RESUMO
Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid-the apicoplast-which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.
Assuntos
Apicoplastos/genética , Genes Essenciais , Mutação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Triose-Fosfato Isomerase/genética , Apicoplastos/metabolismo , Sistemas CRISPR-Cas , Eritrócitos/parasitologia , Ontologia Genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Anotação de Sequência Molecular , Mutagênese , Biogênese de Organelas , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Triose-Fosfato Isomerase/metabolismo , Sequenciamento Completo do Genoma , Proteína Vermelha FluorescenteRESUMO
A series of novel thiazole-containing amides were synthesized. A structure-activity relationship study of these compounds led to the identification of potent and selective PfFPPS/GGPPS inhibitors with good in vitro ADME profiles. The most promising candidate molecules were progressed to mouse in vivo PK studies and demonstrated adequate free drug exposure to warrant further investigation.
Assuntos
Antimaláricos/farmacologia , Difosfonatos/farmacologia , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Geraniltranstransferase/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/síntese química , Antimaláricos/química , Difosfonatos/síntese química , Difosfonatos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Farnesiltranstransferase/metabolismo , Geraniltranstransferase/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Relação Estrutura-AtividadeRESUMO
Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.
Assuntos
Lipídeos/química , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Sítios de Ligação , Membrana Celular/metabolismo , Medição da Troca de Deutério , Células HCT116 , Humanos , Lisofosfolipídeos/biossíntese , Espectrometria de Massas , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Transdução de Sinais , Esfingosina/biossíntese , Esfingosina/metabolismoRESUMO
Sphingolipids represent an important class of bioactive signaling lipids which have key roles in numerous cellular processes. Over the last few decades, the levels of bioactive sphingolipids and/or their metabolizing enzymes have been realized to be important factors involved in disease development and progression, most notably in cancer. Targeting sphingolipid-metabolizing enzymes in disease states has been the focus of many studies and has resulted in a number of pharmacological inhibitors, with some making it into the clinic as therapeutics. In order to better understand the regulation of sphingolipid-metabolizing enzymes as well as to develop much more potent and specific inhibitors, the field of sphingolipids has recently taken a turn toward structural biology. The last decade has seen the structural determination of a number of sphingolipid enzymes and effector proteins. In these terms, one of the most complete arms of the sphingolipid pathway is the sphingosine-1-phosphate (S1P) arm. The structures of proteins involved in the function and regulation of S1P are being used to investigate further the regulation of said proteins as well as in the design and development of inhibitors as potential therapeutics.
Assuntos
Lisofosfolipídeos/metabolismo , Modelos Moleculares , Sistemas do Segundo Mensageiro , Esfingosina/análogos & derivados , Aldeído Liases/antagonistas & inibidores , Aldeído Liases/química , Aldeído Liases/metabolismo , Animais , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Lisofosfolipídeos/química , Moduladores de Transporte de Membrana , Conformação Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/química , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/química , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMO
Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite which has been implicated in many diseases including cancer and inflammatory diseases. Recently, sphingosine kinase 1 (SK1), one of the isozymes which generates S1P, has been implicated in the development and progression of inflammatory bowel disease (IBD). Based on our previous work, we set out to determine the efficacy of a novel SK1 selective inhibitor, LCL351, in a murine model of IBD. LCL351 selectively inhibits SK1 both in vitro and in cells. LCL351, which accumulates in relevant tissues such as colon, did not have any adverse side effects in vivo. In mice challenged with dextran sodium sulfate (DSS), a murine model for IBD, LCL351 treatment protected from blood loss and splenomegaly. Additionally, LCL351 treatment reduced the expression of pro-inflammatory markers, and reduced neutrophil infiltration in colon tissue. Our results suggest inflammation associated with IBD can be targeted pharmacologically through the inhibition and degradation of SK1. Furthermore, our data also identifies desirable properties of SK1 inhibitors.
Assuntos
Colite/tratamento farmacológico , Colite/imunologia , Sulfato de Dextrana/efeitos adversos , Guanidinas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Esfingosina/farmacologia , Células A549 , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Colite/induzido quimicamente , Colite/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Guanidinas/uso terapêutico , Humanos , Esfingosina/uso terapêutico , Fator de Necrose Tumoral alfa/genéticaRESUMO
Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was â¼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an â¼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an â¼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/etiologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Quinase 1 de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Renais/etiologia , Neoplasias Renais/patologia , Lisofosfolipídeos/biossíntese , Invasividade Neoplásica , Neovascularização Patológica , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Receptores de Esfingosina-1-Fosfato , Regulação para CimaRESUMO
The bioactive sphingolipid sphingosine-1-phosphate (S1P) mediates cellular proliferation, mitogenesis, inflammation, and angiogenesis. These biologies are mediated through S1P binding to specific GPCRs [sphingosine-1-phosphate receptor (S1PR)1-5] and some other less well-characterized intracellular targets. Ezrin-radixin-moesin (ERM) proteins, a family of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, are emerging as critical regulators of cancer invasion via regulation of cell morphology and motility. Recently, we identified S1P as an acute ERM activator (via phosphorylation) through its action on S1PR2. In this work, we dissect the mechanism of S1P generation downstream of epidermal growth factor (EGF) leading to ERM phosphorylation and cancer invasion. Using pharmacologic inhibitors, small interfering RNA technologies, and genetic approaches, we demonstrate that sphingosine kinase (SK)2, and not SK1, is essential and sufficient in EGF-mediated ERM phosphorylation in HeLa cells. In fact, knocking down SK2 decreased ERM activation 2.5-fold. Furthermore, we provide evidence that SK2 is necessary to mediate EGF-induced invasion. In addition, overexpressing SK2 causes a 2-fold increase in HeLa cell invasion. Surprisingly, and for the first time, we find that this event, although dependent on S1PR2 activation, does not generate and does not require extracellular S1P secretion, therefore introducing a potential novel model of autocrine/intracrine action of S1P that still involves its GPCRs. These results define new mechanistic insights for EGF-mediated invasion and novel actions of SK2, therefore setting the stage for novel targets in the treatment of growth factor-driven malignancies.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Lisofosfolipídeos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Comunicação Autócrina/genética , Proteínas do Citoesqueleto/genética , Fator de Crescimento Epidérmico/genética , Células HeLa , Humanos , Lisofosfolipídeos/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMO
Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently identified, in a DNA-templated macrocycle library, inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated 'gatekeeper' mutant. We solved two cocrystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bisubstrate-competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance.
Assuntos
Compostos Macrocíclicos/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Células 3T3 , Animais , Ligação Competitiva , Cristalografia por Raios X , DNA/química , Humanos , Camundongos , Estrutura Molecular , Mutação , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-hck/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Sphingolipids and their synthetic enzymes have emerged as critical mediators in numerous diseases including inflammation, aging, and cancer. One enzyme in particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P), has been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. In this review, we will discuss the contributions from the laboratory of Dr. Lina M. Obeid that have defined the roles for several bioactive sphingolipids in signaling and disease with an emphasis on her work defining SK1 in cellular fates and pathobiologies including proliferation, senescence, apoptosis, and inflammation.
Assuntos
Envelhecimento/metabolismo , Lisofosfolipídeos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Envelhecimento/genética , Envelhecimento/patologia , Animais , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Laboratórios , Lisofosfolipídeos/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Esfingolipídeos/genética , Esfingosina/genética , Esfingosina/metabolismoRESUMO
Plasmodium parasites and related apicomplexans contain an essential "complex plastid" organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX protease and bacteriocin processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX postprenylation processing. Indeed, AMR4 is the only putative CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 (E352) is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX postprenylation processing pathway in Plasmodium falciparum Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead it performs a conserved, uncharacterized chloroplast function that has been retained for complex plastid biogenesis.IMPORTANCEPlasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but nonphotosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hot spot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens.
Assuntos
Biogênese de Organelas , Peptídeo Hidrolases/genética , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plastídeos/fisiologia , Proteínas de Protozoários/genética , Cianobactérias/enzimologia , Cianobactérias/genética , Eritrócitos/parasitologia , Hemiterpenos/metabolismo , Humanos , Malária/parasitologia , Compostos Organofosforados/metabolismo , Peptídeo Hidrolases/classificação , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismoRESUMO
Within the last 3 decades, there has been intense study of bioactive sphingolipids and the enzymes which metabolize those lipids. One enzyme is the critical lipid kinase sphingosine kinase 1 (SK1), which produces the potent and pleiotropic signaling lipid, sphingosine 1-phosphate (S1P). SK1 and S1P have been implicated in a host of different diseases including cancer, chronic inflammation, and metabolic diseases. However, while there is ample knowledge about the importance of these molecules in the development and progression of disease there is a dearth of knowledge of the molecular mechanisms which regulate SK1 function. In this review, we will cover some of the more recent and exciting findings about the different ways SK1 function can be regulated, from transcriptional regulation to protein stability. Finally, we will delve into recent structural insights into SK1 and how they might relate to function at cell membranes.
Assuntos
Membrana Celular/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Animais , Ativação Enzimática , Estabilidade Enzimática , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/química , Biossíntese de Proteínas , Transdução de Sinais , Esfingosina/metabolismo , Transcrição GênicaRESUMO
The recently discovered CHK1-Suppressed (CS) pathway is activated by inhibition or loss of the checkpoint kinase CHK1, promoting an apoptotic response to DNA damage mediated by caspase-2 in p53-deficient cells. Although functions of the CS-pathway have been investigated biochemically, it remains unclear whether and how CHK1 inhibition can be regulated endogenously and whether this constitutes a key component of the DNA damage response (DDR). Here, we present data that define the first endogenous activation of the CS-pathway whereby, upon DNA damage, wild type p53 acts as an endogenous regulator of CHK1 levels that modulates caspase-2 activation. Moreover, we demonstrate that persistence of CHK1 levels in response to DNA damage in p53-deficient cancer cells, leads to CHK1-mediated activation of NF-κB and induction of NF-κB-regulated genes in cells and in associated tumor-derived microvesicles (TMVs), both of which are abrogated by loss or inhibition of CHK1. These data define a novel role for CHK1 in the DDR pathway as a regulator NF-κB activity. Our data provide evidence that targeting CHK1 in p53-deficient cancers may abrogate NF-κB signaling that is associated with increased cellular survival and chemoresistance.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , NF-kappa B/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Caspase 2/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/genética , Cisteína Endopeptidases/metabolismo , Doxorrubicina/farmacologia , Ativação Enzimática , Células HCT116 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Células MCF-7 , Camundongos , Inibidor de NF-kappaB alfa/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate. As an enzyme regulating the balance of ceramide and sphingosine-1-phosphate, nCDase is emerging as a therapeutic target for cancer. Here, we present the 2.6-Å crystal structure of human nCDase in complex with phosphate that reveals a striking, 20-Å deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases. Utilizing flexible ligand docking, we predict a likely binding mode for ceramide that superimposes closely with the crystallographically observed transition state analog phosphate. Our results suggest that nCDase uses a new catalytic strategy for Zn(2+)-dependent amidases, and generates ceramide specificity by sterically excluding sphingolipids with bulky headgroups and specifically recognizing the small hydroxyl head group of ceramide. Together, these data provide a foundation to aid drug development and establish common themes for how proteins recognize the bioactive lipid ceramide.