RESUMEN
20(S)-Protopanaxadiol (PPD) is one of the bioactive ingredients in ginseng and possesses neuroprotective properties. Brain-type creatine kinase (CK-BB) is an enzyme involved in brain energy homeostasis via the phosphocreatine-creatine kinase system. We previously identified PPD as directly bound to CK-BB and activated its activity in vitro. In this study, we explored the antidepressive effects of PPD that target CK-BB. First, we conducted time course studies on brain CK-BB, behaviors, and hippocampal structural plasticity responses to corticosterone (CORT) administration. Five weeks of CORT injection reduced CK-BB activity and protein levels and induced depression-like behaviors and hippocampal structural plasticity impairment. Next, a CK inhibitor and an adeno-associated virus-targeting CKB were used to diminish CK-BB activity or its expression in the brain. The loss of CK-BB in the brain led to depressive behaviors and morphological damage to spines in the hippocampus. Then, a polyclonal antibody against PPD was used to determine the distribution of PPD in the brain tissues. PPD was detected in the hippocampus and cortex and observed in astrocytes, neurons, and vascular endotheliocytes. Finally, different PPD doses were used in the chronic CORT-induced depression model. Treatment with a high dose of PPD significantly increased the activity and expression of CK-BB after long-term CORT injection. In addition, PPD alleviated the damage to depressive-like behaviors and structural plasticity induced by repeated CORT injection. Overall, our study revealed the critical role of CK-BB in mediating structural plasticity in CORT-induced depression and identified CK-BB as a therapeutic target for PPD, allowing us to treat stress-related mood disorders.
Asunto(s)
Antidepresivos , Corticosterona , Forma BB de la Creatina-Quinasa , Depresión , Sapogeninas , Animales , Humanos , Masculino , Ratones , Ratas , Antidepresivos/farmacología , Antidepresivos/administración & dosificación , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Forma BB de la Creatina-Quinasa/metabolismo , Forma BB de la Creatina-Quinasa/genética , Depresión/inducido químicamente , Depresión/tratamiento farmacológico , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones Endogámicos C57BL , Panax/química , Extractos Vegetales/farmacología , Extractos Vegetales/administración & dosificación , Ratas Sprague-Dawley , Sapogeninas/farmacologíaRESUMEN
Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease1. Thermogenesis by adipocytes can counteract obesity and metabolic diseases2,3. In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP-purportedly via a phosphorylation cycle4-to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.
Asunto(s)
Tejido Adiposo/metabolismo , Forma BB de la Creatina-Quinasa/metabolismo , Creatina/metabolismo , Termogénesis , Adipocitos/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/enzimología , Animales , Forma BB de la Creatina-Quinasa/deficiencia , Forma BB de la Creatina-Quinasa/genética , AMP Cíclico/metabolismo , Metabolismo Energético/genética , Femenino , Glucosa/metabolismo , Homeostasis , Humanos , Masculino , Ratones , Mitocondrias/metabolismo , Obesidad/enzimología , Obesidad/genética , Obesidad/metabolismo , Transducción de SeñalRESUMEN
Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.
Asunto(s)
Adenosina Trifosfato/metabolismo , MicroARNs/metabolismo , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Apoptosis , Forma BB de la Creatina-Quinasa/genética , Forma BB de la Creatina-Quinasa/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , MicroARNs/genética , eIF-2 Quinasa/genéticaRESUMEN
Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cilios/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Glucosa/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Cerebelo/citología , Cerebelo/metabolismo , Cilios/ultraestructura , Forma BB de la Creatina-Quinasa/genética , Forma BB de la Creatina-Quinasa/metabolismo , Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucosa-6-Fosfatasa/genética , Glucólisis , Masculino , Microsomas/metabolismo , Microsomas/ultraestructura , Neuronas Receptoras Olfatorias/citología , Fosforilación Oxidativa , Ratas , Ratas Sprague-Dawley , Técnicas de Cultivo de TejidosRESUMEN
For maintaining energy homeostasis, creatine kinase (CK) is present at elevated levels in tissues with high and/or fluctuating energy requirements such as muscle, brain, and epithelia, while there is very few CK, if any, in peripheral blood cells. However, an ectopic expression of brain-type creatine kinase (BCK) has been reported for platelets and leukocytes in an autosomal dominant inherited anomaly named CKBE. Here we investigated CK in erythrocytes of CKBE individuals from eight unrelated families. The data revealed a varying but significant increase of CK activity in CKBE individuals as compared to controls, reaching an almost 800-fold increase in two CKBE individuals which also had increased erythrocyte creatine. Immunoblotting with highly specific antibodies confirmed that the expressed CK isoform is BCK. Cell fractionation evidenced soluble BCK, suggesting cytosolic and not membrane localization of erythrocyte CK as reported earlier. These results are discussed in the context of putative CK energy buffering and transfer functions in red blood cells.
Asunto(s)
Forma BB de la Creatina-Quinasa/metabolismo , Eritrocitos/enzimología , Genes Dominantes , Forma BB de la Creatina-Quinasa/genética , Femenino , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , MasculinoRESUMEN
Diet-induced obesity (DIO) has been shown to exacerbate hearing degeneration via increased hypoxia, inflammatory responses, and cell loss via both caspase-dependent and caspase-independent apoptosis signaling pathways. This study aimed to investigate the effects of DIO on the mRNA expressions of protein kinase c-ß (PKC-ß), brain creatine kinase (CKB), transcription modification genes, and autophagy-related genes in the cochlea of CD/1 mice. Sixteen 4-week-old male CD/1 mice were randomly divided into 2 groups. For 16 weeks, the DIO group was fed a high fat diet (60% kcal fat) and the controls were fed a standard diet. Morphometry, biochemistry, auditory brainstem response thresholds, omental fat, and histopathology of the cochlea were compared. Results showed that body weight, body length, body-mass index, omental fat, plasma triglyceride, and auditory brainstem response thresholds were significantly elevated in the DIO group compared with those of the control group. The ratio of vessel wall thickness to radius in the stria vascularis was significantly higher in the DIO group. The cell densities in the spiral ganglion, but not in the spiral prominence, of the cochlea were significantly lower in the DIO group. The expression of histone deacetylation gene 1 (HDAC1) was significantly higher in the DIO group than the control group. However, the expressions of PKC-ß, CKB, HDAC3, histone acetyltransferase gene (P300), lysosome-associated membrane protein 2 (Lamp2), and light chain 3 (Lc3) genes were not significantly different between two groups. These results suggest that DIO might exacerbate hearing degeneration possibly via increased HDAC1 gene expression in the cochlea of CD/1 mice.
Asunto(s)
Autofagia/fisiología , Cóclea/enzimología , Forma BB de la Creatina-Quinasa/metabolismo , Obesidad/metabolismo , Proteína Quinasa C/metabolismo , Transcripción Genética/fisiología , Estimulación Acústica/métodos , Animales , Cóclea/patología , Forma BB de la Creatina-Quinasa/genética , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Masculino , Ratones , Obesidad/genética , Obesidad/patología , Proteína Quinasa C/genéticaRESUMEN
The process of liver colonization in colorectal cancer remains poorly characterized. Here, we addressed the role of microRNA (miRNA) dysregulation in metastasis. We first compared miRNA expression profiles between colorectal cancer cell lines with different metastatic properties and then identified target proteins of the dysregulated miRNAs to establish their functions and prognostic value. We found that 38 miRNAs were differentially expressed between highly metastatic (KM12SM/SW620) and poorly metastatic (KM12C/SW480) cancer cell lines. After initial validation, we determined that three miRNAs (miR-424-3p, -503, and -1292) were overexpressed in metastatic colorectal cancer cell lines and human samples. Stable transduction of non-metastatic cells with each of the three miRNAs promoted metastatic properties in culture and increased liver colonization in vivo. Moreover, miR-424-3p and miR-1292 were associated with poor prognosis in human patients. A quantitative proteomic analysis of colorectal cancer cells transfected with miR-424-3p, miR-503, or miR-1292 identified alterations in 149, 129, or 121 proteins, respectively, with an extensive overlap of the target proteins of the three miRNAs. Importantly, down-regulation of two of these shared target proteins, CKB and UBA2, increased cell adhesion and proliferation in colorectal cancer cells. The capacity of distinct miRNAs to regulate the same mRNAs boosts the capacity of miRNAs to regulate cancer metastasis and underscores the necessity of targeting multiple miRNAs for effective cancer therapy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Neoplasias Colorrectales/genética , MicroARNs/genética , Metástasis de la Neoplasia/genética , ARN Neoplásico/genética , Animales , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Forma BB de la Creatina-Quinasa/biosíntesis , Forma BB de la Creatina-Quinasa/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Ratones Desnudos , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Pronóstico , Proteómica/métodos , Enzimas Activadoras de Ubiquitina/biosíntesis , Enzimas Activadoras de Ubiquitina/genéticaRESUMEN
Creatine kinase (CK) helps maintain homeostasis of intracellular ATP level by catalyzing the reversible phosphotransfer between ATP and phosphocreatine. In humans, there are two cytosolic CK isoforms, the muscle-type (M) and the brain-type (B), which frequently function as homodimers (hMMCK and hBBCK). Interestingly, these isoenzymes exhibit significantly different thermostabilities, despite high similarity in amino acid sequences and tertiary structures. In order to investigate the mechanism of this phenomenon, in this work, we first used domain swapping and site-directed mutagenesis to search for the key residues responsible for the isoenzyme-specific thermostability. Strikingly, the difference in thermostability was found to principally arise from one single residue substitution at position 36 (Pro in hBBCK vs. Leu in hMMCK). We then engaged the molecular dynamics simulations to study the molecular mechanism. The calculations imply that the P36L substitution introduces additional local interactions around residue 36 and thus further stabilizes the dimer interface through a complex interaction network, which rationalizes the observation that hMMCK is more resistant to thermal inactivation than hBBCK. We finally confirmed this molecular explanation through thermal inactivation assays on Asp36 mutants that were proposed to devastate the local interactions and thus the dimer associations in both isoenzymes.
Asunto(s)
Sustitución de Aminoácidos , Forma BB de la Creatina-Quinasa/química , Forma BB de la Creatina-Quinasa/genética , Forma MM de la Creatina-Quinasa/química , Forma MM de la Creatina-Quinasa/genética , Estabilidad Proteica , Humanos , Isoenzimas , Modelos Moleculares , Mutación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , TemperaturaRESUMEN
Peroxiredoxins (Prxs) are versatile enzymes that demonstrate various cell functions as peroxidases, protein chaperones, functions of signal modulators and binding partners. It is well established that Prxs can interact with multiple proteins in cells, such as ASK1, Cdk5-p35, JNK, MIF, PDGF, TK R4 and others. In this study, we attempted to evaluate a possible association between ubiquitous Prx II and ATP/ADP buffering enzyme - brain-type creatine kinase (CK BB). Our co-immunoprecipitation (Co-IP) results from the A549 and HeLa cell lysates with overexpressed HA-Prx II and Flag-CK BB have demonstrated strong association between two proteins under non-stressed conditions. This protein interaction was enhanced by the heat treatment with further HA-Prx II precipitation to the immobilized Flag-CK BB depending on the temperature increase. Temperature induced oligomerization of Prx II may contribute to the formation of Prx II conglomerates, which in turn, can associate with CK BB and increase signal intensities on the blotted membranes. Thus, such association and oligomerization of Prx II could take part in recovery and protection of the CK BB enzyme activity from inactivation during heat-induced stress.
Asunto(s)
Forma BB de la Creatina-Quinasa/metabolismo , Peroxirredoxinas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Estrés Fisiológico/genética , Células A549 , Forma BB de la Creatina-Quinasa/genética , Expresión Génica , Células HeLa , Hemaglutininas/genética , Hemaglutininas/metabolismo , Calor , Humanos , Inmunoprecipitación , Oligopéptidos/genética , Oligopéptidos/metabolismo , Peroxirredoxinas/genética , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Protein oxidation has detrimental effects on the brain functioning, which involves inhibition of the crucial enzyme, brain type creatine kinase (CKBB), responsible for the CK/phosphocreatine shuttle system. Here we demonstrate a susceptibility of CKBB to several ordinary stressors. In our study enzymatic activity of purified recombinant brain-type creatine kinase was evaluated. We assayed 30 nMconcentration of CKBB under normal and stress conditions. In the direction of phosphocreatine formation hydrogen peroxide and heat treatments altered CKBB activity down to 26 and 14%, respectively. Also, examination of immunoblotted membrane patterns by SDS-PAGE electrophoresis and western blot analysis showed a decrease in expression levels of intrinsic CKBB enzyme in HeLa andA549 cells. Hence, our results clearly show that cytosolic CKBB is extremely sensitive to oxidative stress and heat induced inactivation. Therefore, due to its susceptibility, this enzyme may be defined as a potential target in brain damage.
Asunto(s)
Forma BB de la Creatina-Quinasa/química , Expresión Génica , Peróxido de Hidrógeno/química , Proteínas Recombinantes de Fusión/química , Línea Celular Tumoral , Forma BB de la Creatina-Quinasa/antagonistas & inhibidores , Forma BB de la Creatina-Quinasa/genética , Forma BB de la Creatina-Quinasa/aislamiento & purificación , Citosol/metabolismo , Células HeLa , Calor , Humanos , Estrés Oxidativo , Fosfocreatina/química , Plásmidos/química , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , TransfecciónRESUMEN
Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.
Asunto(s)
Forma BB de la Creatina-Quinasa/genética , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/enzimología , Mioblastos/enzimología , Actinas/genética , Actinas/metabolismo , Empalme Alternativo , Animales , Fusión Celular , Forma BB de la Creatina-Quinasa/antagonistas & inhibidores , Forma BB de la Creatina-Quinasa/metabolismo , Forma MM de la Creatina-Quinasa/genética , Forma MM de la Creatina-Quinasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citología , Mioblastos/citología , Polimerizacion , Cultivo Primario de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
The neural substrate of adaptive thermoregulation in mice lacking both brain-type creatine kinase isoforms is further investigated. The cytosolic brain-type creatine kinase (CK-B) and mitochondrial ubiquitous creatine kinase (UbCKmit) are expressed in neural cells throughout the central and peripheral nervous system, where they have an important role in cellular energy homeostasis. Several integral functions appear altered when creatine kinases are absent in the brain (Jost et al., 2002; Streijger et al., 2004, 2005), which has been explained by inefficient neuronal transmission. The CK--/-- double knockout mice demonstrate every morning a body temperature drop of ~1.0 °C, and they have impaired thermogenesis, as revealed by severe hypothermia upon cold exposure. This defective thermoregulation is not associated with abnormal food intake, decreased locomotive activity, or increased torpor sensitivity. Although white and brown adipose tissue fat pads are diminished in CK--/-- mice, intravenous norepinephrine infusion results in a normal brown adipose tissue response with increasing core body temperatures, indicating that the sympathetic innervation functions correctly (Streijger et al., 2009). This study revealed c-fos changes following a cold challenge, and that neuropeptide Y levels were decreased in the paraventricular nucleus of wildtype, but not CK--/--, mice. A reduction in hypothalamic neuropeptide Y is coupled to increased uncoupling protein 1 expression in brown adipose tissue, resulting in thermogenesis. In CK--/-- mice the neuropeptide Y levels did not change. This lack of hypothalamic plasticity of neuropeptide Y might be the result of inefficient neuronal transmission or can be explained by the previous observation of reduced circulating levels of leptin in CK--/-- mice.
Asunto(s)
Regulación de la Temperatura Corporal/genética , Forma BB de la Creatina-Quinasa/deficiencia , Forma Mitocondrial de la Creatina-Quinasa/deficiencia , Técnicas de Inactivación de Genes , Hipotálamo/fisiología , Plasticidad Neuronal/genética , Neuropéptido Y/metabolismo , Animales , Temperatura Corporal/genética , Núcleo Celular/metabolismo , Frío , Forma BB de la Creatina-Quinasa/genética , Forma Mitocondrial de la Creatina-Quinasa/genética , Hipotálamo/citología , Hipotálamo/metabolismo , Masculino , Ratones , Proteínas Proto-Oncogénicas c-fos/metabolismoRESUMEN
Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. Brain-type creatine kinase (CKB) is an enzyme involved in energy homeostasis via the phosphocreatine-creatine kinase system. Although downregulation of CKB was previously reported in brains of HD mouse models and patients, such regulation and its functional consequence in HD are not fully understood. In the present study, we demonstrated that levels of CKB found in both the soma and processes were markedly reduced in primary neurons and brains of HD mice. We show for the first time that mutant HTT (mHTT) suppressed the activity of the promoter of the CKB gene, which contributes to the lowered CKB expression in HD. Exogenous expression of wild-type CKB, but not a dominant negative CKB mutant, rescued the ATP depletion, aggregate formation, impaired proteasome activity, and shortened neurites induced by mHTT. These findings suggest that negative regulation of CKB by mHTT is a key event in the pathogenesis of HD and contributes to the neuronal dysfunction associated with HD. In addition, besides dietary supplementation with the CKB substrate, strategies aimed at increasing CKB expression might lead to the development of therapeutic treatments for HD.
Asunto(s)
Encéfalo/enzimología , Forma BB de la Creatina-Quinasa/biosíntesis , Regulación Enzimológica de la Expresión Génica , Enfermedad de Huntington/enzimología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Animales , Encéfalo/patología , Forma BB de la Creatina-Quinasa/genética , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Enfermedad de Huntington/terapia , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k (2) value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8 × 10(-5) s(-1). The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.
Asunto(s)
Forma BB de la Creatina-Quinasa/química , Forma BB de la Creatina-Quinasa/metabolismo , Dextranos/farmacología , Polietilenglicoles/farmacología , Forma BB de la Creatina-Quinasa/genética , Guanidina/química , Humanos , Cinética , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. When the number of CAG repeats exceeds 36, the translated polyglutamine-expanded Htt protein interferes with the normal functions of many types of cellular machinery and causes cytotoxicity. Clinical symptoms include progressive involuntary movement disorders, psychiatric signs, cognitive decline, dementia, and a shortened lifespan. The most severe brain atrophy is observed in the striatum and cortex. Besides the well-characterized neuronal defects, recent studies showed that the functions of mitochondria and several key players in energy homeostasis are abnormally regulated during HD progression. Energy dysregulation thus is now recognized as an important pathogenic pathway of HD. This review focuses on the importance of three key molecular determinants (peroxisome proliferator-activated receptor-γ coactivator-1α, AMP-activated protein kinase, and creatine kinase B) of cellular energy homeostasis and their possible involvement in HD pathogenesis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Forma BB de la Creatina-Quinasa/fisiología , Metabolismo Energético , Proteínas de Choque Térmico/fisiología , Enfermedad de Huntington/metabolismo , Factores de Transcripción/fisiología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Creatina/uso terapéutico , Forma BB de la Creatina-Quinasa/genética , Forma BB de la Creatina-Quinasa/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Ratones , Modelos Biológicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon-gamma knockout (Ifng-KO) mice. Five upregulated and five downregulated proteins were identified with 2D gels and MALDI-TOF/TOF MS analyses in Ifng-KO mouse brain. Of the identified proteins, we focused on creatine kinase brain (CKB) and triose phosphate isomerase 1 (TPI1). Consistent with the proteomic data, reverse transcriptase-mediated PCR, immunoblotting, and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.
Asunto(s)
Forma BB de la Creatina-Quinasa/metabolismo , Interferón gamma/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Triosa-Fosfato Isomerasa/metabolismo , Animales , Encéfalo/metabolismo , Estudios de Casos y Controles , Forma BB de la Creatina-Quinasa/genética , Regulación hacia Abajo , Femenino , Humanos , Interferón gamma/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Proteoma/análisis , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , República de Corea , Genética Inversa , Triosa-Fosfato Isomerasa/genética , Regulación hacia ArribaRESUMEN
Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9) is a specific substrate-recognition subunit of an elongin C-cullin-SOCS box E3 ubiquitin ligase complex. It recognizes its substrate, brain type creatine kinase (CKB), using the ankyrin repeat domain; and facilitates the polyubiquitination of CKB to mediate proteasomal degradation through the SOCS box domain. HASB9-2 is an isoform of hASB9 that contains one ankyrin repeat domain. In this study, the crystal structure of hASB9-2 is shown at 2.2-Å resolution using molecular replacement. Overall, hASB9-2 forms a slightly curved arch with a characteristic L-shaped cross-section. Amino acid substitution analysis based on docking experiments revealed that His103 and Phe107 in hASB9-2 are essential for binding to CKB. Analysis of truncation mutants demonstrated that the first six ankyrin repeats along with the N-terminal region of hASB9-2 contribute to the interaction with CKB.
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Forma BB de la Creatina-Quinasa/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/química , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Secuencia de Aminoácidos , Repetición de Anquirina , Sitios de Unión , Forma BB de la Creatina-Quinasa/química , Forma BB de la Creatina-Quinasa/genética , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Especificidad por Sustrato , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0-P14) and adult (P22-P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K(+) cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine-phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.
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Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Forma BB de la Creatina-Quinasa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Forma BB de la Creatina-Quinasa/análisis , Forma BB de la Creatina-Quinasa/biosíntesis , Forma BB de la Creatina-Quinasa/genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Masculino , Proteínas de Transporte de Membrana/análisis , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Distribución TisularRESUMEN
To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.
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Biomarcadores de Tumor/metabolismo , Detección Precoz del Cáncer , Neoplasias Pulmonares/metabolismo , Neoplasias de Células Escamosas/metabolismo , Secuencia de Aminoácidos , Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Bronquios/patología , Línea Celular , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/metabolismo , Análisis por Conglomerados , Forma BB de la Creatina-Quinasa/química , Forma BB de la Creatina-Quinasa/genética , Forma BB de la Creatina-Quinasa/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Gutatión-S-Transferasa pi/química , Gutatión-S-Transferasa pi/genética , Gutatión-S-Transferasa pi/metabolismo , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Captura por Microdisección con Láser , Neoplasias Pulmonares/diagnóstico , Chaperonas Moleculares , Datos de Secuencia Molecular , Neoplasias de Células Escamosas/diagnóstico , Proteómica , Curva ROC , Estadísticas no Paramétricas , Espectrometría de Masas en TándemRESUMEN
Cigarette smoke induces damage to proteins and organelles by oxidative stress, resulting in accelerated epithelial cell senescence in the lung, which is implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Although the detailed molecular mechanisms are not fully understood, cellular energy status is one of the most crucial determinants for cell senescence. Creatine kinase (CK) is a constitutive enzyme, playing regulatory roles in energy homeostasis of cells. Among two isozymes, brain-type CK (CKB) is the predominant CK in lung tissue. In this study, we investigated the role of CKB in cigarette smoke extract (CSE)-induced cellular senescence in human bronchial epithelial cells (HBECs). Primary HBECs and Beas2B cells were used. Protein carbonylation was evaluated as a marker of oxidative protein damage. Cellular senescence was evaluated by senescence-associated ß-galactosidase staining. CKB inhibition was examined by small interfering RNA and cyclocreatine. Secretion of IL-8, a hallmark of senescence-associated secretary phenotype, was measured by ELISA. CKB expression levels were reduced in HBECs from patients with COPD compared with that of HBECs from nonsmokers. CSE induced carbonylation of CKB and subsequently decreased CKB protein levels, which was reversed by a proteasome inhibitor. CKB inhibition alone induced cell senescence, and further enhanced CSE-induced cell senescence and IL-8 secretion. CSE-induced oxidation of CKB is a trigger for proteasomal degradation. Concomitant loss of enzymatic activity regulating energy homeostasis may lead to the acceleration of bronchial epithelial cell senescence, which is implicated in the pathogenesis of COPD.