RESUMEN
Brucellosis is the most common zoonotic disease that caused by intracellular parasitic bacteria Brucella. The survival and replication of Brucella in the host depend on the type IV secretion system (T4SS). The T4SS system of Brucella has many components and secreted proteins. But the mechanism helped Brucella to evade the host defense is still not clear. The objective of the present study was to investigate the effects of VceA on autophagy and apoptosis in Brucella-infected embryonic trophoblast cells. We constructed the VceA mutant strain (2308ΔVceA) and complementary strain (2308ΔVceA-C) of Brucella abortus 2308 (S2308). The human trophoblast cells (HPT-8 cells) and mice were infected by S2308, 2308ΔVceA and 2308ΔVceA-C. The cell autophagy and apoptosis were detected. The Atg5, LC3-II and Bcl-2 mRNA expression were significantly increased in 2308ΔVceA group than the S2308 group, and mRNA expression of P62 and Caspase-3 were significantly decreased than the S2308 group. Western blotting, qPCR and flow cytometry analysis showed that 2308ΔVceA promoted autophagy and inhibited apoptosis. Mouse immunohistochemistry experiments showed that P62 protein was scattered coloring and Cytochrome C protein was scarcely in 2308ΔVceA group at the myometrium. These results indicated that 2308ΔVceA promoted autophagy and inhibited apoptosis in HPT-8 cells during Brucella infection.
Asunto(s)
Apoptosis/genética , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucelosis/microbiología , Brucelosis/patología , Trofoblastos/patología , Sistemas de Secreción Tipo IV/genética , Animales , Autofagia/genética , Proteínas Bacterianas/metabolismo , Brucella abortus/metabolismo , Brucelosis/metabolismo , Línea Celular , Femenino , Prueba de Complementación Genética , Humanos , Inmunohistoquímica , Ratones Endogámicos BALB C , Miometrio/metabolismo , Miometrio/patología , ARN Mensajero/metabolismo , Eliminación de Secuencia , Trofoblastos/metabolismoRESUMEN
Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , Ácido Glutámico/metabolismo , Plasticidad Neuronal , Neuronas/metabolismo , Factor de Transcripción Sp1/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Ciclooxigenasa 2/genética , Regulación de la Expresión Génica , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuronas/citología , Regiones Promotoras Genéticas/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/genética , Transcripción Genética/genéticaRESUMEN
System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1ß, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1ß increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1ß induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1ß-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1ß-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1ß-induced stabilization of astrocyte xCT mRNA.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Astrocitos/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Regulación de la Expresión Génica , Interleucina-1beta/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Sistema de Transporte de Aminoácidos y+/genética , Animales , Células Cultivadas , Proteína 1 Similar a ELAV/genética , Ácido Glutámico/metabolismo , Interleucina-1beta/genética , Ratones , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Regulación hacia ArribaRESUMEN
Brucella spp. are Gram-negative intracellular pathogens of both humans and animals that cause great economic burdens in developing countries. Live attenuated vaccines are the most efficient means for the prevention and control of animal Brucellosis. However, Brucella vaccines (strain M5-90 and others) have several drawbacks and do not allow serological differentiation between vaccinated and infected animals. A wboA mutant was derived from Brucella melitensis (B. melitensis) vaccine strain M5-90 and tested for virulence and protective efficiency. T-cell responses (CD4(+), CD8(+)), levels of immunoglobulin G (IgG), and cytokine production were observed. WboA was also assessed as a diagnostic marker for Brucellosis. B. melitensis strain M5-90ΔwboA exhibited reduced survival in murine macrophages (RAW 264.7) and BALB/c mice and induced protective immunity in mice comparable to that from the parental strain M5-90. In mice, the wboA mutant elicited an anti-Brucella-specific IgG response and induced the secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2). In sheep, M5-90ΔwboA immunization induced the secretion of IFN-γ, and serum samples from sheep inoculated with M5-90ΔwboA were negative by Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). In mice, probes against WboA antigen allowed for serological differentiation between natural infection and vaccination. The M5-90ΔwboA mutant is a potential attenuated live vaccine candidate against virulent B. melitensis 16M infection. It will be further evaluated in livestock.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/genética , Vacuna contra la Brucelosis/genética , Brucella melitensis/inmunología , Brucelosis/prevención & control , Eliminación de Gen , Animales , Anticuerpos Antibacterianos/biosíntesis , Proteínas Bacterianas/inmunología , Vacuna contra la Brucelosis/administración & dosificación , Vacuna contra la Brucelosis/inmunología , Brucella melitensis/genética , Brucelosis/inmunología , Brucelosis/microbiología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/microbiología , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Interferón gamma/sangre , Interleucina-2/biosíntesis , Interleucina-2/sangre , Ratones , Ratones Endogámicos BALB C , Vacunación , Vacunas AtenuadasRESUMEN
We had previously reported that RBEL1A, a novel Ras-like GTPase, was overexpressed in multiple human malignancies and that its depletion suppressed cell growth. However, the underlying molecular mechanism remained to be elucidated. Here we report that depletion of endogenous RBEL1A results in p53 accumulation due to increased p53 half-life whereas increased expression of RBEL1A reduces p53 levels under unstressed and genotoxic stress conditions. RBEL1A directly interacts with p53 and MDM2, and strongly enhances MDM2-dependent p53 ubiquitylation and degradation. We also found that RBEL1A modulation of p53 ubiquitylation by MDM2 does not depend on its GTPase activity. We have also defined the p53 oligomeric domain and RBEL1A GTPase domain to be the crucial regions for p53-RBEL1A interactions. Importantly, we have found that RBEL1A strongly interferes with p53 transactivation function; thus our results indicate that RBEL1A appears to function as a novel p53 negative regulator that facilitates MDM2-dependent p53 ubiquitylation and degradation.
Asunto(s)
Daño del ADN , Proteolisis , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación , Proteínas ras/metabolismo , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteínas ras/genéticaRESUMEN
Proteasome inhibitors such as bortezomib constitute novel therapeutic agents that are currently in clinical use and in clinical trials. In some neoplasms, cyclin-dependent kinase inhibitors (CKI) such as p21(WAF1) have been proposed as key targets of proteasome inhibitors. p21(WAF1) expression can be modulated by p53, a tumor suppressor, and especially in breast cancer cells, by estrogen receptor alpha (ERα), which is highly relevant to cancer growth. We investigated the effects of bortezomib using a panel of six cancer cell lines with variable status of ERα or p53 and found that bortezomib inhibited the growth of all cell lines in the same concentration range irrespective of the ERα expression or the mutational status of p53. Bortezomib treatment significantly enhanced p21(WAF1) protein levels in all cell lines but with different mechanisms according to ERα status. In ERα-positive cells, bortezomib treatment caused a strong increase in p21(WAF1) mRNA, whereas in ERα-negative cells it predominantly enhanced p21(WAF1) protein levels suggesting a posttranslational mechanism of p21(WAF1) regulation in the ERα-negative cells. Moreover, the antiproliferative activity of bortezomib was prevented by ERα silencing or p21(WAF1) knockdown in ERα-positive cells. Collectively, our results highlight the potential roles of ERα and p21(WAF1) in growth inhibition of cancer cells mediated by proteasome inhibitors, such as bortezomib.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Receptor alfa de Estrógeno/metabolismo , Pirazinas/farmacología , Antineoplásicos/efectos adversos , Ácidos Borónicos/efectos adversos , Bortezomib , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Células HCT116 , Humanos , Células MCF-7 , Pirazinas/efectos adversos , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The structural integrity of mitochondrial cristae is crucial for mitochondrial functions; however, the molecular events controlling the structural integrity and biogenesis of mitochondrial cristae remain to be fully elucidated. Here, we report the functional characterization of a novel mitochondrial protein named CHCM1 (coiled coil helix cristae morphology 1)/CHCHD6. CHCM1/CHCHD6 harbors a coiled coil helix-coiled coil helix domain at its C-terminal end and predominantly localizes to mitochondrial inner membrane. CHCM1/CHCHD6 knockdown causes severe defects in mitochondrial cristae morphology. The mitochondrial cristae in CHCM1/CHCHD6-deficient cells become hollow with loss of structural definitions and reduction in electron-dense matrix. CHCM1/CHCHD6 depletion also leads to reductions in cell growth, ATP production, and oxygen consumption. CHCM1/CHCHD6 through its C-terminal end strongly and directly interacts with the mitochondrial inner membrane protein mitofilin, which is known to also control mitochondrial cristae morphology. CHCM1/CHCHD6 also interacts with other mitofilin-associated proteins, including DISC1 and CHCHD3. Knockdown of CHCM1/CHCHD6 reduces mitofilin protein levels; conversely, mitofilin knockdown leads to reduction in CHCM1 levels, suggesting coordinate regulation between these proteins. Our results further indicate that genotoxic anticancer drugs that induce DNA damage down-regulate CHCM1/CHCHD6 expression in multiple human cancer cells, whereas mitochondrial respiratory chain inhibitors do not affect CHCM1/CHCHD6 levels. CHCM1/CHCHD6 knockdown in human cancer cells enhances chemosensitivity to genotoxic anticancer drugs, whereas its overexpression increases resistance. Collectively, our results indicate that CHCM1/CHCHD6 is linked to regulation of mitochondrial cristae morphology, cell growth, ATP production, and oxygen consumption and highlight its potential as a possible target for cancer therapeutics.
Asunto(s)
Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/biosíntesis , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Adenosina Trifosfato/biosíntesis , Adenosina Trifosfato/genética , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Mitocondrias/genética , Mitocondrias/patología , Membranas Mitocondriales/patología , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Proteínas Musculares/genética , Proteínas de Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/genética , Estructura Terciaria de ProteínaRESUMEN
We have previously cloned and characterized a novel p53 and DNA damage-regulated gene named PDRG1. PDRG1 was found to be differentially regulated by ultraviolet (UV) radiation and p53. In this study, we further investigated stress regulation of PDRG1 and found it to be selectively regulated by agents that induce genotoxic stress (DNA damage). Using cancer profiling arrays, we also investigated PDRG1 expression in matching normal and tumor samples representing various malignancies and found its expression to be upregulated in multiple malignancies including cancers of the colon, rectum, ovary, lung, stomach, breast and uterus when compared to their respective matched normal tissues. Western blot and immunohistochemical analyses were also performed on select specimen sets of colon cancers and matching normal tissues and the results also indicated PDRG1 overexpression in tumors relative to normal tissues. To gain insight into the function of PDRG1, we performed PDRG1 knockdown in human colon cancer cells and found its depletion to result in marked slowdown of tumor cell growth. These results suggest that PDGR1 may be linked to cell growth regulation. Yeast two-hybrid screen also led to the identification of PDCD7, CIZ1 and MAP1S as PDRG1-interacting proteins that are involved in apoptosis and cell cycle regulation which further implicate PDRG1 in controlling cell growth regulation. Taken together, our results indicate that PDRG1 expression is increased in multiple human malignancies suggesting it to be a high-value novel tumor marker that could play a role in cancer development and/or progression.
Asunto(s)
Biomarcadores de Tumor/genética , Daño del ADN , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Daño del ADN/genética , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Homeodomain-interacting protein kinases including HIPK1, HIPK2 and HIPK3 are serine/threonine kinases that form a family of highly conserved kinases. HIPKs are involved in diverse cellular functions including regulation cell death, survival, proliferation and differentiation. Here we report the characterization of a human HIPK4 that we identified in a proteomic screen during our efforts to unravel novel markers linked to cell death and survival. Human HIPK4 protein is composed of 616 residues with predicted molecular mass of 69.425 kDa and harbors a serine/threonine protein kinase catalytic domain at its N-terminal end. In the in vitro kinase assay, HIPK4 exhibits kinase activity and mutation of the conserved lysine 40 or aspartic acid 136 residue in its catalytic domain inactivates its kinase function. Human HIPK4 harbors multiple putative serine/threonine- and tyrosine-specific phsophorylation sites and also contains four high probability sumoylation sites, findings that suggest its function to be modulated by post-translational modifications. HIPK4 has been so named in the database because of its sequence homology to HIPK1, 2 and 3 predominantly within its catalytic domain. However, HIPK4 is smaller in size than the known HIPKs and has additional distinct features suggesting it to be a unique member of the HIPK family. Further functional characterization of HIPK4 is needed and will prove valuable to ascertain whether it performs distinct functions or share overlapping functions with other HIPKs.
RESUMEN
Pirh2 is an E3 ubiquitin ligase that promotes tumor suppressor p53 ubiquitination and proteasomal degradation. Recently, we have reported the identification and characterization of two novel isoforms of Pirh2 named Pirh2B and Pirh2C and accordingly, reclassified the full-length Pirh2 as Pirh2A. Both Pirh2B and C negatively regulate p53 and also exhibit interactions with MDM2. Here, we report the existence of an additional Pirh2 isoform that we have named Pirh2D. Translation of nucleotide sequence predicts Pirh2D to be composed of 75 amino acids with a molecular mass of 8493.74 Da. Thus, Pirh2D is a truncated protein that harbors 67 amino-terminal amino acids identical to those in Pirh2A, Pirh2B and Pirh2C and has 8 additional unique amino acids at the carboxyl-terminal end. Further studies are needed to determine whether Pirh2D also functions in a manner similar to Pirh2B and Pirh2C.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Metaloproteinasa 2 de la Matriz/metabolismo , Neoplasias/tratamiento farmacológico , Oligopéptidos/farmacología , Paclitaxel/administración & dosificación , Profármacos/farmacología , Animales , Antineoplásicos Fitogénicos/farmacología , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Paclitaxel/farmacologíaRESUMEN
In this article, we report the characterization of a novel DNA damage-regulated gene, named DNA damage-regulated overexpressed in cancer 45 (DOC45). Our results indicate that DNA damage-inducing agents, including doxorubicin (adriamycin), etoposide, and ionizing and UV radiation, strongly downregulate DOC45 expression, whereas endoplasmic reticulum stress-inducing agents do not. Our results also indicate that DOC45 is overexpressed in several human malignancies, including cancers of the colon, rectum, ovary, lung, stomach, and uterus. DOC45 harbors conserved nucleotide triphosphate-binding motifs and is capable of ATP hydrolysis, findings that highlight its function as a novel ATPase. Although predominantly cytoplasmic, DOC45 exhibits a characteristic nucleocytoplasmic distribution and, on inhibition of nuclear export, predominantly accumulates in the nucleoli. These results suggest that DOC45 may shuttle between nucleus and cytoplasm to carry out its function. Our results also indicate that DOC45 expression is enhanced during oncogenic Ras-mediated transformation and that its expression is linked to phosphoinositide 3-kinase signaling pathway. Furthermore, short hairpin RNA-mediated knockdown of DOC45 in human colon cancer cells inhibits their proliferation and enhances cellular sensitivity to doxorubicin-induced cell death, suggesting that DOC45 plays an important role in cell proliferation and survival. Collectively, our results indicate that DOC45 is a novel ATPase that is linked to cellular stress response and tumorigenesis, and may also serve as a valuable tumor marker.
Asunto(s)
Adenosina Trifosfatasas/genética , Neoplasias/genética , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Clonación Molecular , Citoplasma/genética , Citoplasma/metabolismo , Daño del ADN/genética , Proteínas de Unión al GTP , Regulación Neoplásica de la Expresión Génica , Células HT29 , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Regulación hacia ArribaRESUMEN
Sulindac, the non-steroidal anti-inflammatory drug has shown promise in the prevention of colon cancer but the molecular mechanisms by which it mediates such effects remain to be elucidated. Sulindac sulfide is the major active metabolite of sulindac and believed to be responsible for mediating the effects of sulindac. Previously, our group and others have shown that sulindac sulfide induces apoptosis by engaging death receptor and mitochondrial pathways and that a cross-talk exists between these two pathways during sulindac sulfide-induced apoptosis. Second mitochondrial-derived activator (Smac) is an important pro-apoptotic molecule that activates caspases by antagonizing the inhibitors of apoptosis (IAPs). In this study, we have utilized Smac-proficient and -deficient human colon cancer cells to investigate the role of Smac during sulindac sulfide-induced apoptosis and found that Smac deficiency affects sulindac sulfide-induced apoptosis in human colon cancer cells. Sulindac sulfide-induced apoptosis is coupled with upregulation of death receptor 5 (DR5), and activation of caspases 3, 9 and 8 in Smac-proficient cells. In Smac-deficient cells, although sulindac sulfide-induced DR5 upregulation is not altered, activation of caspases 3, 9 and 8 is affected. Smac deficiency also abrogates sulindac sulfide-induced cytochrome c release from mitochondria into cytosol. Our results, therefore, demonstrate that Smac is involved in sulindac sulfide-induced apoptotic signal transduction in human colon cancer cells and highlight the existence of a potential cross-talk between Smac and cytochrome c.
RESUMEN
Thapsigargin (TG) is a sesquiterpen lactone that inhibits the endoplasmic reticulum (ER) calcium ATPases to disrupt calcium homeostasis and consequently induces ER stress. We have previously reported that TG induces apoptosis by engaging the death receptor 5 (DR5) and the intrinsic pathways. Second mitochondrial-derived activator (Smac) is an important modulator of apoptosis that induces activation of caspases by antagonizing inhibitors of apoptosis (IAPs). In this study, we have utilized Smac-proficient and -deficient human colon cancer cells to investigate the effects of Smac deficiency during ER-stress-induced apoptosis. Our results indicate that Smac deficiency considerably affects ER stress-induced apoptosis in human colon cancer cells. For example, ER stress inducing agent TG upregulates DR5, and activates caspases 3, 9 and 8 in Smac-proficient cells. In Smac-deficient cells, although TG-induced DR5 upregulation is not affected, activation of caspases 3, 9 and 8 is affected. Smac deficiency also affects TG-induced cytochrome c release from mitochondria into cytosol suggesting the existence of a potential cross-talk between Smac and cytochrome c. Thus, our results indicate that ER stress-induced apoptosis also engages Smac for transduction of apoptotic signals in human colon cancer cells and that a potential feedback signaling between Smac and cytochrome c appears to modulate the intrinsic pathway of apoptosis.
RESUMEN
Homogeneity is proposed for evaluation of the quality of analytical biodevices, such as biosensors and biochips. As a demonstration, glucose oxidase (GOx) was modified at its C-terminal with a linker peptide with a cysteine residue at the end. The fusion structure (GOx-linker-cysteine) enables the enzyme to immobilize on gold surfaces with a Cys-S-Au bond or to immobilize on a silanized glass surface via disulfide chemistry. With this fusion structure, the enzyme can be anchored onto the substrate with well-controlled orientation, thus forming a homogeneous biological layer on biodevices. The linker peptide between GOx and the cysteine acts as a spacer to reduce the steric hindrance caused by the bulky body of the enzyme. Biochemistry experiments showed that this genetically modified glucose oxidase (shortened to GOxm) retained most of its catalytic characteristics, with K(m) and K(cat) similar to those of the wild-type GOx. Electrochemistry experiments showed that GOxm-modified electrode gave higher and more stable current responses than the electrode modified with GOx which has no free -SH on its surface. The coefficients of variation (used for evaluation of the interchangeability of the enzyme device from the same batch preparation) were 9.5% for the GOxm gold electrode and 20.0% for the GOx gold electrode and the GOxm oxygen electrode. The relative errors (used for evaluation of the precision of the individual enzyme device) were 2.9% for the GOxm gold electrode, 12.0% for the GOx gold electrode, and 11.2% for the GOxm oxygen electrode. Atomic force microscopy images revealed that GOxm formed a self-assembled monolayer in a hexagonal-like lattice packing arrangement on the gold surface, while GOx formed multilayer assembling or aggregated particles. The homogeneity of the protein chips, the GOxm array that was prepared through -S-S- formation, and the GOx array that was prepared through nonspecific adsorption was evaluated. The coefficients of variation, calculated with the signal level of all dots, were 5.4% for the GOxm array and 81.8% for the GOx array. All experimental results pointed to the fact that the homogeneity of the analytical biodevices could be considerably improved by using the proposed method.