RESUMO
TFEB is a master regulator of autophagy, lysosome biogenesis, mitochondrial metabolism, and immunity that works primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomics analysis reveals extensive TFEB co-immunoprecipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. High resolution confocal microscopy and biochemistry confirms TFEB localization in the mitochondrial matrix. TFEB translocation depends on a conserved N-terminal TOMM20-binding motif and is enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.
Assuntos
Autofagia , Inflamação , Humanos , Inflamação/metabolismo , Citosol/metabolismo , Transporte Ativo do Núcleo Celular , Lisossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/metabolismoRESUMO
Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Antineoplásicos/uso terapêutico , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Ceramidas/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identifies a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the apical ectodermal ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate the coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisofosfolipídeos , Mesoderma/metabolismo , Esfingosina/análogos & derivados , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Acute myeloid leukemia (AML) kills 75% of patients and represents a major clinical challenge with a need to improve on current treatment approaches. Targeting sphingosine kinase 1 with a novel ATP-competitive-inhibitor, MP-A08, induces cell death in AML. However, limitations in MP-A08's "drug-like properties" (solubility, biodistribution, and potency) hinder its pathway to the clinic. This study demonstrates a liposome-based delivery system of MP-A08 that exhibits enhanced MP-A08 potency against AML cells. MP-A08-liposomes increased MP-A08 efficacy against patient AML cells (>140-fold) and significantly prolonged overall survival of mice with human AML disease (P = 0.03). The significant antileukemic property of MP-A08-liposomes could be attributed to its enhanced specificity, bioaccessibility, and delivery to the bone marrow, as demonstrated in the pharmacokinetic and biodistribution studies. Our findings indicate that MP-A08-liposomes have potential as a novel treatment for AML.
Assuntos
Leucemia Mieloide Aguda , Lipossomos , Humanos , Camundongos , Animais , Lipossomos/uso terapêutico , Distribuição Tecidual , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Linhagem Celular TumoralRESUMO
PCDH19 is a nonclustered protocadherin molecule involved in axon bundling, synapse function, and transcriptional coregulation. Pathogenic variants in PCDH19 cause infantile-onset epilepsy known as PCDH19-clustering epilepsy or PCDH19-CE. Recent advances in DNA-sequencing technologies have led to a significant increase in the number of reported PCDH19-CE variants, many of uncertain significance. We aimed to determine the best approaches for assessing the disease relevance of missense variants in PCDH19. The application of the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines was only 50% accurate. Using a training set of 322 known benign or pathogenic missense variants, we identified MutPred2, MutationAssessor, and GPP as the best performing in silico tools. We generated a protein structural model of the extracellular domain and assessed 24 missense variants. We also assessed 24 variants using an in vitro reporter assay. A combination of these tools was 93% accurate in assessing known pathogenic and benign PCDH19 variants. We increased the accuracy of the ACMG-AMP classification of 45 PCDH19 variants from 50% to 94%, using these tools. In summary, we have developed a robust toolbox for the assessment of PCDH19 variant pathogenicity to improve the accuracy of PCDH19-CE variant classification.
Assuntos
Caderinas , Epilepsia , Caderinas/genética , Humanos , Mutação de Sentido Incorreto , Protocaderinas , Análise de Sequência de DNARESUMO
Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.
Assuntos
Proteínas de Transporte/metabolismo , Lisina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Motivos de Aminoácidos , Proteínas de Transporte/genética , Proteínas Culina/genética , Proteínas Culina/metabolismo , Humanos , Lisina/genética , Proteínas dos Microfilamentos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , UbiquitinaçãoRESUMO
There is growing evidence of the influence of sphingosine kinase (SK) enzymes on viral infection. Here, the role of sphingosine kinase 2 (SK2), an isoform of SK prominent in the brain, was defined during dengue virus (DENV) infection. Chemical inhibition of SK2 activity using two different SK2 inhibitors, ABC294640 and K145, had no effect on DENV infection in human cells in vitro. In contrast, DENV infection was restricted in SK2-/- immortalized mouse embryonic fibroblasts (iMEFs) with reduced induction of IFN-ß mRNA and protein, and mRNA for the IFN-stimulated genes (ISGs) viperin, IFIT1, IRF7 and CXCL10 in DENV-infected SK2-/- compared to WT iMEFs. Intracranial (ic) DENV injection in C57BL/6 SK2-/- mice induced body weight loss earlier than in WT mice but DENV RNA levels were comparable in the brain. Neither SK1 mRNA or sphingosine-1-phosphate (S1P) levels were altered following ic DENV infection in WT or SK2-/- mice but brain S1P levels were reduced in all SK2-/- mice, independent of DENV infection. CD8 mRNA was induced in the brains of both DENV-infected WT and SK2-/- mice, suggesting normal CD8+ T-cell infiltration into the DENV-infected brain independent of SK2 or S1P. Thus, although SK2 may be important for replication of some viruses SK2 activity does not affect DENV infection in vitro and SK2 or S1P levels do not influence DENV infection or T-cell infiltration in the context of infection in the brain.
Assuntos
Vírus da Dengue/patogenicidade , Dengue/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Linhagem Celular Tumoral , Dengue/tratamento farmacológico , Vírus da Dengue/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Interferon beta/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Tiazolidinedionas/farmacologiaRESUMO
Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34+ progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective downregulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Clorometilcetonas de Aminoácidos/farmacologia , Amino Álcoois/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Inibidores de Caspase/farmacologia , Caspases/genética , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The prevalence of allergies, including rhinitis, eczema, and anaphylaxis, is rising dramatically worldwide. This increase is especially problematic in children who bear the greatest burden of this rising trend. Increasing evidence identifies neutrophils as primary perpetrators of the more severe and difficult to manage forms of inflammation. A newly recognized mechanism by which neutrophils are recruited during the early phase of histamine-induced inflammation involves the sphingosine kinase (SK)/sphingosine-1-phosphate axis. This study examines whether topical application of fingolimod, an established SK/sphingosine-1-phosphate antagonist already in clinical use to treat multiple sclerosis, may be repurposed to treat cutaneous inflammation. Using two mouse models of ear skin inflammation (histamine- and IgE-mediated passive cutaneous anaphylaxis) we topically applied fingolimod prophylactically, as well as after establishment of the inflammatory response, and examined ear swelling, SK activity, vascular permeability, leukocyte recruitment, and production of proinflammatory mediators. The present study reveals that when applied topically, fingolimod attenuates both immediate and late-phase responses to histamine with reduced extravasation of fluid, SK-1 activity, proinflammatory cytokine and chemokine production, and neutrophil influx and prevents ear swelling. Intravital microscopy demonstrates that histamine-induced neutrophil rolling and adhesion to the postcapillary venules in the mouse ears is significantly attenuated even after 24 h. More importantly, these effects are achievable even once inflammation is established. Translation into humans was also accomplished with epicutaneous application of fingolimod resolving histamine-induced and allergen-induced inflammatory reactions in forearm skin. Overall, this study demonstrates, to our knowledge for the first time, that fingolimod may be repurposed to treat cutaneous inflammation.
Assuntos
Dermatite/tratamento farmacológico , Cloridrato de Fingolimode/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pele/efeitos dos fármacos , Administração Tópica , Animais , Permeabilidade Capilar/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Cloridrato de Fingolimode/farmacologia , Histamina/metabolismo , Humanos , Imunoglobulina E/sangue , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Pele/imunologiaRESUMO
Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.
Assuntos
Adenilato Quinase/metabolismo , Calreticulina/metabolismo , Dipeptidases/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Cátions , Linhagem Celular Tumoral , Separação Celular , Citoplasma/metabolismo , Metabolismo Energético , Citometria de Fluxo , Homeostase , Humanos , Marcação por Isótopo , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors. Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P2 and S1P4) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P2/4, inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P2/4.
Assuntos
Pirazóis/química , Piridinas/química , Esfingolipídeos/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inibidores , Receptores de Esfingosina-1-Fosfato/metabolismo , Células HEK293 , Humanos , Cinética , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores de Esfingosina-1-Fosfato/genéticaRESUMO
The introduction of the proteasome inhibitor bortezomib into treatment regimens for myeloma has led to substantial improvement in patient survival. However, whilst bortezomib elicits initial responses in many myeloma patients, this haematological malignancy remains incurable due to the development of acquired bortezomib resistance. With other patients presenting with disease that is intrinsically bortezomib resistant, it is clear that new therapeutic approaches are desperately required to target bortezomib-resistant myeloma. We have previously shown that targeting sphingolipid metabolism with the sphingosine kinase 2 (SK2) inhibitor K145 in combination with bortezomib induces synergistic death of bortezomib-naïve myeloma. In the current study, we have demonstrated that targeting sphingolipid metabolism with K145 synergises with bortezomib and effectively resensitises bortezomib-resistant myeloma to this proteasome inhibitor. Notably, these effects were dependent on enhanced activation of the unfolded protein response, and were observed in numerous separate myeloma models that appear to have different mechanisms of bortezomib resistance, including a new bortezomib-resistant myeloma model we describe which possesses a clinically relevant proteasome mutation. Furthermore, K145 also displayed synergy with the next-generation proteasome inhibitor carfilzomib in bortezomib-resistant and carfilzomib-resistant myeloma cells. Together, these findings indicate that targeting sphingolipid metabolism via SK2 inhibition may be effective in combination with a broad spectrum of proteasome inhibitors in the proteasome inhibitor resistant setting, and is an approach worth clinical exploration.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Bortezomib/química , Bortezomib/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/uso terapêutico , Técnicas de Inativação de Genes , Humanos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/uso terapêutico , Relação Estrutura-Atividade , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8(D772), DP8(Y315), DP8(H434) and DP8(D435) and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8(D772A/E/S/V) affected catalysis but did not confer endopeptidase activity. Mutations of DP8(H434F), DP8(D435F) and DP8(Y315F) reduced catalytic activity. Furthermore, mutations to DP8(D772A/E/S/V), DP8(H434F), DP8(D435F) and DP8(Y315F) affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8(D772), DP8(H434) and DP8(D435) were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.
Assuntos
Biocatálise , Domínio Catalítico , Dimerização , Dipeptidases/química , Dipeptidases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Bases de Dados de Proteínas , Dipeptidases/genética , Dipeptidases/isolamento & purificação , Dipeptidil Peptidase 4/química , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Endopeptidases , Gelatinases/química , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Especificidade por SubstratoRESUMO
While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.
Assuntos
Dineínas do Citoplasma/genética , Genes Supressores de Tumor , Glioblastoma/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Apoptose/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Membrana Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Células HEK293 , Humanos , Lisofosfolipídeos/genética , Camundongos , Esfingosina/análogos & derivados , Esfingosina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
N-terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G-protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N-terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF-1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.
Assuntos
Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Quimiocina CXCL12/metabolismo , Dipeptidases/metabolismo , Quimiocina CXCL10/química , Quimiocina CXCL11/química , Quimiocina CXCL12/química , Dipeptidases/isolamento & purificação , Dipeptidil Peptidase 4/isolamento & purificação , Dipeptidil Peptidase 4/metabolismo , Meia-Vida , Humanos , Cinética , Peso Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
Sphingosine kinases (SK) are the sole enzymes responsible for the production of sphingosine 1-phosphate (S1P). S1P is a signaling molecule with a plethora of targets, acting as both a second messenger intracellularly and extracellularly via a family of cell surface G-protein-coupled S1P receptors. The two sphingosine kinases, SK1 and SK2, arise from different genes and have some distinct and overlapping cellular functions that are regulated in part by differential cellular localization, developmental expression, and catalytic properties. Here, we describe an improved method for selectively detecting SK1 activity in vitro and cell lysates via the use of the zwitterionic detergent CHAPS, which effectively inhibits SK2 activity and thus allows selective analysis of SK1 activity in a range of cell samples. The assay measures the production of 32P-labeled S1P following the addition of exogenous sphingosine and [γ32P]ATP. The S1P product can be purified by Bligh-Dyer solvent extraction, separated by thin layer chromatography (TLC), and the radiolabeled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 activity in tissue, cell, and recombinant protein samples.
Assuntos
Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Animais , Ácidos Cólicos/química , Cromatografia em Camada Fina , Detergentes/química , Humanos , Transdução de Sinais , Esfingosina/metabolismoRESUMO
INTRODUCTION: We have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from COPD subjects. We hypothesize that this defect may include a disruption of epithelial-macrophage crosstalk via Spns2-mediated intercellular S1P signaling. METHODS: Primary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Cells were exposed to 10% cigarette smoke extract, or vehicle control. Spns2 expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RT-PCR. Phagocytosis was assessed by flow-cytometry. Levels of intra- and extracellular S1P were measured by S1P [3H]-labeling. RESULTS: Spns2 expression was significantly increased (p<0.05) in alveolar macrophages from current-smokers/COPD patients (n = 5) compared to healthy nonsmokers (n = 8) and non-smoker lung transplant patients (n = 4). Consistent with this finding, cigarette smoke induced a significant increase in Spns2 expression in both human alveolar and THP-1 macrophages. In contrast, a remarkable Spns2 down-regulation was noted in response to cigarette smoke in 16HBE14o- cell line (p<0.001 in 3 experiments), primary nasal epithelial cells (p<0.01 in 2 experiments), and in smoke-exposed mice (p<0.001, n = 6 animals per group). Spns2 was localized to cilia in primary bronchial epithelial cells. In both macrophage and epithelial cell types, Spns2 was also found localized to cytoplasm and the nucleus, in line with a predicted bipartile Nuclear Localization Signal at the position aa282 of the human Spns2 sequence. In smoke-exposed mice, alveolar macrophage phagocytic function positively correlated with Spns2 protein expression in bronchial epithelial cells. CONCLUSION: Our data suggest that the epithelium may be the major source for extracellular S1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via Spns2-mediated S1P signaling in COPD and in response to cigarette smoke exposure.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Lisofosfolipídeos/metabolismo , Macrófagos Alveolares/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Animais , Estudos de Casos e Controles , Células Cultivadas , Fumar Cigarros , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Camundongos , Fagocitose , Esfingosina/metabolismo , Frações Subcelulares/metabolismoRESUMO
The proteasome inhibitor bortezomib has proven to be invaluable in the treatment of myeloma. By exploiting the inherent high immunoglobulin protein production of malignant plasma cells, bortezomib induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in myeloma cell death. In most cases, however, the disease remains incurable highlighting the need for new therapeutic targets. Sphingosine kinase 2 (SK2) has been proposed as one such therapeutic target for myeloma. Our observations that bortezomib and SK2 inhibitors independently elicited induction of ER stress and the UPR prompted us to examine potential synergy between these agents in myeloma. Targeting SK2 synergistically contributed to ER stress and UPR activation induced by bortezomib, as evidenced by activation of the IRE1 pathway and stress kinases JNK and p38MAPK, thereby resulting in potent synergistic myeloma apoptosis in vitro. The combination of bortezomib and SK2 inhibition also exhibited strong in vivo synergy and favourable effects on bone disease. Therefore, our studies suggest that perturbations of sphingolipid signalling can synergistically enhance the effects seen with proteasome inhibition, highlighting the potential for the combination of these two modes of increasing ER stress to be formally evaluated in clinical trials for the treatment of myeloma patients.
Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mieloma Múltiplo/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Modelos Biológicos , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Inibidores de Proteassoma/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sphingosine kinase 1 (SK1) is a key regulator of the cellular balance between proapoptotic and prosurvival sphingolipids. Oncogenic signaling by SK1 relies on its localization to the plasma membrane, which is mediated by the calcium and integrin binding protein CIB1 via its Ca2+-myristoyl switch function. Here we show that another member of the CIB family, CIB2, plays a surprisingly opposite role to CIB1 in the regulation of SK1 signaling. CIB2 bound SK1 on the same site as CIB1, yet it lacks the Ca2+-myristoyl switch function. As a result, CIB2 blocked translocation of SK1 to the plasma membrane and inhibited its subsequent signaling, which included sensitization to TNFα-induced apoptosis and inhibition of Ras-induced neoplastic transformation. CIB2 was significantly downregulated in ovarian cancer and low CIB2 expression was associated with poor prognosis in ovarian cancer patients. Notably, reintroduction of CIB2 in ovarian cancer cells blocked plasma membrane localization of endogenous SK1, reduced in vitro neoplastic growth and tumor growth in mice, and suppressed cell motility and invasiveness both in vitro and in vivo Consistent with the in vitro synergistic effects between the SK1-specific inhibitor SK1-I and standard chemotherapeutics, expression of CIB2 also sensitized ovarian cancer cells to carboplatin. Together, these findings identify CIB2 as a novel endogenous suppressor of SK1 signaling and potential prognostic marker and demonstrate the therapeutic potential of SK1 in this gynecologic malignancy. Cancer Res; 77(18); 4823-34. ©2017 AACR.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proteínas de Ligação ao Cálcio/genética , Movimento Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sphingosine kinase (SK) 1 and 2 are lipid kinases that catalyse the formation of sphingosine 1-phosphate (S1P), a potent signalling molecule with a wide array of cellular effects. SK1 and 2 have been shown to be up-regulated in tumours and their genetic ablation or inhibition has been shown to slow tumour growth as well as sensitise cancer cells to chemotherapeutics. The SKs have been extensively studied, with a plethora of inhibitors developed that target the sphingosine-binding pocket of the enzyme, some with nanomolar affinities. Recently, inhibitors targeting the ATP pocket of SK have also been described. Here we discuss the development of these new small molecule SK inhibitors, summarise the recent discovery of off-targets effects of many current SK inhibitors, and provide an overview of the usefulness of these inhibitors as in vitro tools and therapeutic agents.