RESUMO
BACKGROUND: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs. METHODS AND RESULTS: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes. CONCLUSIONS: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment.
Assuntos
Aciltransferases , Neoplasias , Fosforilação Oxidativa , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genética , Linhagem Celular Tumoral , Fosforilação Oxidativa/efeitos dos fármacos , Aciltransferases/metabolismo , Ácido Mirístico/metabolismo , Proteômica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , MultiômicaRESUMO
Myristoylation, the N-terminal addition of the fatty acid myristate to proteins, regulates membrane-bound signal transduction pathways important in cancer cell biology. This modification is catalyzed by two N-myristoyltransferases, NMT1 and NMT2. Zelenirstat is a first-in-class potent oral small molecule inhibitor of both NMT1 and NMT2 proteins. Patients with advanced solid tumors and relapsed/refractory (R/R) B-cell lymphomas were enrolled in an open label, phase I dose escalation trial of oral daily zelenirstat, administered in 28-day cycles until progression or unacceptable toxicity. The endpoints were to evaluate dose-limiting toxicities (DLT) to establish a maximum tolerated dose (MTD), pharmacokinetic parameters, and anticancer activity. Twenty-nine patients were enrolled (25 advanced solid tumor; 4 R/R B-cell lymphoma) and 24 were DLT-evaluable. Dosing ranged from 20 mg once daily (OD) to 210 mg OD without DLT, but gastrointestinal DLTS were seen in the 280 mg cohort. MTD and recommended phase 2 dose were 210 mg OD. Common adverse events were predominantly Gr ≤ 2 nausea, vomiting, diarrhea, and fatigue. Plasma concentrations peaked at 2 h with terminal half-lives averaging 10 h. Steady state was achieved by day 15, and higher doses achieved trough concentrations predicted to be therapeutic. Stable disease as best response was seen in eight (28%) patients. Progression-free survival and overall survival were significantly better in patients receiving 210 mg OD compared to those receiving lower doses. Zelenirstat is well-tolerated, achieves plasma exposures expected for efficacy, and shows early signs of anticancer activity. Further clinical development of zelenirstat is warranted.
Assuntos
Aciltransferases , Linfoma de Células B , Dose Máxima Tolerável , Humanos , Pessoa de Meia-Idade , Feminino , Masculino , Idoso , Adulto , Administração Oral , Linfoma de Células B/tratamento farmacológico , Aciltransferases/antagonistas & inibidores , Antineoplásicos/farmacocinética , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Antineoplásicos/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Recidiva Local de Neoplasia/tratamento farmacológicoRESUMO
PURPOSE: N-myristoyltransferases 1 and 2 (NMT1 and NMT2) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. Myristoylation regulates numerous membrane-bound signal transduction pathways important in cancer biology and the pan-NMT inhibitor PCLX-001 is approaching clinical development as a cancer therapy. The tissue distribution, relative abundances, and prognostic value of the two human NMTs remain poorly understood. METHODS: We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to human NMT1 and NMT2. These mAbs were used to perform immunohistochemical analysis of the abundance and distribution of NMT1 and NMT2 in normal breast epithelial samples and a large cohort of primary breast adenocarcinomas from the BCIRG001 clinical trial (n = 706). RESULTS: NMT1 protein was readily quantified in normal and most transformed breast epithelial tissue and was associated with higher overall histologic grade, higher Ki67, and lower hormone receptor expression. While NMT2 protein was readily detected in normal breast epithelial tissue, it was undetectable in the majority of breast cancers. Detectable NMT2 protein correlated with significantly poorer overall survival (hazard ratio 1.36; P = 0.029) and worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. Treatment of cultured breast cancer cells with PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts produced significant dose-dependent tumor growth inhibition in vivo. CONCLUSIONS: These results support further evaluation of NMT immunohistochemistry for patient selection and clinical trials of NMT inhibition in breast cancer patients.
Assuntos
Neoplasias da Mama , Preparações Farmacêuticas , Aciltransferases/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Camundongos , PrognósticoRESUMO
BACKGROUND: Multiple organ dysfunction is a common cause of morbidity and mortality in intensive care units (ICUs). Original development of the Sequential Organ Failure Assessment (SOFA) score was not to predict outcome, but to describe temporal changes in organ dysfunction in critically ill patients. Organ dysfunction scoring may be a reasonable surrogate outcome in clinical trials but further exploration of the impact of case mix on the temporal sequence of organ dysfunction is required. Our aim was to compare temporal changes in SOFA scores between hospital survivors and non-survivors. METHODS: We performed a population-based observational retrospective cohort study of critically ill patients admitted from January 1, 2004, to December 31, 2013, to 4 multisystem adult intensive care units (ICUs) in Calgary, Canada. The primary outcome was temporal changes in daily SOFA scores during the first 14 days of ICU admission. SOFA scores were modeled between hospital survivors and non-survivors using generalized estimating equations (GEE) and were also stratified by admission SOFA (≤ 11 versus > 11). RESULTS: The cohort consisted of 20,007 patients with at least one SOFA score and was mostly male (58.2%) with a median age of 59 (interquartile range [IQR] 44-72). Median ICU length of stay was 3.5 (IQR 1.7-7.5) days. ICU and hospital mortality were 18.5% and 25.5%, respectively. Temporal change in SOFA scores varied by survival and admission SOFA score in a complicated relationship. Area under the receiver operating characteristic (ROC) curve using admission SOFA as a predictor of hospital mortality was 0.77. The hospital mortality rate was 5.6% for patients with an admission SOFA of 0-2 and 94.4% with an admission SOFA of 20-24. There was an approximately linear increase in hospital mortality for SOFA scores of 3-19 (range 8.7-84.7%). CONCLUSIONS: Examining the clinical course of organ dysfunction in a large non-selective cohort of patients provides insight into the utility of SOFA. We have demonstrated that hospital outcome is associated with both admission SOFA and the temporal rate of change in SOFA after admission. It is necessary to further explore the impact of additional clinical factors on the clinical course of SOFA with large datasets.
Assuntos
Insuficiência de Múltiplos Órgãos/classificação , Insuficiência de Múltiplos Órgãos/fisiopatologia , Projetos de Pesquisa/normas , Fatores de Tempo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alberta , Estudos de Coortes , Feminino , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos/complicações , Escores de Disfunção Orgânica , Projetos de Pesquisa/tendências , Estudos Retrospectivos , Análise de Sobrevida , Sobreviventes/estatística & dados numéricosRESUMO
BACKGROUND: Admission to the intensive care unit (ICU) outside daytime hours has been shown to be variably associated with increased morbidity and mortality. We aimed to describe the characteristics and outcomes of patients admitted to the ICU afterhours (22:00-06:59 h) in a large Canadian health region. We further hypothesized that the association between afterhours admission and mortality would be modified by indicators of strained ICU capacity. METHODS: This is a population-based cohort study of 12,265 adults admitted to nine ICUs in Alberta from June 2012 to December 2014. We used a path-analysis modeling strategy and mixed-effects multivariate regression analysis to evaluate direct and integrated associations (mediated through Acute Physiology and Chronic Health Evaluation (APACHE) II score) between afterhours admission (22:00-06:59 h) and ICU mortality. Further analysis examined the effects of strained ICU capacity and varied definitions of afterhours and weekend admissions. ICU occupancy ≥ 90% or clustering of admissions (≥ 0.15, defined as number of admissions 2 h before or after the index admission, divided by the number of ICU beds) were used as indicators of strained capacity. RESULTS: Of 12,265 admissions, 34.7% (n = 4251) occurred afterhours. The proportion of afterhours admissions varied amongst ICUs (range 26.7-37.8%). Patients admitted afterhours were younger (median (IQR) 58 (44-70) vs 60 (47-70) years, p < 0.0001), more likely to have a medical diagnosis (75.9% vs 72.1%, p < 0.0001), and had higher APACHE II scores (20.9 (8.6) vs 19.9 (8.3), p < 0.0001). Crude ICU mortality was greater for those admitted afterhours (15.9% vs 14.1%, p = 0.007), but following multivariate adjustment there was no direct or integrated effect on ICU mortality (odds ratio (OR) 1.024; 95% confidence interval (CI) 0.923-1.135, p = 0.658). Furthermore, direct and integrated analysis showed no association of afterhours admission and hospital mortality (p = 0.90) or hospital length of stay (LOS) (p = 0.27), although ICU LOS was shorter (p = 0.049). Early-morning admission (00:00-06:59 h) with ICU occupancy ≥ 90% was associated with short-term (≤ 7 days) and all-cause ICU mortality. CONCLUSIONS: One-third of critically ill patients are admitted to the ICU afterhours. Afterhours ICU admission was not associated with greater mortality risk in most circumstances but was sensitive to strained ICU capacity.
Assuntos
Plantão Médico/normas , Número de Leitos em Hospital/estatística & dados numéricos , Mortalidade Hospitalar , APACHE , Plantão Médico/estatística & dados numéricos , Alberta , Estudos de Coortes , Número de Leitos em Hospital/normas , Hospitalização/estatística & dados numéricos , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Estudos Retrospectivos , Fatores de TempoRESUMO
Transient receptor potential polycystin-3 (TRPP3) is a cation channel activated by calcium and proton and is involved in hedgehog signaling, intestinal development, and sour tasting. How TRPP3 channel function is regulated remains poorly understood. By N-terminal truncation mutations, electrophysiology, and Xenopus oocyte expression, we first identified fragment Asp-21-Ser-42 to be functionally important. We then found that deletion mutant Δ1-36 (TRPP3 missing fragment Met-1-Arg-36) has a similar function as wild-type TRPP3, whereas Δ1-38 is functionally dead, suggesting the importance of Val-37 or Cys-38. Further studies found that Cys-38, but not Val-37, is functionally critical. Cys-38 is a predicted site of palmitoylation, and indeed TRPP3 channel activity was inhibited by palmitoylation inhibitor 2-bromopalmitate and rescued by palmitoylation substrate palmitic acid. The TRPP3 N terminus (TRPP3NT, Met-1-Leu-95) localized along the plasma membrane of HEK293 cells but stayed in the cytoplasm with 2-bromopalmitate treatment or C38A mutation, indicating that TRPP3NT anchors to the surface membrane through palmitoylation at Cys-38. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When putative phosphorylation sites near Cys-38 were mutated to Asp or Glu to mimic phosphorylation, only T39D and T39E reduced TRPP3 function. Furthermore, TRPP3NT displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. However, palmitoylation at Cys-38 and phosphorylation at Thr-39 independently regulated TRPP3 channel function, in contrast to previous reports about correlated palmitoylation with a proximate phosphorylation. Palmitoylation at Cys-38 represents a novel mechanism of functional regulation for TRPP3.
Assuntos
Canais de Cálcio/metabolismo , Lipoilação/fisiologia , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Canais de Cálcio/genética , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Fosforilação/fisiologia , Domínios Proteicos , Receptores de Superfície Celular/genética , Deleção de Sequência , Xenopus laevisRESUMO
Acyl CoA:2-monoacylglycerol acyltransferase (MGAT)-2 has an important role in dietary fat absorption in the intestine. MGAT2 resides in the endoplasmic reticulum and catalyzes the synthesis of diacylglycerol which is then utilized as a substrate for triacylglycerol synthesis. This triacylglycerol is then incorporated into chylomicrons which are released into the circulation. In this study, we determined the membrane topology of human MGAT2. Protease protection experiments showed that the C-terminus is exposed to the cytosol, while the N-terminus is partially buried in the ER membrane. MGAT2, like murine DGAT2, was found to have two transmembrane domains. We also identified a region of MGAT2 associated with the ER membrane that contains the histidine-proline-histidine-glycine sequence present in all DGAT2 family members that is thought to comprise the active site. Proteolysis experiments demonstrated that digestion of total cellular membranes from cells expressing MGAT2 with trypsin abolished MGAT activity, indicating that domains that are important for catalysis face the cytosol. We also explored the role that the five cysteines residues present in MGAT2 have in catalysis. MGAT activity was sensitive to two thiol modifiers, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). Furthermore, mutation of four cysteines resulted in a reduction in MGAT activity. However, when the C-terminal cysteine (C334) was mutated, MGAT activity was actually higher than that of wild-type FL-MGAT2. Lastly, we determined that both transmembrane domains of MGAT2 are important for its ER localization, and that MGAT2 is present in mitochondrial-associated membranes.
Assuntos
Retículo Endoplasmático/metabolismo , Mucosa Intestinal/metabolismo , Lipogênese/genética , N-Acetilglucosaminiltransferases/genética , Acil Coenzima A/metabolismo , Animais , Células COS , Chlorocebus aethiops , Diglicerídeos/biossíntese , Retículo Endoplasmático/enzimologia , Humanos , Intestinos/enzimologia , Membranas/enzimologia , Membranas/metabolismo , Camundongos , Mitocôndrias/metabolismo , N-Acetilglucosaminiltransferases/biossíntese , Triglicerídeos/biossínteseRESUMO
Huntington disease (HD) is a debilitating neurodegenerative disease characterized by the loss of motor control and cognitive ability that ultimately leads to death. It is caused by the expansion of a polyglutamine tract in the huntingtin (HTT) protein, which leads to aggregation of the protein and eventually cellular death. Both the wild-type and mutant form of the protein are highly regulated by post-translational modifications including proteolysis, palmitoylation and phosphorylation. We now demonstrate the existence of a new post-translational modification of HTT: the addition of the 14 carbon fatty acid myristate to a glycine residue exposed on a caspase-3-cleaved fragment (post-translational myristoylation) and that myristoylation of this fragment is altered in a physiologically relevant model of mutant HTT. Myristoylated HTT553-585-EGFP, but not its non-myristoylated variant, initially localized to the ER, induced the formation of autophagosomes and accumulated in abnormally large autophagolysosomal/lysosomal structures in a variety of cell types, including neuronal cell lines under nutrient-rich conditions. Our results suggest that accumulation of myristoylated HTT553-586 in cells may alter the rate of production of autophagosomes and/or their clearance through the heterotypic autophagosomal/lysosomal fusion process. Overall, our novel observations establish a role for the post-translational myristoylation of a caspase-3-cleaved fragment of HTT, highly similar to the Barkor/ATG14L autophagosome-targeting sequence domain thought to sense, maintain and/or promote membrane curvature in the regulation of autophagy. Abnormal processing or production of this myristoylated HTT fragment might be involved in the pathophysiology of HD.
Assuntos
Caspase 3/metabolismo , Glicina/metabolismo , Ácido Mirístico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Proteína Huntingtina , Lisossomos/metabolismo , Proteínas do Tecido Nervoso/química , Fagossomos/metabolismoRESUMO
The mitochondria-associated membrane (MAM) is a domain of the endoplasmic reticulum (ER) that mediates the exchange of ions, lipids and metabolites between the ER and mitochondria. ER chaperones and oxidoreductases are critical components of the MAM. However, the localization motifs and mechanisms for most MAM proteins have remained elusive. Using two highly related ER oxidoreductases as a model system, we now show that palmitoylation enriches ER-localized proteins on the MAM. We demonstrate that palmitoylation of cysteine residue(s) adjacent to the membrane-spanning domain promotes MAM enrichment of the transmembrane thioredoxin family protein TMX. In addition to TMX, our results also show that calnexin shuttles between the rough ER and the MAM depending on its palmitoylation status. Mutation of the TMX and calnexin palmitoylation sites and chemical interference with palmitoylation disrupt their MAM enrichment. Since ER-localized heme oxygenase-1, but not cytosolic GRP75 require palmitoylation to reside on the MAM, our findings identify palmitoylation as key for MAM enrichment of ER membrane proteins.
Assuntos
Calnexina/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Calnexina/química , Calnexina/genética , Linhagem Celular Tumoral , Cisteína/metabolismo , Cães , Células HeLa , Heme Oxigenase-1/metabolismo , Humanos , Lipoilação , Melanoma/patologia , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.
Assuntos
Diacilglicerol O-Aciltransferase/metabolismo , Retículo Endoplasmático/metabolismo , Triglicerídeos/química , Animais , Células COS , Fracionamento Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Diacilglicerol O-Aciltransferase/química , Diacilglicerol O-Aciltransferase/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Ácido Oleico/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Triglicerídeos/biossínteseRESUMO
The palmitoylation of calnexin serves to enrich calnexin on the mitochondria-associated membrane (MAM). Given a lack of information on the significance of this finding, we have investigated how this endoplasmic reticulum (ER)-internal sorting signal affects the functions of calnexin. Our results demonstrate that palmitoylated calnexin interacts with sarcoendoplasmic reticulum (SR) Ca(2+) transport ATPase (SERCA) 2b and that this interaction determines ER Ca(2+) content and the regulation of ER-mitochondria Ca(2+) crosstalk. In contrast, non-palmitoylated calnexin interacts with the oxidoreductase ERp57 and performs its well-known function in quality control. Interestingly, our results also show that calnexin palmitoylation is an ER-stress-dependent mechanism. Following a short-term ER stress, calnexin quickly becomes less palmitoylated, which shifts its function from the regulation of Ca(2+) signaling towards chaperoning and quality control of known substrates. These changes also correlate with a preferential distribution of calnexin to the MAM under resting conditions, or the rough ER and ER quality control compartment (ERQC) following ER stress. Our results have therefore identified the switch that assigns calnexin either to Ca(2+) signaling or to protein chaperoning.
Assuntos
Calnexina/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Lipoilação/fisiologia , Membranas Mitocondriais/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Células 3T3 , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Retículo Endoplasmático/metabolismo , Fibroblastos , Células HEK293 , Células HeLa , Humanos , Camundongos , Mitocôndrias/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismoRESUMO
Calnexin is a type 1 integral endoplasmic reticulum (ER) membrane molecular chaperone with a highly conserved C-terminal domain oriented to the cytoplasm. Protein N-myristoylation plays an important role in a wide variety of cellular signal transduction pathways and it is catalyzed by N-myristoyltransferase (NMT), a cytoplasmic and ER associated enzyme. Here using yeast two-hybrid screen, Western blot analysis, immunoprecipitation, immunolocalization and cellular fractionation we discovered that N-myristoyltransferase 1 interacts with calnexin at the ER. These observations point at a previously unrecognized contribution of calnexin to the retention of NMT1 at the ER membrane.
Assuntos
Aciltransferases/metabolismo , Calnexina/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Frações Subcelulares/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Células Cultivadas , Ativação Enzimática , Camundongos , Ligação Proteica , Especificidade por Substrato , Distribuição TecidualRESUMO
ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol efflux onto lipidated apolipoprotein A-I and HDL and plays a role in various important physiological functions. However, the mechanism by which ABCG1 mediates cholesterol translocation is unclear. Protein palmitoylation regulates many functions of proteins such as ABCA1. Here we investigated if ABCG1 is palmitoylated and the subsequent effects on ABCG1-mediated cholesterol efflux. We demonstrated that ABCG1 is palmitoylated in both human embryonic kidney 293 cells and in mouse macrophage, J774. Five cysteine residues located at positions 26, 150, 311, 390 and 402 in the NH2-terminal cytoplasmic region of ABCG1 were palmitoylated. Removal of palmitoylation at Cys311 by mutating the residue to Ala (C311A) or Ser significantly decreased ABCG1-mediated cholesterol efflux. On the other hand, removal of palmitoylation at sites 26, 150, 390 and 402 had no significant effect. We further demonstrated that mutations of Cys311 affected ABCG1 trafficking from the endoplasmic reticulum. Therefore, our data suggest that palmitoylation plays a critical role in ABCG1-mediated cholesterol efflux through the regulation of trafficking.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Rim/metabolismo , Lipoproteínas/metabolismo , Lipoilação , Macrófagos/metabolismo , Processamento de Proteína Pós-Traducional , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Células Cultivadas , Colesterol/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Imunofluorescência , Humanos , Rim/citologia , Lipoproteínas/genética , Macrófagos/citologia , Camundongos , Mutagênese Sítio-Dirigida , Mutação/genética , Transporte ProteicoRESUMO
Myristoylation occurs cotranslationally on nascent proteins and post-translationally during apoptosis after caspase cleavages expose cryptic myristoylation sites. We demonstrate a drastic change in the myristoylated protein proteome in apoptotic cells, likely as more substrates are revealed by caspases. We show for the first time that both N-myristoyltransferases (NMTs) 1 and 2 are cleaved during apoptosis and that the caspase-3- or -8-mediated cleavage of NMT1 at Asp-72 precedes the cleavage of NMT2 by caspase-3 mainly at Asp-25. The cleavage of NMTs did not significantly affect their activity in apoptotic cells until the 8 h time point. However, the cleavage of the predominantly membrane bound NMT1 (64%) removed a polybasic domain stretch and led to a cytosolic relocalization (>55%), whereas predominantly cytosolic NMT2 (62%) relocalized to membranes when cleaved (>80%) after the removal of a negatively charged domain. The interplay between caspases and NMTs during apoptosis is of particular interest since caspases may not only control the rates of substrate production but also their myristoylation rate by regulating the location and perhaps the specificity of NMTs. Since apoptosis is often suppressed in cancer, the reduced caspase activity seen in cancer cells might also explain the higher NMT levels observed in many cancers.
Assuntos
Aciltransferases/metabolismo , Apoptose/fisiologia , Caspases/metabolismo , Ácidos Mirísticos/metabolismo , Aciltransferases/química , Aciltransferases/genética , Substituição de Aminoácidos , Animais , Células COS , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Caspases/química , Chlorocebus aethiops , Células HeLa , Humanos , Células Jurkat , Células MCF-7 , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Modificação Traducional de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismo , Especificidade por SubstratoRESUMO
Myristoylation, the addition of a 14-carbon fatty acid to the N-terminal glycine of a protein, is key to protein-membrane and protein-protein interactions. Typically, myristoylation occurs cotranslationally; however, post-translational myristoylation of caspase-cleaved proteins is now emerging as a well-established protein modification and as a novel regulator of apoptosis. To identify additional post-translationally myristoylated proteins, we engineered a plasmid vector encoding for a caspase-cleavable reporter protein named tandem reporter assay for myristoylation of proteins post-translationally (TRAMPP). pTRAMPP consists of tdTomato-DEVD-"test myristoylation sequence"-enhanced green fluorescent protein (EGFP). After induction of apoptosis, the reporter protein is cleaved by caspases, which frees a new N-terminal glycine residue attached to EGFP that can be myristoylated. We used pTRAMPP in appropriately transfected cells to identify 7 post-translationally myristoylated proteins. First, we confirmed the post-translational myristoylation of two previously identified putative substrates, cytoplasmic dynein intermediate chain 2A and PKCε (ctPKCε), and identified 5 more caspase-cleaved potential substrates for myristoylation that include the antiapoptotic regulator of apoptosis, Mcl-1, and the causative agent of Huntington's disease, huntingtin protein. Further investigation revealed that post-translationally myristoylated ctPKCε localized to membranes and increased Erk signaling and degradation of the proapoptotic protein Bim, which prevented a significant loss of mitochondrial potential of 17% over nonmyristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Taken together, these findings suggest a possible antiapoptotic role for post-translationally myristoylated caspase-cleaved ctPKCε.
Assuntos
Clonagem Molecular/métodos , Proteínas de Fluorescência Verde/genética , Ácido Mirístico/metabolismo , Plasmídeos/genética , Proteína Quinase C-épsilon/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Apoptose/fisiologia , Células COS , Caspases/metabolismo , Chlorocebus aethiops , Genes Reporter/genética , Vetores Genéticos/genética , Células HeLa , Humanos , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Transfecção/métodos , Quinases Ativadas por p21/metabolismoRESUMO
Cullin (Cul)-based E3 ubiquitin ligases are activated through the attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 E3-ligase by interacting with its Cul substrates and the Nedd8 E2 Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each containing a C-terminal potentiating neddylation domain but distinct amino-terminal extensions. Although the UBA-containing DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, DCNL3 also interacts with human Culs and is able to complement the neddylation defect of yeast dcn1Delta cells. DCNL3 down-regulation by RNAi decreases Cul neddylation, and overexpression of a Cul3 mutant deficient in DCNL3 binding interferes with Cul3 function in vivo. Interestingly, DCNL3 accumulates at the plasma membrane through a conserved, lipid-modified motif at the N terminus. Membrane-bound DCNL3 is able to recruit Cul3 to membranes and is functionally important for Cul3 neddylation in vivo. We conclude that DCNL proteins function as nonredundant Cul Nedd8-E3 ligases. Moreover, the diversification of the N termini in mammalian Dcn1 homologues may contribute to substrate specificity by regulating their subcellular localization.
Assuntos
Membrana Celular/metabolismo , Proteínas Culina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas Culina/genética , Imunofluorescência , Teste de Complementação Genética , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Mutação , Proteína NEDD8 , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas de Saccharomyces cerevisiae/genética , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) have limited treatment options, particularly if they are transplantation or chimeric antigen receptor (CAR) T-cell ineligible, and novel therapeutics are needed. An 86-year-old woman with relapsed DLBCL received a novel, first-in-class small molecule inhibitor of N-myristoyltransferase (NMT) as the initial patient on a phase I dose escalation trial. Daily oral administration of 20 mg PCLX-001 tablets produced a pharmacokinetic profile suitable for single daily dosing: rapid oral absorption, followed by an apparent elimination half-life of 16 h, without systemic accumulation of drug by day 15. Pharmacodynamic tests showed no clear change in NMT1 and NMT2 levels or selected NMT substrate Lyn and HGAL protein levels in normal circulating blood mononuclear cells, suggesting a higher dose will be required for normal tissue toxicity. The patient did not experience any dose-limiting toxicities but had disease progression after 28 days of study therapy. Dose escalation continues in other patients in this first-in-human study of a new class of anticancer drug. We conclude that PCLX-001 oral monotherapy has suitable pharmacokinetic parameters for dose escalation, and that higher doses are required to achieve pharmacodynamic evidence of on-target activity in normal tissues. The current protocol is appropriately designed to achieve these ends, and the study proceeds without modification.
Assuntos
Linfoma Difuso de Grandes Células B , Idoso de 80 Anos ou mais , Feminino , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológicoRESUMO
Excessive liver production of ketone bodies is one of many metabolic complications that can arise from diabetes, and in severe untreated cases, it can result in ketoacidosis, coma, and death. Mitochondrial HMG-CoA synthase (HMGCS2), the rate-limiting enzyme in ketogenesis, has been shown to interact with PPARalpha and act as a coactivator to up-regulate transcription from the PPRE of its own gene. Although protein palmitoylation is typically a cytosolic process that promotes membrane association, we recently identified 21 palmitoylated proteins in rat liver mitochondria, including HMGCS2. Herein, our data support a mechanism whereby palmitate is first added onto HMGCS2 active site Cys166 and then transacylated to Cys305. Palmitoylation promotes the HMGCS2/PPARalpha interaction, resulting in transcriptional activation from the Hmgcs2 PPRE. These results, together with the fact that 8 of the 21 palmitoylated mitochondrial proteins that we previously identified have nuclear receptor interacting motifs, demonstrate a novel--and perhaps ubiquitous--role for palmitoylation as a modulator of transcription.
Assuntos
Ácidos Graxos/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Lipoilação , PPAR alfa/metabolismo , Acilação , Western Blotting , Domínio Catalítico , Cisteína/genética , Cisteína/metabolismo , Humanos , Imunoprecipitação , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.