RESUMEN
AIMS: Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that was reported to target ion channel proteins. L-type voltage-dependent Ca2+ channel (LTCC) density and dysfunction is a critical player in heart failure with reduced ejection fraction (HFrEF). However, the underlying cellular mechanisms by which CRBN regulates LTCC subtype Cav1.2α during cardiac dysfunction remain unclear. Here, we explored the role of CRBN in HFrEF by investigating the direct regulatory role of CRBN in Cav1.2α activity and examining how it can serve as a target to address myocardial dysfunction. METHODS AND RESULTS: Cardiac tissues from HFrEF patients exhibited increased levels of CRBN compared with controls. In vivo and ex vivo studies demonstrated that whole-body CRBN knockout (CRBN-/-) and cardiac-specific knockout mice (Crbnfl/fl/Myh6Cre+) exhibited enhanced cardiac contractility with increased LTCC current (ICaL) compared with their respective controls, which was modulated by the direct interaction of CRBN with Cav1.2α. Mechanistically, the Lon domain of CRBN directly interacted with the N-terminal of Cav1.2α. Increasing CRBN levels enhanced the ubiquitination and proteasomal degradation of Cav1.2α and decreased ICaL. In contrast, genetic or pharmacological depletion of CRBN via TD-165, a novel PROTAC-based CRBN degrader, increased surface expression of Cav1.2α and enhanced ICaL. Low CRBN levels protected the heart against cardiomyopathy in vivo. CONCLUSION: Cereblon selectively degrades Cav1.2α, which in turn facilitates cardiac dysfunction. A targeted approach or an efficient method of reducing CRBN levels could serve as a promising strategy for HFrEF therapeutics.
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Insuficiencia Cardíaca , Ubiquitina-Proteína Ligasas , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Humanos , Ratones , Volumen Sistólico , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Echinochrome A (Ech A, 7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone) has been known to exhibit anti-oxidative and anti-inflammatory effects. However, no study has been carried out on the efficacy of Ech A against skin photoaging; this process is largely mediated by oxidative stress. Six-week-old male SKH-1 hairless mice (n = 36) were divided into five groups. Except for a group that were not treated (n = 4), all mice underwent ultraviolet-B (UVB) exposure for 8 weeks while applying phosphate-buffered saline or Ech A through intraperitoneal injection. UVB impaired skin barrier function, showing increased transepidermal water loss and decreased stratum corneum hydration. UVB induced dermal collagen degeneration and mast cell infiltration. Ech A injection was found to significantly lower transepidermal water loss while attenuating tissue inflammatory changes and collagen degeneration compared to the control. Furthermore, Ech A was found to decrease the relative expression of matrix metalloproteinase, tryptase, and chymase. Taken together, these results suggest that Ech A protects against UVB-induced photoaging in both functional and histologic aspects, causing a lowering of collagen degradation and inflammatory cell infiltration.
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Colágeno/metabolismo , Naftoquinonas/farmacología , Sustancias Protectoras/farmacología , Envejecimiento de la Piel/efectos de los fármacos , Animales , Organismos Acuáticos , Modelos Animales de Enfermedad , Masculino , Mastocitos/efectos de los fármacos , Ratones , Ratones Pelados , Naftoquinonas/administración & dosificación , Sustancias Protectoras/administración & dosificación , Rayos Ultravioleta , Pérdida Insensible de Agua/efectos de los fármacosRESUMEN
We previously demonstrated that CPNE1 induces neuronal differentiation and identified two binding proteins of CPNE1 (14-3-3γ and Jab1) as potential regulators of CPNE1-mediated neuronal differentiation in hippocampal progenitor cells. To better understand the cellular processes in which CPNE1 participates in neuronal differentiation, we here carried out a yeast two-hybrid screening to find another CPNE1 binding protein. Among the identified proteins, HCLS1-related protein X-1 (HAX1) directly interacts with CPNE1. Immunostaining experiments showed that a fraction of CPNE1 and HAX1 co-localized in the cytosol, particularly in the plasma membrane. In addition, the physical interaction as well as the specific binding regions between CPNE1 and HAX1 were confirmed in vitro and in vivo. Moreover, AKT phosphorylation, Tuj1 (neuronal marker protein) expression, and neurite outgrowth are all reduced in CPNE1/HAX1 overexpressing cells compared to CPNE1 only overexpressing HiB5 cells. Conversely, the HAX1 mutant that does not bind to CPNE1 was unable to inhibit the CPNE1-mediated neuronal differentiation. Together these results indicate that HAX1 is a binding partner of CPNE1 and CPNE1-mediated neuronal differentiation is negatively affected through the binding of HAX1, especially its N-terminal region, with CPNE1.
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Proteínas de Unión al Calcio/genética , Diferenciación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Tubulina (Proteína)/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/metabolismo , Células COS , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Regulación de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Neuronas/citología , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal , Tubulina (Proteína)/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4-PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in naïve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of naïve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4.
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Membrana Celular/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Canales Catiónicos TRPM/metabolismo , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Transporte de ProteínasRESUMEN
Copine1 (CPNE1), has tandem C2 domains and an A domain. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Protein kinase B (AKT) phosphorylation in the hippocampal progenitor cell line, HiB5. To better understand its cellular function, we carried out a yeast two-hybrid screening to find CPNE1 binding partners. Among the identified proteins, Jun activation domain-binding protein 1 (JAB1) appears to directly interact with CPNE1. Between CPNE1 and JAB1, the physical interaction was confirmed in vitro and in vivo. In addition the specific binding regions of CPNE1 and JAB1 was confirmed with truncated mutant assay. Furthermore, our results also demonstrate that AKT phosphorylation and expression of the neuronal marker protein are increased when JAB1 is overexpressed in CPNE1 high expressed HiB5 cells. Moreover, overexpression of both CPNE1 and JAB1 in HiB5 cells effectively increased neurite outgrowth. Collectively, our findings suggest that JAB1 activates the neuronal differentiation ability of CPNE1 through the binding of C2A domain in CPNE1 with MPN domain in JAB1.
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Complejo del Señalosoma COP9/metabolismo , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular/fisiología , Hipocampo/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Péptido Hidrolasas/metabolismo , Sitios de Unión , Línea Celular , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/metabolismo , Humanos , Células-Madre Neurales/citología , Neuronas/citología , Unión ProteicaRESUMEN
Unlike stable and immobile cell line conditions, animal hearts contract and relax to pump blood throughout the body. Mitochondria play an essential role by producing biological energy molecules to maintain heart function. In this study, we assessed the effect of heart mimetic cyclic stretch on mitochondria in a cardiac cell line. To mimic the geometric and biomechanical conditions surrounding cells in vivo, cyclic stretching was performed on HL-1 murine cardiomyocytes seeded onto an elastic micropatterned substrate (10% elongation, 0.5â¯Hz, 4â¯h/day). Cell viability, semi-quantitative Q-PCR, and western blot analyses were performed in non-stimulated control and cyclic stretch stimulated HL-1â¯cell lines. Cyclic stretch significantly increased the expression of mitochondria biogenesis-related genes (TUFM, TFAM, ERRα, and PGC1-α) and mitochondria oxidative phosphorylation-related genes (PHB1 and CYTB). Western blot analysis confirmed that cyclic stretch increased protein levels of mitochondria biogenesis-related proteins (TFAM, and ERRα) and oxidative phosphorylation-related proteins (NDUFS1, UQCRC, and PHB1). Consequently, cyclic stretch increased mitochondrial mass and ATP production in treated cells. Our results suggest that cyclic stretch transcriptionally enhanced mitochondria biogenesis and oxidative phosphorylation without detrimental effects in a cultured cardiac cell line.
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Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Biogénesis de Organelos , Estrés Mecánico , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Supervivencia Celular , Expresión Génica , Ratones , Mitocondrias Cardíacas/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/citología , Fosforilación OxidativaRESUMEN
Copine1 (CPNE1), known as a calcium-dependent membrane-binding protein, has tandem C2 domains and an A domain. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Protein kinase B (AKT) phosphorylation in the hippocampal progenitor cell line, HiB5. To better understand its cellular function, we carried out a yeast two-hybrid screening to find CPNE1 binding partners. Among the identified proteins, 14-3-3γ appears to directly interact with CPNE1. Between CPNE1 and 14-3-3γ, the physical interaction as well as the specific binding regions of CPNE1 was confirmed in vitro and in vivo. Furthermore, among the seven 14-3-3 isotypes, only 14-3-3γ directly interacts with CPNE1. Our results also demonstrate that AKT phosphorylation, neurite outgrowth and expression of the neuronal marker protein are increased when 14-3-3γ is overexpressed in CPNE1 high expressed HiB5 cells. Furthermore, the neighboring Ser54 amino acids residue of C2A domain in CPNE1 has an important role in binding with 14-3-3γ, and in differentiation-related function of CPNE1. Moreover, mutation of Ser54 amino acids residue in CPNE1 effectively decreased association with 14-3-3γ and neuronal differentiation of HiB5 cells. Collectively, our findings indicate that 14-3-3γ regulates the differentiation ability of CPNE1 through the binding with C2A domain of CPNE1 in HiB5 cells.
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Proteínas 14-3-3/metabolismo , Proteínas de Unión al Calcio/metabolismo , Hipocampo/citología , Neurogénesis/genética , Células Madre/citología , Proteínas 14-3-3/genética , Animales , Células COS , Proteínas de Unión al Calcio/genética , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Fosforilación , Unión Proteica/genética , Dominios Proteicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Anoctamin-1 (ANO1) is a Ca(2+)-activated chloride channel (CaCC) that plays important physiological roles in normal and cancerous tissues. However, the plasma membrane trafficking mechanisms of ANO1 remain poorly characterized. In yeast two-hybrid screening experiments, we observed direct interactions of ANO1 with ß-COP, which is a subunit of Coat Protein Complex I (COPI). This interaction was then confirmed using several in vitro and in vivo binding assays. Moreover, the cotransfection of ß-COP with ANO1 into HEK293T cells led to decreased the surface expression and the channel activity of ANO1. Accordingly, endogenous ANO1 was associated with ß-COP in U251 glioblastoma cells, and silencing of ß-COP enhanced surface expression and whole-cell currents of ANO1 in these cells. Taken together, these data suggest that ß-COP negatively regulates ANO1 surface expression.
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Canales de Cloruro/metabolismo , Proteína Coatómero/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Anoctamina-1 , Transporte Biológico , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteína Coatómero/análisis , Glioblastoma/metabolismo , Células HEK293 , HumanosRESUMEN
Copine1 (CPNE1) has tandem C2 domains and an A domain and is known as a calcium-dependent membrane-binding protein that regulates signal transduction and membrane trafficking. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Akt phosphorylation in the hippocampal progenitor cell line, HiB5. To determine which region of CPNE1 is related to HiB5 cell neurite outgrowth, we constructed several mutants. Our results show that over-expression of each C2 domain of CPNE1 increased neurite outgrowth and expression of the neuronal marker protein neurofilament (NF). Even though protein localization of the calcium binding-deficient mutant of CPNE1 was not affected by ionomycin, this mutant increased neurite outgrowth and NF expression in HiB5 cells. Furthermore, Akt phosphorylation was increased by over-expression of the calcium binding-deficient CPNE1 mutant. These results suggest that neither cellular calcium levels nor the localization of CPNE1 affect its function in neuronal differentiation. Collectively, our findings indicating that the C2 domains of CPNE1 play a calcium-independent role in regulating the neuronal differentiation of HiB5 cells.
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Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Animales , Células COS , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Chlorocebus aethiops , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/genética , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estructura Terciaria de Proteína , Ratas , Eliminación de Secuencia , Transducción de SeñalRESUMEN
PURPOSE: To evaluate characteristics of delayed ischemic stroke after stent-assisted coil placement in cerebral aneurysms and to determine the optimal duration of dual antiplatelet therapy for its prevention. MATERIALS AND METHODS: This retrospective study was approved by the institutional review board, and the requirement to obtain written informed consent was waived. Of 1579 patients with 1661 aneurysms, 395 patients (25.0%) with 403 aneurysms (24.3%) treated with stent-assisted coil placement were included and assigned to groups stratified as early (126 patients [31.9%]; 3 months of coil placement), midterm (160 patients [40.5%]; 6 months), or late (109 patients [27.6%]; ≥ 9 months), according to the time points of switching dual antiplatelet therapy to monotherapy from coil placement. Cumulative rates of delayed ischemic stroke in each group were calculated by using Kaplan-Meier estimates that were compared with log-rank tests. Risk factors of delayed ischemic stroke were identified by using Cox proportional hazard analysis. RESULTS: Delayed ischemic stroke occurred in 3.5% of all cases (embolism, 3.0%; thrombotic occlusion, 0.5%) within 2 months following the switch. Late switch yielded no delayed ischemic stroke, unlike early (seven of 126 patients [5.6%]; P = .013) or midterm (seven of 160 patients [4.4%]; P = .028) switch. Incomplete occlusion (hazard ratio, 6.68 [95% confidence interval: 1.490, 29.900]) was identified as a risk factor. CONCLUSION: Delayed ischemic stroke after stent-assisted coil placement is caused by embolism from or thrombotic occlusion of stent-containing vessels after switching from dual antiplatelet therapy to monotherapy. The stent-containing vessel with incomplete aneurysm occlusion presents as a long-term thromboembolic source. Therefore, dual antiplatelet therapy for more than 9 months and late switch to monotherapy are recommended for its prevention.
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Aspirina/administración & dosificación , Embolización Terapéutica/efectos adversos , Aneurisma Intracraneal/terapia , Inhibidores de Agregación Plaquetaria/administración & dosificación , Stents/efectos adversos , Accidente Cerebrovascular/etiología , Ticlopidina/análogos & derivados , Clopidogrel , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistema de Registros , Estudios Retrospectivos , Accidente Cerebrovascular/prevención & control , Ticlopidina/administración & dosificación , Resultado del TratamientoRESUMEN
INTRODUCTION: The novel Low-profile Visualized Intraluminal Support (LVIS™, LVIS and LVIS Jr.) device was recently introduced for stent-supported coil embolization of intracranial aneurysms. Periprocedural and midterm follow-up results for its use in stent-supported coil embolization of unruptured aneurysms are presented herein. METHODS: In this prospective multicenter study, clinical and radiologic outcomes were analyzed for 55 patients with saccular aneurysms undergoing LVIS-assisted coil embolization between October 2012 and February 2013. Magnetic resonance angiography or digital subtraction angiography was performed to evaluate midterm follow-up results. RESULTS: The standard LVIS device, deployed in 27 patients, was more often used in internal carotid artery (ICA) aneurysms (n=19), whereas the LVIS Jr. (a lower profile stent, n=28) was generally reserved for anterior communicating artery (n=14) and middle cerebral artery (n=8) aneurysms. With LVIS-assisted coil embolization, successful occlusion was achieved in 45 aneurysms (81.8 %). Although no instances of navigation failure or stent malposition occurred, segmentally incomplete stent expansion was seen in five patients where the higher profile LVIS was applied to ICA including carotid siphon. Procedural morbidity was low (2/55, 3.6 %), limited to symptomatic thromboembolism. In the imaging of lesions (54/55, 98.2 %) at 6-month follow-up, only a single instances of major recanalization (1.9 %) occurred. Follow-up angiography of 30 aneurysms (54.5 %) demonstrated in-stent stenosis in 26 (86.7 %), with no instances of stent migration. Only one patient suffered late delayed infarction (modified Rankin Scale 1). CONCLUSION: The LVIS device performed acceptably in stent-assisted coil embolization of non-ruptured aneurysms due to easy navigation and precise placement, although segmentally incomplete stent expansion and delayed in-stent stenosis were issues.
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Catéteres de Permanencia , Angiografía Cerebral/instrumentación , Embolización Terapéutica/instrumentación , Aneurisma Intracraneal/diagnóstico por imagen , Aneurisma Intracraneal/terapia , Radiografía Intervencional/instrumentación , Anciano , Angiografía Cerebral/métodos , Embolización Terapéutica/métodos , Análisis de Falla de Equipo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miniaturización , Diseño de Prótesis , Radiografía Intervencional/métodosRESUMEN
Cells can resist and even recover from stress induced by acute hypoxia, whereas chronic hypoxia often leads to irreversible damage and eventually death. Although little is known about the response(s) to acute hypoxia in neuronal cells, alterations in ion channel activity could be preferential. This study aimed to elucidate which channel type is involved in the response to acute hypoxia in rat pheochromocytomal (PC12) cells as a neuronal cell model. Using perfusing solution saturated with 95% N(2) and 5% CO(2), induction of cell hypoxia was confirmed based on increased intracellular Ca(2+) with diminished oxygen content in the perfusate. During acute hypoxia, one channel type with a conductance of about 30 pS (2.5 pA at -80 mV) was activated within the first 2~3 min following onset of hypoxia and was long-lived for more than 300 ms with high open probability (P(o), up to 0.8). This channel was permeable to Na(+) ions, but not to K(+), Ca(+), and Cl(-) ions, and was sensitively blocked by amiloride (200 nM). These characteristics and behaviors were quite similar to those of epithelial sodium channel (ENaC). RT-PCR and Western blot analyses confirmed that ENaC channel was endogenously expressed in PC12 cells. Taken together, a 30-pS ENaC-like channel was activated in response to acute hypoxia in PC12 cells. This is the first evidence of an acute hypoxia-activated Na(+) channel that can contribute to depolarization of the cell.
RESUMEN
Small G protein superfamily consists of more than 150 members, and is classified into six families: the Ras, Rho, Rab, Arf, Ran, and RGK families. They regulate a wide variety of cell functions such as cell proliferation/differentiation, cytoskeletal reorganization, vesicle trafficking, nucleocytoplasmic transport and microtubule organization. The small G proteins have also been shown to regulate cell death/survival and cell shape. In this study, we compared the role of representative members of the six families of small G proteins in cell migration and cell death/survival, two cellular phenotypes that are associated with inflammation, tumorigenesis, and metastasis. Our results show that small G proteins of the six families differentially regulate cell death and cell cycle distribution. In particular, our results indicate that Rho family of small G proteins is antiapoptotic. Ras, Rho, and Ran families promoted cell migration. There was no significant correlation between the cell death- and cell migration-regulating activities of the small G proteins. Nevertheless, RalA was not only cytoprotective against multiple chemotherapeutic drugs, but also promigratory inducing stress fiber formation, which was accompanied by the activation of Akt and Erk pathways. Our study provides a framework for further systematic investigation of small G proteins in the perspectives of cell death/survival and motility in inflammation and cancer.
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Movimiento Celular , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Animales , Western Blotting , Células COS , Muerte Celular , Supervivencia Celular , Células Cultivadas , Chlorocebus aethiops , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HeLa , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP ral/genéticaRESUMEN
This study evaluated the safety and efficacy of tubeless patch pump called EOPatch in patients with well-controlled type 1 diabetes mellitus (T1DM). This 4-week, two-center, open-label, single-arm study enrolled 10 adult patients diagnosed with T1DM with glycosylated hemoglobin less than 7.5%. The co-primary end points were patch pump usage time for one attachment and number of serious adverse events related to the patch pump. The secondary end points were total amount of insulin injected per patch and changes in glycemic parameters including continuous glucose monitoring data compared to those at study entry. The median usage time per patch was 84.00 hours (interquartile range, 64.50 to 92.50). Serious adverse events did not occur during the trial. Four weeks later, time in range 70 to 180 mg/dL was significantly improved (70.71%±17.14 % vs. 82.96%±9.14%, P=0.01). The times spent below range (<54 mg/dL) and above range (>180 mg/dL) also improved (All P<0.05). Four-week treatment with a tubeless patch pump was safe and led to clinical improvement in glycemic control.
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Diabetes Mellitus Tipo 1 , Sistemas de Infusión de Insulina , Adulto , Humanos , Automonitorización de la Glucosa Sanguínea , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Sistemas de Infusión de Insulina/efectos adversosRESUMEN
OBJECTIVE: Ischemic cardiomyopathy (ICM) is the leading cause of heart failure. Proteomic and genomic studies have demonstrated ischemic preconditioning (IPC) can assert cardioprotection against ICM through mitochondrial function regulation. Considering IPC is conducted in a relatively brief period, regulation of protein expression also occurs very rapidly, highlighting the importance of protein function modulation by post-translational modifications. This study aimed to identify and analyze novel phosphorylated mitochondrial proteins that can be harnessed for therapeutic strategies for preventing ischemia/reperfusion (I/R) injury. METHODS: Sprague-Dawley rat hearts were used in an ex vivo Langendorff system to simulate normal perfusion, I/R, and IPC condition, after which the samples were prepared for phosphoproteomic analysis. Employing human cardiomyocyte AC16 cells, we investigated the cardioprotective role of CKMT2 through overexpression and how site-directed mutagenesis of putative CKMT2 phosphorylation sites (Y159A, Y255A, and Y368A) can affect cardioprotection by measuring CKMT2 protein activity, mitochondrial function and protein expression changes. RESULTS: The phosphoproteomic analysis revealed dephosphorylation of mitochondrial creatine kinase (CKMT2) during ischemia and I/R, while preserving its phosphorylated state during IPC. CKMT2 overexpression conferred cardioprotection against hypoxia/reoxygenation (H/R) by increasing cell viability and mitochondrial adenosine triphosphate level, preserving mitochondrial membrane potential, and reduced reactive oxygen species (ROS) generation, while phosphomutations, especially in Y368, nullified cardioprotection by significantly reducing cell viability and increasing ROS production during H/R. CKMT2 overexpression increased mitochondrial function by mediating the proliferator-activated receptor γ coactivator-1α/estrogen-related receptor-α pathway, and these effects were mostly inhibited by Y368A mutation. CONCLUSION: These results suggest that regulation of quantitative expression and phosphorylation site Y368 of CKMT2 offers a unique mechanism in future ICM therapeutics.
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Transient receptor potential-melastatin 4 (TRPM4) is a Ca(2+)-activated, but Ca(2+)-impermeable, cation channel. Increasing [Ca(2+)](i) induce current activation and reduction through TRPM4 channels. Several TRPM4 isoforms are expressed in mice and humans, but rat TRPM4 (rTRPM4) has not been previously identified. Here, we identified, cloned, and characterized two rTRPM4 isoforms, rTRPM4a and rTRPM4b, using 5'-RACE-PCR. rTRPM4b channel activity increased with [Ca(2+)](i) in a dose-dependent manner. However, the rTRPM4b Ca(2+)-dependent activity at negative potentials differed from that of human TRPM4b (hTRPM4b), even though both represent full-length proteins. Additionally, rTRPM4b showed a slightly different single-channel current amplitude and open time distribution than hTRPM4b. However, rTRPM4a, which lacks the N-terminal region of rTRPM4b, and hTRPM4a had no similar functional channel activities. Furthermore, we characterized splicing regions, tissue distribution, and cellular localization of these isoforms. Unlike rTRPM4a, rTRPM4b was localized to the membrane at high levels, suggesting that rTRPM4b is the functionally active channel.
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Canales Catiónicos TRPM/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/metabolismo , Clonación Molecular , Humanos , Ratones , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Canales Catiónicos TRPM/genética , Distribución TisularRESUMEN
NELL2 is a neuron-specific secreted glycoprotein containing an N-terminal thrombospondin I-like domain (TSP-N). In this study, we describe NELL2-Tsp, a novel alternative splice variant of rat NELL2. NELL2-Tsp uses an alternate stop codon resulting in a C-terminal truncated form of NELL2, containing a signal peptide and a TSP-N domain. NELL2-Tsp is a glycosylated protein specifically expressed in brain tissue. NELL2-Tsp and NELL2 are secreted, likely due to the putative signal peptide. However, due to the truncation, the secreted portion of NELL2-Tsp is smaller than that of NELL2. Immunoprecipitation analysis confirmed that NELL2-Tsp was able to associate with NELL2 and with itself. In addition, expression of NELL2-Tsp notably reduced secretion of NELL2 and inhibited NELL2-mediated neurite outgrowth. These results suggest that NELL2-Tsp may act as a negative regulator of wild-type NELL2.
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Empalme Alternativo , Encéfalo/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Codón de Terminación/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Neuritas/metabolismo , Neuritas/fisiología , Señales de Clasificación de Proteína/genética , Estructura Terciaria de Proteína , RatasRESUMEN
TREK1 belongs to a family of two-pore-domain K(+) (K(2P)) channels and produce background currents that regulate cell excitability. In the present study, we identified a vesicle transport protein, beta-COP, as an interacting partner by yeast two-hybrid screening of a human brain cDNA library with N-terminal region of TREK1 (TREK1-N) as bait. Several in vitro and in vivo binding assays confirmed the protein-protein interaction between TREK1 and beta-COP. We also found that beta-COP was associated with TREK1 in native condition at the PC3 cells. When RFP-beta-COP was co-transfected with GFP-TREK1 into COS-7 cells, both proteins were found localized to the plasma membrane. In addition, the channel activity and surface expression of GFP-TREK1 increased dramatically by co-transfection with RFP-beta-COP. Surface expression of the TREK1 channel was also clearly reduced with the addition of beta-COP-specific shRNA. Collectively, these data suggest that beta-COP plays a critical role in the forward transport of TREK1 channel to the plasma membrane.
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Membrana Celular/metabolismo , Proteína Coatómero/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Células COS , Chlorocebus aethiops , Proteína Coatómero/genética , Humanos , Canales de Potasio de Dominio Poro en Tándem/genética , Mapeo de Interacción de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
TTYH2 is a calcium-activated, inwardly rectifying anion channel that has been shown to be related to renal cancer and colon cancer. Based on the topological prediction, TTYH2 protein has five transmembrane domains with the extracellular N-terminus and the cytoplasmic C-terminus. In the present study, we identified a vesicle transport protein, ß-COP, as a novel specific binding partner of TTYH2 by yeast two-hybrid screening using a human brain cDNA library with the C-terminal region of TTYH2 (TTYH2-C) as a bait. Using in vitro and in vivo binding assays, we confirmed the protein-protein interactions between TTYH2 and ß-COP. We also found that the surface expression and activity of TTYH2 were decreased by co-expression with ß-COP in the heterologous expression system. In addition, ß-COP associated with TTYH2 in a native condition at a human colon cancer cell line, LoVo cells. The over-expression of ß-COP in the LoVo cells led to a dramatic decrease in the surface expression and activity of endogenous TTYH2. Collectively, these data suggested that ß-COP plays a critical role in the trafficking of the TTYH2 channel to the plasma membrane. [BMB Reports 2019; 52(7): 445-450].
Asunto(s)
Proteína Coatómero/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Propiedades de SuperficieRESUMEN
Mitochondrion, a maternally hereditary, subcellular organelle, is the site of the tricarboxylic acid (TCA) cycle, electron transport chain (ETC), and oxidative phosphorylation (OXPHOS)-the basic processes of ATP production. Mitochondrial function plays a pivotal role in the development and pathology of different cancers. Disruption in its activity, like mutations in its TCA cycle enzymes, leads to physiological imbalances and metabolic shifts of the cell, which contributes to the progression of cancer. In this review, we explored the different significant mutations in the mitochondrial enzymes participating in the TCA cycle and the diseases, especially cancer types, that these malfunctions are closely associated with. In addition, this paper also discussed the different therapeutic approaches which are currently being developed to address these diseases caused by mitochondrial enzyme malfunction.