RESUMEN
The lack of coronavirus-specific antiviral drugs has instigated multiple drug repurposing studies to redirect previously approved medicines for the treatment of SARS-CoV-2, the coronavirus behind the ongoing COVID-19 pandemic. A recent, large-scale, retrospective clinical study showed that famotidine, when administered at a high dose to hospitalized COVID-19 patients, reduced the rates of intubation and mortality. A separate, patient-reported study associated famotidine use with improvements in mild to moderate symptoms such as cough and shortness of breath. While a prospective, multi-center clinical study is ongoing, two parallel in silico studies have proposed one of the two SARS-CoV-2 proteases, 3CLpro or PLpro, as potential molecular targets of famotidine activity; however, this remains to be experimentally validated. In this report, we systematically analyzed the effect of famotidine on viral proteases and virus replication. Leveraging a series of biophysical and enzymatic assays, we show that famotidine neither binds with nor inhibits the functions of 3CLpro and PLpro. Similarly, no direct antiviral activity of famotidine was observed at concentrations of up to 200 µM, when tested against SARS-CoV-2 in two different cell lines, including a human cell line originating from lungs, a primary target of COVID-19. These results rule out famotidine as a direct-acting inhibitor of SARS-CoV-2 replication and warrant further investigation of its molecular mechanism of action in the context of COVID-19.
Asunto(s)
Famotidina/farmacología , Péptido Hidrolasas/metabolismo , SARS-CoV-2/enzimología , Replicación Viral/efectos de los fármacos , Células A549 , Animales , COVID-19/virología , Chlorocebus aethiops , Humanos , SARS-CoV-2/efectos de los fármacos , Células VeroRESUMEN
Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) that usually portends a poor prognosis with limited therapeutic options available. Currently, only allogeneic stem cell transplantation is curative in those who are candidates, while administration of the JAK1/2 inhibitor ruxolitinib carries a risk of worsening cytopenia. The limited therapeutic options available highlight the need for the development of novel treatments for PMF. Lysyl oxidase (LOX), an enzyme vital for collagen cross-linking and extracellular matrix stiffening, has been found to be upregulated in PMF. Herein, we evaluate two novel LOX inhibitors, PXS-LOX_1 and PXS-LOX_2, in two animal models of PMF (GATA1low and JAK2V617F-mutated mice). Specifically, PXS-LOX_1 or vehicle was given to 15- to 16-week-old GATA1low mice via intraperitoneal injection at a dose of 15 mg/kg four times a week for 9 weeks. PXS-LOX_1 was found to significantly decrease the bone marrow fibrotic burden and megakaryocyte number compared to vehicle in both male and female GATA1low mice. Given these results, PXS-LOX_1 was then tested in 15- to 17-week-old JAK2V617F-mutated mice at a dose of 30 mg/kg four times a week for 8 weeks. Again, we observed a significant decrease in bone marrow fibrotic burden. PXS-LOX_2, a LOX inhibitor with improved oral bioavailability, was next evaluated in 15- to 17-week-old JAK2V617F-mutated mice at a dose of 5 mg/kg p.o. four times a week for 8 weeks. This inhibitor also resulted in a significant decrease in bone marrow fibrosis, albeit with a more pronounced amelioration in female mice. Taking these results together, PXS-LOX_1 and PXS-LOX_2 appear to be promising new candidates for the treatment of fibrosis in PMF.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Mielofibrosis Primaria/tratamiento farmacológico , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Animales , Disponibilidad Biológica , Neoplasias de la Médula Ósea , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Femenino , Masculino , Ratones , Factores Sexuales , Resultado del TratamientoRESUMEN
Bone marrow (BM) reticulin fibrosis (RF), revealed by silver staining of tissue sections, is associated with myeloproliferative neoplasms, while tools for quantitative assessment of reticulin deposition throughout a femur BM are still in need. Here, we present such a method, allowing via analysis of hundreds of composite images to identify a patchy nature of RF throughout the BM during disease progression in a mouse model of myelofibrosis. To this end, initial conversion of silver stained BM color images into binary images identified two limitations: variable color, owing to polychromatic staining of reticulin fibers, and variable background in different sections of the same batch, limiting application of the color deconvolution method, and use of constant threshold, respectively. By blind coding image identities, to allow for threshold input (still within a narrow range), and using shape filtering to further eliminate background we were able to quantitate RF in myelofibrotic Gata-1low (experimental) and wild type (control) mice as a function of animal age. Color images spanning the whole femur BM were batch-analyzed using ImageJ software, aided by our two newly added macros. The results show heterogeneous RF density in different areas of the marrow of Gata-1low mice, with degrees of heterogeneity reduced upon aging. This method can be applied uniformly across laboratories in studies assessing RF remodeling induced by aging or other conditions in animal models.
RESUMEN
Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall-degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.
Asunto(s)
Regulación de la Temperatura Corporal/fisiología , Endo-1,4-beta Xilanasas/fisiología , Mejoramiento Genético/métodos , Inteínas/genética , Lignina/metabolismo , Plantas Modificadas Genéticamente/fisiología , Zea mays/fisiologíaRESUMEN
Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-ß (PDGFR-ß), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with ß-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with ß-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.
Asunto(s)
Médula Ósea/patología , Megacariocitos/citología , Mielofibrosis Primaria/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Animales , Western Blotting , División Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Masculino , Megacariocitos/enzimología , Ratones , Poliploidía , Mielofibrosis Primaria/terapia , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , ARN Mensajero/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of cellular LOX activity by preincubation of vascular smooth muscle cells (VSMC) with ß-aminopropionitrile (BAPN), the irreversible inhibitor of LOX activity, resulted in the marked suppression of the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. Plasma membranes purified from VSMC not previously exposed to BAPN contained a group of oxidized plasma membrane proteins, including the PDGF receptor, PDGFR-ß. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to BAPN-free cells, which had been previously exposed to BAPN, restored the profile of oxidized proteins towards that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells was significantly diminished when compared with cells in which oxidation by LOX was prevented by BAPN. The chemotactic responses of LOX knock-out mouse embryonic fibroblasts mirrored those obtained with VSMC treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-ß.
Asunto(s)
Membrana Celular/enzimología , Quimiocinas/metabolismo , Quimiotaxis/fisiología , Proteínas de la Membrana/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Animales , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Quimiocinas/agonistas , Quimiocinas/fisiología , Quimiotaxis/efectos de los fármacos , Ratones , Ratones Noqueados , Oxidación-Reducción , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/fisiologíaRESUMEN
Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX-PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full-length LOX cDNA (pre-pro-LOX), the N-glycosylation null pre-[N/Q]pro-LOX cDNA and the deletion mutant pre-LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N-terminal propeptide sequence. The results show that glycosylation of the LOX-PP is not required for secretion and extracellular processing of pro-LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX-PP for pro-LOX exit from the ER and is the first to highlight the influence of LOX-PP glycosylation on LOX enzyme activity.
Asunto(s)
Precursores Enzimáticos/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Eliminación de Secuencia , Animales , Células CHO , Cricetinae , Cricetulus , ADN Complementario , Retículo Endoplásmico/metabolismo , Glicosilación , Humanos , Transporte de Proteínas , Proteína-Lisina 6-Oxidasa/genética , TransfecciónRESUMEN
BACKGROUND: The cardioprotective benefits of bradykinin are attributable to activation of its B(2) receptor (B(2)R)-mediated actions and abolished by B(2)R antagonists. The current experiments evaluated the cardioprotective potential of a potent, long-acting B(2)R-selective agonist peptide analogue of bradykinin, the compound NG291. METHODS: We compared the extent of cardiac tissue damage and remodeling and expression pattern of selected genes in mice submitted to acute myocardial infarct (MI) and treated for 1 week with either NG291 [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin or with saline delivered via osmotic minipump. RESULTS: Active treatment resulted in better ejection fraction (EF) 69 +/- 1% vs. 61 +/- 3.1% (P = 0.01), (vs. 85 +/- 1.3% in sham-operated controls), fractional shortening (FS) 38 +/- 4% vs. 32 +/- 8% (NS) (vs. 53 +/- 1.2 in sham-operated controls), and fewer markers of myocyte apoptosis (TUNEL-positive nuclei 4.9 +/- 1.1% vs. 9.7 +/- 0.03%, P = 0.03). Systolic blood pressure (SBP) at end point was normal at 110 +/- 4.2 in actively treated mice, but tended to be lower at 104 +/- 4.7 mm Hg in saline controls with decreased cardiac systolic capacity. Expression patterns of selected genes to factors related to tissue injury, inflammation, and metabolism (i.e., the B(1)R, B(2)R, endothelial nitric oxide synthase (eNOS), TNF-alpha, cardiomyopathy-associated 3 (Cmya3), and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4)) showed that acute MI induced significant upregulation of these genes, and active treatment prevented or attenuated this upregulation, whereas the B(2)R agonist itself produced no difference in the myocardium of sham-operated mice. CONCLUSIONS: Treatment with a selective B(2)R agonist initiated at the time of induction of acute MI in mice had a beneficial effect on cardiac function, tissue remodeling, and inflammation-related tissue gene expression, which may explain its structural and functional benefits.
Asunto(s)
Bradiquinina/análogos & derivados , Cardiotónicos/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Receptor de Bradiquinina B2/agonistas , Animales , Apoptosis/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Cardiotónicos/farmacología , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Proteínas con Dominio LIM , Masculino , Ratones , Ratones Endogámicos , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Nucleares/metabolismo , Volumen Sistólico/efectos de los fármacos , Volumen Sistólico/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The angiotensin converting enzyme (ACE) catalyzes the extracellular formation of angiotensin II, and degradation of bradykinin, thus regulating blood pressure and renal handling of electrolytes. We have previously shown that exogenously added ACE elicited transcriptional regulation independent of its enzymatic activity. Because transcriptional regulation generates from protein-DNA interactions within the cell nucleus we have investigated the initial cellular response to exogenous ACE and the putative internalization of the enzyme in smooth muscle cells (SMC) and endothelial cells (EC). The following phenomena were observed when ACE was added to cells in culture: 1) it bound to SMC and EC with high affinity (K(d) = 361.5 +/- 60.5 pM) and with a low binding occupancy (B(max) = 335.0 +/- 14.0 molecules/cell); 2) it triggered cellular signaling resulting in late activation of focal adhesion kinase and SHP2; 3) it modulated platelet-derived growth factor receptor-beta signaling; 4) it was endocytosed by SMC and EC; and 5) it transited through the early endosome, partially occupied the late endosome and the lysosome, and was localized to the nuclei. The incorporation of ACE or a fragment of it into the nuclei reached saturation at 120 min, and was preceded by a lag time of 40 min. Internalized ACE was partially cleaved into small fragments. These results revealed that extracellular ACE modulated cell signaling properties, and that SMC and EC have a pathway for delivery of extracellular ACE to the nucleus, most likely involving cell surface receptor(s) and requiring transit through late endosome/lysosome compartments.
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Núcleo Celular/enzimología , Células Endoteliales/enzimología , Músculo Liso/enzimología , Miocitos del Músculo Liso/enzimología , Peptidil-Dipeptidasa A/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal/fisiología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Línea Celular , Endosomas/enzimología , Células Endoteliales/citología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Lisosomas/enzimología , Músculo Liso/citología , Miocitos del Músculo Liso/citología , Peptidil-Dipeptidasa A/farmacología , Ratas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiologíaRESUMEN
The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of proTRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40PGL42, within the proTRH31-52 sequence. The decreased steady state level of the mutant DeltaPGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of DeltaPGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.
Asunto(s)
Precursores de Proteínas/metabolismo , Vías Secretoras , Hormona Liberadora de Tirotropina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Eliminación de Gen , Ratones , Datos de Secuencia Molecular , Mutación/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Ratas , Hormona Liberadora de Tirotropina/química , Hormona Liberadora de Tirotropina/genéticaRESUMEN
Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.
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Aorta/enzimología , Quimiotaxis/fisiología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Proteína-Lisina 6-Oxidasa/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Aminopropionitrilo/farmacología , Animales , Aorta/citología , Becaplermina , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Fibroblastos/citología , Fibroblastos/enzimología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Oxidación-Reducción , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-sis , Ratas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Lipid rafts are membrane microdomains enriched in saturated phospholipids, sphingolipids, and cholesterol. They have a varied but distinct protein composition and have been implicated in diverse cellular processes including polarized traffic, signal transduction, endo- and exo-cytoses, entrance of obligate intracellular pathogens, and generation of pathological forms of proteins associated with Alzheimer's and prion diseases. Raft proteins can be permanently or temporarily associated to lipid rafts. Here, we review recent advances on the biochemical and cell biological characterization of rafts, and on the emerging concept of the temporary residency of proteins in rafts as a regulatory mechanism of their biological activity.
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Homeostasis/fisiología , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Sitios de Unión , Humanos , Fluidez de la Membrana , Conformación Molecular , Unión ProteicaRESUMEN
In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.
Asunto(s)
Pared Celular/metabolismo , Quitina Sintasa/metabolismo , Quitina/biosíntesis , Glucosamina/farmacología , Saccharomyces cerevisiae/genética , Acetilglucosamina/análisis , Acetilglucosamina/farmacología , Quitina Sintasa/genética , Regulación hacia Abajo , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genotipo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Lipoproteínas/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta/genética , Feromonas , Pirofosfatasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Fracciones Subcelulares/química , Regulación hacia Arriba , Uridina Difosfato N-Acetilglucosamina/análisisRESUMEN
Increased sensitivity to ionizing radiation (IR) has been shown to be due to defects in DNA double-strand break repair machinery. The major pathway in mammalian cells dedicated to the repair of DNA double-strand breaks is by the nonhomologous end-joining machinery. Six components function in this pathway, of which three (Ku70, Ku86, and DNA-PKcs) constitute a protein complex known as DNA-dependent protein kinase (DNA-PK). However, it is now recognized that the cellular radiation response is complex, and radiosensitivity may be also regulated at different levels in the radiation signal transduction pathway. In addition to DNA damage, exposure to IR triggers intracellular signaling cascades that overlap with pathways initiated by ligand engagement to a receptor. In this study, we provide evidence for the novel localization of the DNA-PK complex in lipid rafts. We also show this property is not a generalized characteristic of all DNA repair proteins. Furthermore, we have detected Ku86 in yeast lipid rafts. Our results suggest that the components of this complex might be recruited separately to the plasma membrane by tethering with raft-resident proteins. In addition, we found an irradiation-induced differential protein phosphorylation pattern dependent upon DNA-PKcs in lipid rafts. Thus, we speculate that another role for the DNA-PKcs subunit and perhaps for the holoenzyme is in the signal transduction of IR response.
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ADN Helicasas , Microdominios de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos Nucleares/metabolismo , Western Blotting , Células CHO , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Cricetinae , Reparación del ADN , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel Bidimensional , Humanos , Autoantígeno Ku , Microdominios de Membrana/efectos de la radiación , Microsomas/metabolismo , Proteínas Nucleares , Fosforilación , Unión Proteica , Radiación Ionizante , Transducción de Señal , Temperatura , Células Tumorales CultivadasRESUMEN
Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.