Fe import is well understood, its export is thought to be limited to transmembrane extrusion through ferroportin (also known as Slc40a1), the only known mammalian Fe exporter. Utilizing primary cells and cell lines (including those with no discernible expression of ferroportin on their surface), we demonstrate that upon Fe loading, the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is recruited to the cell surface, 'treadmills' apotransferrin in and out of the cell. Kinetic analysis utilizing labeled ligand, GAPDH-knockdown cells, (55)Fe-labeled cells and pharmacological inhibitors of endocytosis confirmed GAPDH-dependent apotransferrin internalization as a prerequisite for cellular Fe export. These studies define an unusual rapid recycling process of retroendocytosis for cellular Fe extrusion, a process mirroring receptor mediated internalization that has never before been considered for maintenance of cellular cationic homeostasis. Modulation of this unusual pathway could provide insights for management of Fe overload disorders.
Assuntos
Apoproteínas/metabolismo , Endocitose , Ferro/metabolismo , Transferrina/metabolismo , Animais , Linhagem Celular , Camundongos , Transporte ProteicoRESUMO
Prokaryotic pathogens establish infection in mammals by capturing the proteolytic enzyme plasminogen (Plg) onto their surface to digest host extracellular matrix (ECM). One of the bacterial surface Plg receptors is the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a defensive response, the host mounts an inflammatory response, which involves infiltration of leukocytes to sites of inflammation. This requires macrophage exit from the blood and migration across basement membranes, a phenomenon dependent on proteolytic remodeling of the ECM utilizing Plg. The ability of Plg to facilitate inflammatory cell recruitment critically depends on receptors on the surface of phagocyte cells. Utilizing a combination of biochemical, cellular, knockdown, and in vivo approaches, we demonstrated that upon inflammation, macrophages recruit GAPDH onto their surface to carry out the same task of capturing Plg to digest ECM to aid rapid phagocyte migration and combat the invading pathogens. We propose that GAPDH is an ancient, evolutionarily conserved receptor that plays a key role in the Plg-dependent regulation of macrophage recruitment in the inflammatory response to microbial aggression, thus pitting prokaryotic GAPDH against mammalian GAPDH, with both involved in a conserved role of Plg activation on the surface of their respective cells, to conflicting ends.-Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Dhiman, A., Sheokand, N., Malhotra, H., Raje, C. I., Raje, M. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.
Assuntos
Evolução Molecular , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Macrófagos/metabolismo , Plasminogênio/metabolismo , Animais , Linhagem Celular , Movimento Celular , Regulação Enzimológica da Expressão Gênica/fisiologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Camundongos , Receptores de Superfície Celular , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Regulação para CimaRESUMO
RIG-I-like receptor (RLR)-mediated interferon production is critical for antiviral responses. A recent study (Zhang et al., Cell, 2019) uncovered a reciprocal inhibition between RLR signaling and glycolysis: lactate produced by glycolysis inhibits RLR signaling by binding to RLR signaling component mitochondrial antiviral-signaling (MAVS), whereas RLR activation suppresses glycolysis through inhibiting glycolysis enzyme hexokinase.
Assuntos
Antivirais , Glicólise , Proteínas Adaptadoras de Transdução de Sinal , Ácido Láctico , Transdução de SinaisRESUMO
Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages.