RESUMEN
Protein S-acylation is a reversible post-translational modification (PTM). It is present on diverse proteins and has important roles in regulating protein function. Aminolysis with hydroxylamine is widely used in the global identification of the PTM. However, the identification is indirect. Distinct criteria have been used for identification, and the false discovery rate has not been addressed. Here, we report a site-specific method for S-acylation identification based on tagging of S-acylation sites with iodoTMT0. Efforts to improve the performance of the method and confidence of identification are discussed, highlighting the importance of reducing contaminant peptides and keeping the recovery rate consistent between aliquots with or without hydroxylamine treatment. With very stringent criteria, presumptive S-acylation sites of 269, 684, 695, and 780 were identified from HK2 cells, HK11 cells, mouse brain, and mouse liver samples, respectively. Among them, the newly identified protein S-acylation sites are equivalent to 34% of human and 24% of mouse S-acylation sites reported previously. In addition, false-positive rates for S-acylation identification and S-acylation abundances were estimated. Significant differences in S-acylation abundance were found from different samples (from 0.08% in HK2 cells to 0.76% in mouse brain), and the false-positive rates were significantly higher for samples with a low abundance of S-acylation.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas , Animales , Ratones , Humanos , Acilación , Lipoilación , Hidroxilamina , HidroxilaminasRESUMEN
Antibody-drug conjugates (ADCs) have achieved great success in cancer therapy in recent years. Some peptidyl microtubule inhibitors consisting of natural and unnatural amino acids, such as monomethyl auristatin E (MMAE) and F (MMAF), are extremely cytotoxic and have been used as a payload in ADCs. However, their precise molecular interaction with tubulin and microtubules remains unclear. We determined the crystal structures of tubulin in complex with three ultra-potent peptidyl microtubule inhibitors [MMAE, taltobulin (HTI- 286), and tubulysin M] at 2.5 Å. Our data showed that the three peptides bound to the vinca domain and shared a common and key pharmacophore containing two consecutive hydrophobic groups (Val, Ile-like side chain). These groups protruded in opposite directions into hydrophobic pockets on the tubulin ß and α subunits. Nitrogen and oxygen atoms from the same backbone formed hydrogen bonds with Asn329 from the α subunit and Asp179 from the ß subunit in a direction normal to the surface formed by the aforementioned hydrophobic groups. In addition, our crystal structure data indicated that tubulysin M bound to the ß subunit alone, providing a structural explanation for its higher affinity. We also compared the conformations of two representative structurally different vinca domain compounds, ustiloxin D and vinblastine, with those of the aforementioned peptidyl ligands, and found that they shared a similar pharmacophore. Our findings lay a foundation for the rational design of novel vinca domain ligands and may facilitate the development of microtubule inhibitors with high specificity, affinity, and efficiency as payloads for ADCs in cancer therapy.
Asunto(s)
Microtúbulos/química , Microtúbulos/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Animales , Microtúbulos/efectos de los fármacos , Conformación Molecular , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Porcinos , Moduladores de Tubulina/farmacología , Difracción de Rayos XRESUMEN
The interactions of different dietary doses of copper with fructose contribute to the development of metabolic dysfunction-associated steatotic liver disease (MASLD) via the gut-liver axis. The underlying mechanisms remain elusive. The aim of this study was to identify the specific pathways leading to gut barrier dysfunction in the ileum using a proteomics approach in a rat model. Male weanling Sprague Dawley rats were fed diets with adequate copper (CuA), marginal copper (CuM), or supplemented copper (CuS) in the absence or presence of fructose supplementation (CuAF, CuMF, and CuSF) for 4 weeks. Ileum protein was extracted and analyzed with an LC-MS. A total of 2847 differentially expressed proteins (DEPs) were identified and submitted to functional enrichment analysis. As a result, the ileum proteome and signaling pathways that were differentially altered were revealed. Of note, the CuAF is characterized by the enrichment of oxidative phosphorylation and ribosome as analyzed with the KEGG; the CuMF is characterized by an enriched arachidonic acid metabolism pathway; and focal adhesion, the regulation of the actin cytoskeleton, and tight junction were significantly enriched by the CuSF. In conclusion, our proteomics analysis identified the specific pathways in the ileum related to the different dietary doses of copper-fructose interactions, suggesting that distinct mechanisms in the gut are involved in the development of MASLD.
Asunto(s)
Cobre , Fructosa , Íleon , Hígado , Proteómica , Ratas Sprague-Dawley , Animales , Fructosa/administración & dosificación , Fructosa/efectos adversos , Masculino , Cobre/metabolismo , Proteómica/métodos , Íleon/metabolismo , Íleon/efectos de los fármacos , Hígado/metabolismo , Hígado/efectos de los fármacos , Ratas , Dieta , Proteoma/metabolismo , Transducción de Señal/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Suplementos DietéticosRESUMEN
Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. An unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines were performed. Among 4,890 proteins identified, 737 and 651 proteins were found significantly (p < 0.01) upregulated and downregulated, respectively, in NAT1 KO cells, compared to the parental cells. Each set of proteins was analyzed to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in the set. Among the proteins upregulated in NAT1 KO cells, processes associated with MHC major histocompatibility complex I-mediated antigen presentation were significantly enriched. Multiple processes involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells. The current dataset contains additional representations of the biological processes and components that are differentially enriched in NAT1 KO MDA-MB-231 cells and will serve as a basis for generating novel hypotheses regarding the role of NAT1 in breast cancer. Data are available via ProteomeXchange with identifier PXD035953.
RESUMEN
Previous studies have shown that inhibition or depletion of N-acetyltransferase 1 (NAT1) in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. To understand molecular and cellular processes that NAT1 contributes to and generate novel hypotheses in regard to NAT1's role in breast cancer, we performed an unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines. Among 4890 proteins identified, 737 proteins were found significantly (p < 0.01) upregulated, and 651 proteins were significantly (p < 0.01) downregulated in both NAT1 KO cell lines. We performed enrichment analyses to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set. Among the proteins upregulated in NAT1 KO cells, pathways associated with MHC (major histocompatibility complex) I-mediated antigen presentation were significantly enriched. This raises an interesting and new hypothesis that upregulation of NAT1 in breast cancer cells may aid them evade immune detection. Multiple pathways involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells, consistent with reported observations that NAT1 KO cells exhibit a slower growth rate both in vitro and in vivo. Thus, mitochondrial dysfunction in NAT1 KO cells likely contributes to growth retardation.
RESUMEN
Slc7a11 is the key component of system Xc -, an antiporter that imports cystine (CySS) and exports glutamate. It plays an important role in cellular defense against oxidative stress because cysteine (Cys), reduced from CySS, is used for and limits the synthesis of glutathione (GSH). We have shown that downregulation of Slc7a11 is responsible for oxidation of extracellular Cys/CySS redox potential in lung fibroblasts from old mice. However, how age-related change of Slc7a11 expression affects the intracellular redox environment of mouse lung fibroblasts remains unexplored. The purpose of this study is to evaluate the effects of aging on the redox states of intracellular proteins and to examine whether Slc7a11 contributes to the age-dependent effects. Iodoacetyl Tandem Mass Tags were used to differentially label reduced and oxidized forms of Cys residues in primary lung fibroblasts from young and old mice, as well as old fibroblasts transfected with Slc7a11. The ratio of oxidized/reduced forms (i.e., redox state) of a Cys residue was determined via multiplexed tandem mass spectrometry. Redox states of 151 proteins were different in old fibroblasts compared to young fibroblasts. Slc7a11 overexpression restored redox states of 104 (69%) of these proteins. Ingenuity Pathway Analysis (IPA) showed that age-dependent Slc7a11-responsive proteins were involved in pathways of protein translation initiation, ubiquitin-proteasome-mediated degradation, and integrin-cytoskeleton-associated signaling. Gene ontology analysis showed cell adhesion, protein translation, and organization of actin cytoskeleton were among the top enriched terms for biological process. Protein-protein interaction network demonstrated the interactions between components of the three enriched pathways predicted by IPA. Follow-up experiments confirmed that proteasome activity was lower in old cells than in young cells and that upregulation of Slc7a11 expression by sulforaphane restored this activity. This study finds that aging results in changes of redox states of proteins involved in protein turnover and cytoskeleton dynamics, and that upregulating Slc7a11 can partially restore the redox states of these proteins.
Asunto(s)
Envejecimiento/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Cistina/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Pulmón/citología , Animales , Senescencia Celular , Femenino , Ontología de Genes , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Mapas de Interacción de ProteínasRESUMEN
Acrylonitrile (AN) is an organic compound produced in large quantities by the chemical industry and is acutely toxic. One mechanism proposed to explain the toxicity of AN is metabolism by P450 into cyanide (CN). Although blood and brain levels of CN in rats following an LD90 dose of AN are consistent with acute toxicity, blocking CN formation with P450 inhibitors does not prevent lethality. Another mechanism implicated in toxicity is covalent binding of AN to cysteine residues in tissue proteins. Previous work in our laboratory has shown that AN can irreversibly inactivate the catalytically active cysteine-149 in glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and, when coupled to the inhibition of mitochondrial ATP synthesis by the AN metabolite CN, would result in metabolic arrest, particularly in brain. In this study we have measured the high energy metabolites phosphocreatine (PCr), ATP, ADP and AMP by HPLC and compared their levels in the brains of rats treated with an LD90 dose of AN, when respiration ceased, vs. controls. Two methods of rapid brain freezing in liquid nitrogen were used: funnel freezing (FF) and head immersion (HI). AN administration resulted in large decreases in PCr of 74% (FF) and 80% (HI) but relatively minor decreases in ATP of 5% (FF) and 21% (HI) and Energy Charge of 6% (FF) and 10% (HI). Thus, although substantial depletion of PCr was observed, possibly due to inhibition of creatine kinase by AN, we found no evidence that brain ATP is depleted when respiration ceases in AN-intoxicated rats.
Asunto(s)
Acrilonitrilo/toxicidad , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Metabolismo Energético/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Creatina Quinasa/metabolismo , Técnicas de Preparación Histocitológica , Masculino , Fosfocreatina/metabolismo , Ratas , Ratas Sprague-Dawley , Pruebas de ToxicidadRESUMEN
Acrylonitrile (AN) is a vinyl monomer used in the production of synthetic fibers, rubber and plastics. AN is acutely toxic but its mechanism of toxicity remains to be established. AN is metabolized to cyanide in vivo but cyanide production alone cannot explain acute AN toxicity. Previous work in our laboratory has shown that AN can alkylate highly reactive cysteine residues in proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a critical enzyme involved in glycolysis, has a catalytically active cysteine 149 in its active site. We report that AN irreversibly inhibits GAPDH with second-order rate constants, at pH 7.4, of 3.7 and 9.2 M(-1) s(-1) at 25 and 37 degrees C, respectively. A combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and electrospray ionization-mass spectrometry-mass spectrometry (ESI-MS-MS) was used to show that AN inactivates GAPDH by covalently binding to cysteine 149 in the active site of the enzyme. Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and when coupled with the inhibition of mitochondrial ATP synthesis by the AN metabolite cyanide would result in metabolic arrest. The brain can withstand metabolic arrest for only a few minutes thus these combined actions may account for the acute toxicity of AN in vivo.
Asunto(s)
Acrilonitrilo/toxicidad , Carcinógenos/toxicidad , Cisteína/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Acrilonitrilo/metabolismo , Animales , Carcinógenos/metabolismo , Activación Enzimática/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cinética , Conejos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónAsunto(s)
Aminas , Óxidos , Parasimpaticomiméticos , Acetilcolina/farmacología , Aminas/análisis , Aminas/farmacología , Animales , Anuros , Inhibidores de la Colinesterasa/farmacología , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Músculos/efectos de los fármacos , Óxidos/análisis , Óxidos/farmacología , Parasimpaticomiméticos/farmacologíaRESUMEN
Metallothionein (MT) releases zinc under oxidative stress conditions in cultured cells. The change in the MT molecule after zinc release in vivo is unknown although in vitro studies have identified MT disulfide bond formation. The present study was undertaken to test the hypothesis that MT disulfide bond formation occurs in vivo. A cardiac-specific MT-overexpressing transgenic mouse model was used. Mice were administered saline as a control or doxorubicin (20 mg/kg), which is an effective anticancer drug but with severe cardiac toxicity at least partially because of the generation of reactive oxygen species. A differential alkylation of cysteine residues in MT of the heart extracts was performed. Free and metal-bound cysteines were first trapped by N-ethylmaleimide and the disulfide bonds were reduced by dithiothreitol followed by alkylation with radiolabeled iodoacetamide. Analyses of the differentially alkylated MTs in the heart extract by high performance liquid chromatography, SDS-PAGE, Western blot, and mass spectrometry revealed that disulfide bonds were present in MT in vivo under both physiological and oxidative stress conditions. More disulfide bonds were found in MT under the oxidative stress conditions. The MT disulfide bonds were likely intramolecular and both alpha- and beta-domains were involved in the disulfide bond formation, although the alpha-domain appeared to be more easily oxidized than the beta-domain. The results suggest that under physiological conditions, the formation of MT disulfide bonds is involved in the regulation of zinc homeostasis. Additional zinc release from MT under oxidative stress conditions is accompanied by more MT disulfide bond formation.
Asunto(s)
Metalotioneína/química , Metalotioneína/genética , Miocardio/metabolismo , Estrés Oxidativo , Animales , Antineoplásicos/farmacología , Western Blotting , Cromatografía Líquida de Alta Presión , Cisteína/química , Disulfuros/química , Ditiotreitol/farmacología , Doxorrubicina/farmacología , Electroforesis en Gel de Poliacrilamida , Etilmaleimida/farmacología , Yodoacetamida/farmacología , Espectrometría de Masas , Metalotioneína/fisiología , Ratones , Ratones Transgénicos , Miocardio/patología , Péptidos/química , Estructura Terciaria de Proteína , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , Tripsina/química , Tripsina/farmacología , Zinc/químicaRESUMEN
Some of the more striking expressions of toxicity are the tremors and seizures observed approximately 100 min after exposure of rats to an acutely toxic dose of acrylonitrile (AN). These early events are followed by a second wave of severe clonic convulsions that occur just prior to death at about 3-4 h. For AN, at least two chemical entities could produce these toxic effects, namely the parent AN molecule, the metabolically-released cyanide, or both. Which of these two agents is responsible for each of the symptoms of acute intoxication is not known. To help dissect the toxicity, it was anticipated that an effective inhibitor of the oxidative metabolism of AN to cyanide could help us to understand which toxic symptoms might be associated with each agent. Three inhibitors of oxidative metabolism were tested, namely SKF-525A, 1-benzylimidazole and metyrapone and one alternative substrate, ethanol. As compared to SKF-525A and metyrapone, both 1-benzylimidazole and ethanol were highly effective in reducing blood cyanide levels to insignificant levels in rats treated with an LD90 dose of AN. In addition, both agents abolished the early seizure activity, suggesting that this first phase of seizures is due to cyanide and not the parent molecule. 1-Benzylimidazole did not prevent the severe clonic convulsive phase preceding death, suggesting that these terminal convulsions are due to the toxic effects of the parent AN molecule. The CNS depressant ethanol was only partially effective in attenuating the terminal convulsions. None of these agents affected the incidence of AN-induced mortality, clearly establishing that, even in the absence of cyanide, the parent AN molecule is acutely toxic. The partial effectiveness of ethanol suggested that anticonvulsants might be of benefit. Both phenobarbital and phenytoin protected rats from both the early and terminal convulsions, while valproic acid was ineffective. These effects were not related to a reduction in blood cyanide levels but rather due to their inherent anticonvulsant activity.
Asunto(s)
Acrilonitrilo/toxicidad , Anticonvulsivantes/farmacología , Carcinógenos/toxicidad , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Convulsiones/prevención & control , Acrilonitrilo/metabolismo , Animales , Carcinógenos/metabolismo , Depresores del Sistema Nervioso Central/metabolismo , Depresores del Sistema Nervioso Central/farmacología , Cianuros/sangre , Etanol/metabolismo , Etanol/farmacología , Imidazoles/farmacología , Masculino , Fenobarbital/farmacología , Fenitoína/farmacología , Proadifeno/farmacología , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Factores de TiempoRESUMEN
Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), and detected by autoradiography. A unique 65Zn-binding band was observed from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.
Asunto(s)
Aconitato Hidratasa/metabolismo , Metalotioneína/metabolismo , Miocardio/metabolismo , Zinc/metabolismo , Aconitato Hidratasa/química , Aconitato Hidratasa/genética , Secuencia de Aminoácidos , Animales , Técnicas In Vitro , Transporte Iónico , Metalotioneína/deficiencia , Metalotioneína/genética , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Covalent binding of reactive chemical species to tissue proteins is a common, but poorly understood, mechanism of toxicity. Identification of the proteins and the specific amino acid residues within the proteins that are chemically modified will aid our understanding of the toxification/detoxification mechanisms involved in covalent binding. Acrylonitrile (AN) is a commercial vinyl monomer that is acutely toxic and readily binds to tissue proteins. Total covalent binding of AN to tissue proteins is highly correlated with acute toxicity. Two-dimensional PAGE and autoradiography were used to locate proteins in male rat liver cytosol that are radiolabeled following administration of [2,3-(14)C]AN in vivo. Four intensely labeled spots were prominent in the autoradiogram and formed an apparent "charge-train" at approximately 30 kDa. Tryptic peptide mapping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used to identify all of the spots as carbonic anhydrase III (CAIII). HPLC of the tryptic digests combined with MALDI-TOF MS was used to localize the radiolabel to tryptic fragment T22 containing amino acids 171-187. This tryptic fragment contains two Cys residues (Cys181 and Cys186) in the rat CAIII sequence. Electrospray ionization ion-trap MS was used to sequence the peptide and establish that only Cys186 was labeled. Thus, although AN is considered to be highly reactive, our data indicate that it does not react indiscriminately with rat CAIII but rather is selective for one out of five Cys residues. Rat liver CAIII has previously been shown to protect cells against oxidative stress. Our data suggest that CAIII is also capable of scavenging reactive xenobiotics and may help prevent covalent binding to more critical macromolecules.