RESUMO
Caloric restriction is known to improve inflammatory and autoimmune diseases. However, the mechanisms by which reduced caloric intake modulates inflammation are poorly understood. Here we show that short-term fasting reduced monocyte metabolic and inflammatory activity and drastically reduced the number of circulating monocytes. Regulation of peripheral monocyte numbers was dependent on dietary glucose and protein levels. Specifically, we found that activation of the low-energy sensor 5'-AMP-activated protein kinase (AMPK) in hepatocytes and suppression of systemic CCL2 production by peroxisome proliferator-activator receptor alpha (PPARα) reduced monocyte mobilization from the bone marrow. Importantly, we show that fasting improves chronic inflammatory diseases without compromising monocyte emergency mobilization during acute infectious inflammation and tissue repair. These results reveal that caloric intake and liver energy sensors dictate the blood and tissue immune tone and link dietary habits to inflammatory disease outcome.
Assuntos
Restrição Calórica , Monócitos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adulto , Animais , Antígenos Ly/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , PPAR alfa/deficiência , PPAR alfa/genética , PPAR alfa/metabolismoRESUMO
Much more than the "powerhouse" of the cell, mitochondria have emerged as critical hubs involved in metabolism, cell death, inflammation, signaling, and stress responses. To open our mitochondria focus issue, we asked several scientists to share the unanswered questions, emerging themes, and topics of investigation that excite them.
Assuntos
Mitocôndrias , Transdução de Sinais , Humanos , Mitocôndrias/metabolismo , Morte Celular , Inflamação/metabolismoRESUMO
Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.
Assuntos
Apoptose , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Esfingolipídeos/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Feminino , Células HeLa , Humanos , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/metabolismo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
In this issue of Molecular Cell, Cho et al. (2019) identify a mechanism by which the mitochondrial division machinery provides selective pressure to identify dysfunctional organelles through the coordinated action of DRP1, Zip1, and Zn2+ transport into mitochondria.
Assuntos
Mitocôndrias , Zinco , Dinâmica MitocondrialRESUMO
Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.
Assuntos
Mitocôndrias/metabolismo , NADH Desidrogenase/genética , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Animais , Apoptose/genética , Respiração Celular/genética , Citosol/metabolismo , Drosophila melanogaster/genética , Complexo I de Transporte de Elétrons/genética , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Transdução de Sinais/genéticaRESUMO
Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P1 receptor (S1P1R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P1R on T cells, and that the requirement for S1P1R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.
Assuntos
Células Endoteliais/metabolismo , Tecido Linfoide/citologia , Lisofosfolipídeos/metabolismo , Mitocôndrias/metabolismo , Esfingosina/análogos & derivados , Linfócitos T/citologia , Animais , Proteínas de Transporte de Ânions/metabolismo , Movimento Celular , Sobrevivência Celular , Feminino , Linfonodos/citologia , Linfonodos/imunologia , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/metabolismo , Baço/citologia , Baço/imunologia , Linfócitos T/imunologiaRESUMO
The cytosolic fraction of the tumor suppressor p53 activates the apoptotic effector protein BAX to trigger apoptosis. Here we report that p53 activates BAX through a mechanism different from that associated with activation by BH3 only proteins (BIM and BID). We observed that cis-trans isomerization of proline 47 (Pro47) within p53, an inherently rare molecular event, was required for BAX activation. The prolyl isomerase Pin1 enhanced p53-dependent BAX activation by catalyzing cis-trans interconversion of p53 Pro47. Our results reveal a signaling mechanism whereby proline cis-trans isomerization in one protein triggers conformational and functional changes in a downstream signaling partner. Activation of BAX through the concerted action of cytosolic p53 and Pin1 may integrate cell stress signals to induce a direct apoptotic response.
Assuntos
Apoptose , Peptidilprolil Isomerase/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Linhagem Celular Tumoral , Humanos , Cinética , Peptidilprolil Isomerase de Interação com NIMA , Prolina/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estereoisomerismo , Proteína Supressora de Tumor p53/química , Proteína X Associada a bcl-2/químicaRESUMO
Mitochondrial division is essential for mitosis and metazoan development, but a mechanistic role in cancer biology remains unknown. Here, we examine the direct effects of oncogenic RAS(G12V)-mediated cellular transformation on the mitochondrial dynamics machinery and observe a positive selection for dynamin-related protein 1 (DRP1), a protein required for mitochondrial network division. Loss of DRP1 prevents RAS(G12V)-induced mitochondrial dysfunction and renders cells resistant to transformation. Conversely, in human tumor cell lines with activating MAPK mutations, inhibition of these signals leads to robust mitochondrial network reprogramming initiated by DRP1 loss resulting in mitochondrial hyper-fusion and increased mitochondrial metabolism. These phenotypes are mechanistically linked by ERK1/2 phosphorylation of DRP1 serine 616; DRP1(S616) phosphorylation is sufficient to phenocopy transformation-induced mitochondrial dysfunction, and DRP1(S616) phosphorylation status dichotomizes BRAF(WT) from BRAF(V600E)-positive lesions. These findings implicate mitochondrial division and DRP1 as crucial regulators of transformation with leverage in chemotherapeutic success.
Assuntos
Transformação Celular Neoplásica/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Células HT29 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Serina/metabolismo , Proteínas ras/genéticaRESUMO
Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.
Assuntos
GTP Fosfo-Hidrolases/genética , Mitocôndrias Hepáticas/genética , Membranas Mitocondriais/metabolismo , Forma das Organelas/genética , Proteína X Associada a bcl-2/genética , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Dinâmica Mitocondrial/genética , Membranas Mitocondriais/ultraestrutura , Permeabilidade , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
GM-CSF (Csf-2) is a critical cytokine for the in vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103(+) DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103(+) and CD11b(+) DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8(+) T cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8(+) T cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in vivo.
Assuntos
Subunidade beta Comum dos Receptores de Citocinas/fisiologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Inflamação/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Linhagem da Célula , Subunidade beta Comum dos Receptores de Citocinas/antagonistas & inibidores , Subunidade beta Comum dos Receptores de Citocinas/deficiência , Subunidade beta Comum dos Receptores de Citocinas/genética , Células Dendríticas/classificação , Células Dendríticas/citologia , Encefalomielite Autoimune Experimental/imunologia , Endotoxemia/imunologia , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Homeostase , Lipopolissacarídeos/toxicidade , Listeriose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/transplante , Especificidade de Órgãos , Infecções por Orthomyxoviridae/imunologia , Infecções Pneumocócicas/imunologia , Quimera por Radiação , Baço/imunologia , Tamoxifeno/farmacologiaRESUMO
Aging of established antiviral T cell memory can foster a series of progressive adaptations that paradoxically improve rather than compromise protective CD8+ T cell immunity. We now provide evidence that this gradual evolution, the pace of which is contingent on the precise context of the primary response, also impinges on the molecular mechanisms that regulate CD8+ memory T cell (TM) homeostasis. Over time, CD8+ TM generated in the wake of an acute infection with the natural murine pathogen lymphocytic choriomeningitis virus become more resistant to apoptosis and acquire enhanced cytokine responsiveness without adjusting their homeostatic proliferation rates; concurrent metabolic adaptations promote increased CD8+ TM quiescence and fitness but also impart the reacquisition of a partial effector-like metabolic profile; and a gradual redistribution of aging CD8+ TM from blood and nonlymphoid tissues to lymphatic organs results in CD8+ TM accumulations in bone marrow, splenic white pulp, and, particularly, lymph nodes. Altogether, these data demonstrate how temporal alterations of fundamental homeostatic determinants converge to render aged CD8+ TM poised for greater recall responses.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/fisiologia , Memória Imunológica/imunologia , Linfonodos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Animais , Antígenos Virais/imunologia , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAFV600E), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAFV600E is pharmacologically inhibited by targeted therapies (e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAFV600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.
Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Lignanas/farmacologia , Mitocôndrias/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Proteína Quinase CDC2 , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Fase G1/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Desacopladores/farmacologiaRESUMO
B cell CLL/lymphoma-2 (BCL-2) and its relatives comprise the BCL-2 family of proteins, which were originally characterized with respect to their roles in controlling outer mitochondrial membrane integrity and apoptosis. Current observations expand BCL-2 family function to include numerous cellular pathways. Here we will discuss the mechanisms and functions of the BCL-2 family in the context of these pathways, highlighting the complex integration and regulation of the BCL-2 family in cell fate decisions.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Sequência de Aminoácidos , Apoptose , Autofagia , Retículo Endoplasmático/metabolismo , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Alinhamento de Sequência , Transdução de SinaisRESUMO
Mitochondria are dynamic organelles that supply energy required to drive key cellular processes, such as survival, proliferation, and migration. Critical to all of these processes are changes in mitochondrial architecture, a mechanical mechanism encompassing both fusion and fragmentation (fission) of the mitochondrial network. Changes to mitochondrial shape, size, and localization occur in a regulated manner to maintain energy and metabolic homeostasis, while deregulation of mitochondrial dynamics is associated with the onset of metabolic dysfunction and disease. In cancers, oncogenic signals that drive excessive proliferation, increase intracellular stress, and limit nutrient supply are all able to alter the bioenergetic and biosynthetic requirements of cancer cells. Consequently, mitochondrial function and shape rapidly adapt to these hostile conditions to support cancer cell proliferation and evade activation of cell death programs. In this review, we will discuss the molecular mechanisms governing mitochondrial dynamics and integrate recent insights into how changes in mitochondrial shape affect cellular migration, differentiation, apoptosis, and opportunities for the development of novel targeted cancer therapies.
Assuntos
Dinâmica Mitocondrial , Neoplasias/metabolismo , Animais , Humanos , Fusão de Membrana , Membranas Mitocondriais/metabolismo , Metástase Neoplásica , Neoplasias/patologia , Transdução de SinaisRESUMO
Hematopoietic stem cells (HSC) are primarily dormant but have the potential to become highly active on demand to reconstitute blood. This requires a swift metabolic switch from glycolysis to mitochondrial oxidative phosphorylation. Maintenance of low levels of reactive oxygen species (ROS), a by-product of mitochondrial metabolism, is also necessary for sustaining HSC dormancy. Little is known about mechanisms that integrate energy metabolism with hematopoietic stem cell homeostasis. Here, we identify the transcription factor FOXO3 as a new regulator of metabolic adaptation of HSC. ROS are elevated in Foxo3(-/-) HSC that are defective in their activity. We show that Foxo3(-/-) HSC are impaired in mitochondrial metabolism independent of ROS levels. These defects are associated with altered expression of mitochondrial/metabolic genes in Foxo3(-/-) hematopoietic stem and progenitor cells (HSPC). We further show that defects of Foxo3(-/-) HSC long-term repopulation activity are independent of ROS or mTOR signaling. Our results point to FOXO3 as a potential node that couples mitochondrial metabolism with HSC homeostasis. These findings have critical implications for mechanisms that promote malignant transformation and aging of blood stem and progenitor cells.
Assuntos
Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento/genética , Animais , Proteína Forkhead Box O3 , Homeostase/genética , Homeostase/fisiologia , Camundongos , Mitocôndrias/genética , Estresse Oxidativo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Mitochondria are an essential component of multicellular life - from primitive organisms, to highly complex entities like mammals. The importance of mitochondria is underlined by their plethora of well-characterized essential functions such as energy production through oxidative phosphorylation (OX-PHOS), calcium and reactive oxygen species (ROS) signaling, and regulation of apoptosis. In addition, novel roles and attributes of mitochondria are coming into focus through the recent years of mitochondrial research. In particular, over the past decade the study of mitochondrial shape and dynamics has achieved special significance, as they are found to impact mitochondrial function. Recent advances indicate that mitochondrial function and dynamics are inter-connected, and maintain the balance between health and disease at a cellular and an organismal level. For example, excessive mitochondrial division (fission) is associated with functional defects, and is implicated in multiple human diseases from neurodegenerative diseases to cancer. In this chapter we examine the recent literature on the mitochondrial dynamics-function relationship, and explore how it impacts on the development and progression of human diseases. We will also highlight the implications of therapeutic manipulation of mitochondrial dynamics in treating various human pathologies.
Assuntos
Dinâmica Mitocondrial/fisiologia , Animais , Modelos Animais de Doenças , Dinaminas , GTP Fosfo-Hidrolases/fisiologia , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/fisiologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/fisiologia , Doenças Neurodegenerativas/etiologiaRESUMO
Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(ßTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.
Assuntos
Lisina/metabolismo , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Substituição de Aminoácidos , Apoptose/genética , Biocatálise , Linhagem Celular Tumoral , Células HEK293 , Humanos , Ligação de Hidrogênio , Immunoblotting , Lisina/química , Lisina/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Poliubiquitina/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/química , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Understanding the impact of the p53 tumor suppressor pathway on the regulation of genome integrity, cancer development, and cancer treatment has intrigued scientists and clinicians for decades. It appears that the p53 pathway is a central node for nearly all cell stress responses, including: gene expression, DNA repair, cell cycle arrest, metabolic adjustments, apoptosis, and senescence. In the past decade, it has become increasingly clear that p53 function is directly regulated by poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair signaling. Here, we will discuss the impact of PARP-1 on p53 function, along with a recently described novel role for the reciprocal regulation of p53 regulated, PARP-1 dependent necrosis following DNA damage.