RESUMEN
Parkinson's disease (PD) is characterized by slow, progressive degeneration of dopaminergic neurons in the substantia nigra. The cause of neuronal death in PD is largely unknown, but several genetic loci, including leucine-rich repeat kinase 2 (LRRK2), have been identified. LRRK2 has guanosine triphosphatase (GTPase) and kinase activities, and mutations in LRRK2 are the major cause of autosomal-dominant familial PD. Histone deacetylases (HDACs) remove acetyl groups from lysine residues on histone tails, promoting transcriptional repression via condensation of chromatin. Here, we demonstrate that LRRK2 binds to and directly phosphorylates HDAC3 at Ser-424, thereby stimulating HDAC activity. Specifically, LRRK2 promoted the deacetylation of Lys-5 and Lys-12 on histone H4, causing repression of gene transcription. Moreover, LRRK2 stimulated nuclear translocation of HDAC3 via the phoshorylation of karyopherin subunit α2 and α6. HDAC3 phosphorylation and its nuclear translocation were increased in response to 6-hydroxydopamine (6-OHDA) treatment. LRRK2 also inhibited myocyte-specific enhancer factor 2D activity, which is required for neuronal survival. LRRK2 ultimately promoted 6-OHDA-induced cell death via positive modulation of HDAC3. These findings suggest that LRRK2 affects epigenetic histone modification and neuronal survival by facilitating HDAC3 activity and regulating its localization.
Asunto(s)
Encéfalo/patología , Neuronas Dopaminérgicas/patología , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Neuritas/patología , Neuroblastoma/patología , Acetilación , Animales , Encéfalo/metabolismo , Supervivencia Celular , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Células HEK293 , Histona Desacetilasas/genética , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuritas/metabolismo , Neuroblastoma/metabolismo , Fosforilación , RatasRESUMEN
The proto-oncogene c-Myc has a pivotal function in growth control, differentiation, and apoptosis and is frequently affected in human cancer, including breast cancer. Ubiquitin-specific protease 22 (USP22), a member of the USP family of deubiquitinating enzymes (DUBs), mediates deubiquitination of target proteins, including histone H2B and H2A, telomeric repeat binding factor 1, and cyclin B1. USP22 is also a component of the mammalian SAGA transcriptional co-activating complex. In this study, we explored the functional role of USP22 in modulating c-Myc stability and its physiological relevance in breast cancer progression. We found that USP22 promotes deubiquitination of c-Myc in several breast cancer cell lines, resulting in increased levels of c-Myc. Consistent with this, USP22 knockdown reduces c-Myc levels. Furthermore, overexpression of USP22 stimulates breast cancer cell growth and colony formation, and increases c-Myc tumorigenic activity. In conclusion, the present study reveals that USP22 in breast cancer cell lines increases c-Myc stability through c-Myc deubiquitination, which is closely correlated with breast cancer progression.
Asunto(s)
Neoplasias de la Mama/enzimología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Tioléster Hidrolasas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Semivida , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Células MCF-7 , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteolisis , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Tioléster Hidrolasas/genética , Factores de Tiempo , Transfección , Ubiquitina Tiolesterasa , UbiquitinaciónRESUMEN
PTEN-induced putative kinase 1 (PINK1) is a serine/threonine kinase that phosphorylates several substrates and exerts neuroprotective effects against stress-induced apoptotic cell death. Mutations in PINK1 have been linked to autosomal recessive forms of Parkinson's disease (PD). Mitophagy is a type of autophagy that selectively promotes mitochondrial turnover and prevents the accumulation of dysfunctional mitochondria to maintain cellular homeostasis. Toll-interacting protein (Tollip) was initially identified as a negative regulator of IL-1ß receptor signaling, suppressing inflammatory TLR signaling cascades. Recently, Tollip has been reported to play a role in autophagy and is implicated in neurodegeneration. In this study, we determined whether Tollip was functionally linked to PINK1-mediated mitophagy. Our results demonstrated that Tollip promoted the mitochondrial processing of PINK1 and altered the localization of PINK1, predominantly to the cytosol. This action was attributed to increased binding of PINK1 to mitochondrial processing peptidase ß (MPPß) and the subsequent increase in MPPß-mediated mitochondrial PINK1 cleavage. Furthermore, Tollip suppressed mitophagy following carbonyl cyanide m-chlorophenylhydrazoneinduced mitochondrial dysfunction. These findings suggest that Tollip inhibits mitophagy via the PINK1/parkin pathway upon mitochondrial damage, leading to the blockade of PINK1-mediated neuroprotection. [BMB Reports 2022; 55(10): 494-499].
Asunto(s)
Mitofagia , Proteínas Quinasas , Mitofagia/genética , Citosol/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Mitocondrias/metabolismo , AutofagiaRESUMEN
The formation of Lewy bodies (LBs), intracellular filamentous inclusions, is one of the hallmarks of Parkinson's disease (PD). α-Synuclein is the main component of LBs and its abnormal accumulation contributes to the pathogenesis of PD. Direct phosphorylation of α-synuclein at multiple Ser/Tyr residues is known to induce its aggregation, consequently promoting LB formation. Death-associated protein kinase 1 (DAPK1), originally identified as a positive mediator of γ-interferon-induced programmed cell death, possesses tumor-suppressive activity and mediates a wide range of cellular processes, including apoptosis and autophagy. Accumulating evidence suggests that DAPK1 is also associated with neuronal cell death and neurodegeneration. For example, DAPK1 phosphorylates tau and amyloid precursor protein, and induces tau aggregation and amyloid ß production, respectively, in Alzheimer's disease. DAPK1 is also accumulated to a larger extent in a mouse model of PD, causing synucleinopathy and dopaminergic neuron degeneration. In this study, we attempted to determine whether DAPK1 phosphorylates α-synuclein and affects cell viability in human dopaminergic neuroblastoma SH-SY5Y cells. We demonstrated that DAPK1 directly phosphorylates α-synuclein at Ser129, and induces the formation of insoluble α-synuclein aggregates. We also showed that DAPK1 enhances rotenone-induced aggregation of α-synuclein, potentiating neuronal cell death. Taken together, these findings suggest that DAPK1 acts as a novel regulator of toxic α-synuclein aggregation, possibly affecting and playing a role in the development of PD.
RESUMEN
Mutations in the phosphatase and tensin homologue-induced putative kinase 1 (PINK1) gene have been linked to an early-onset autosomal recessive form of familial Parkinson's disease (PD). PINK1, a mitochondrial serine/threonine-protein kinase, plays an important role in clearing defective mitochondria by mitophagy - the selective removal of mitochondria through autophagy. Evidence suggests that alteration of the PINK1 pathway contributes to the pathogenesis of PD, but the mechanisms by which the PINK1 pathway regulates mitochondrial quality control through mitophagy remain unclear. Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase that functions in telomere maintenance as well as several non-telomeric activities. For example, hTERT has been associated with cellular immortalization, cell growth control, and mitochondrial regulation. We determined that hTERT negatively regulates the cleavage and cytosolic processing of PINK1 and enhances its mitochondrial localization by inhibiting mitochondrial processing peptidase ß (MPPß). Consequently, hTERT promotes mitophagy following carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondrial dysfunction and improves the function of damaged mitochondria by modulating PINK1. These findings suggest that hTERT positively regulates PINK1 function, leading to increased mitophagy following mitochondrial damage.
Asunto(s)
Citoplasma/metabolismo , Mitocondrias/metabolismo , Mitofagia/fisiología , Proteínas Quinasas/metabolismo , Telomerasa/metabolismo , Animales , Células HEK293 , Humanos , Ratones , TransfecciónRESUMEN
The ubiquitin-proteasome system (UPS) and autophagy are two major degradative pathways of proteins in eukaryotic cells. As about 30% of newly synthesized proteins are known to be misfolded under normal cell conditions, the precise and timely operation of the UPS and autophagy to remove them as well as their tightly controlled regulation, is so important for proper cell function and survival. In the UPS, target proteins are labeled by small proteins called ubiquitin, which are then transported to the proteasome complex for degradation. Alternatively, many greatly damaged proteins are believed to be delivered to the lysosome for autophagic degradation. Although these autophagy and UPS pathways have not been considered to be directly related, many recent studies proposed their close link and dynamic interconversion. In this review, we'll focus on the several regulatory molecules that function in both UPS and autophagy and their crosstalk. Among the proposed multiple modulators, we will take a closer look at the so-called main connector of UPS-autophagy regulation, p62. Last, the functional role of p62 in the mitophagy and its implication for the pathogenesis of Parkinson's disease, one of the major neurodegenerative diseases, will be briefly reviewed. [BMB Reports 2020; 53(1): 56-63].
Asunto(s)
Autofagia/genética , Mitofagia/genética , Enfermedad de Parkinson/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Sequestosoma-1/metabolismo , Ubiquitina/metabolismo , Animales , Autofagia/efectos de los fármacos , Progresión de la Enfermedad , Humanos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacosRESUMEN
In this study, we developed pH and redox-responsive crosslinked polypeptide-based combination micelles for enhanced chemotherapeutic efficacy and minimized side effects. The stability and drug release properties of the polypeptide micelles were efficiency balanced by the corona-crosslinking of the triblock copolymer, poly(ethylene glycol)-b-poly(aspartic acid)-b-poly(tyrosine) (PEG-b-pAsp-b-pTyr) with coordinated redox and pH dual-sensitivity by introducing disulfide crosslinkages. Because of the crosslinking of the middle shell of the triblock polypeptide micelles, their robust structure was maintained in strong destabilization conditions and exhibited excellent stability. GSH concentrations were significantly higher in tumor tissue than in normal tissue, which formed the basis for our design. Drug release was elevated under redox and low acidic conditions. Furthermore, crosslinked micelles showed a superior anticancer effect compared to that of non-crosslinked micelles. Incorporation of docetaxel (DTX) and lonidamine (LND) in crosslinked polypeptide micelles increased the intracellular reactive oxygen species (ROS) level and oxidative stress and caused damage to intracellular components that resulted in greater apoptosis of cancer cells than when DTX or LND was used alone. The combination of DTX and LND in crosslinked micelles exhibited efficacious inhibition of tumor growth with an excellent safety profile compared to that reported for drug cocktail combinations and non-crosslinked micelles. Overall, redox/pH-responsive polypeptide micelles could be an interesting platform for efficient chemotherapy. STATEMENT OF SIGNIFICANCE: We have synthesized a biodegradable polypeptide block copolymer to construct a facile pH and redox-responsive polymeric micelle asan advanced therapeutic system for cancer therapy. We have designed a corona-crosslinked triblock copolymer (poly (ethylene glycol)-b-poly(aspartic acid)-b-poly(tyrosine) (PEG-b-pAsp-b-pTyr)) micelles co-loaded with docetaxel and lonidamine (cl-M/DL). The corona of triblock polymer was crosslinked to maintain its structural integrity in the physiological environment. The mitochondrial targeting LND is expected to generate ROS, oxidative stress and thereby synergize the chemotherapeutic efficacy of DTX in killing cancer cells. Consistently, cl-M/DL exhibited excellent antitumor efficacy in xenograft tumor model with remarkable tumor regression. Overall, we demonstrated the construction of bioreducible nanosystem for the effective synergistic delivery of DTX/LND in tumor tissues towards cancer treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Materiales Biocompatibles/química , Reactivos de Enlaces Cruzados/química , Micelas , Neoplasias/tratamiento farmacológico , Péptidos/uso terapéutico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Rastreo Diferencial de Calorimetría , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Liberación de Fármacos , Endocitosis/efectos de los fármacos , Glutatión/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones Desnudos , Neoplasias/patología , Oxidación-Reducción , Péptidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de Fourier , Distribución Tisular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Since breast cancer is one of the most lethal malignancies, targeted strategies are urgently needed. In this study, we report the enhanced therapeutic efficacy of docetaxel (DTX) when combined with polyunsaturated fatty acids (PUFA) for effective treatment of multi-resistant breast cancers. Folic acid (FA)-conjugated PUFA-based lipid nanoparticles (FA-PLN/DTX) was developed. The physicochemical properties, in vitro uptake, in vitro cytotoxicity, and in vivo anticancer activity of FA-PLN/DTX were evaluated. FA-PLN/DTX could efficiently target and treat human breast tumor xenografts in vivo. They showed high payload carrying capacity with controlled release characteristics and selective endocytic uptake in folate receptor-overexpressing MCF-7 and MDA-MB-231 cells. PUFA synergistically improved the anticancer efficacy of DTX in both tested cancer cell lines by inducing a G2/M phase arrest and cell apoptosis. Combination of PUFA and DTX remarkably downregulated the expression levels of pro-apoptotic and anti-apoptotic markers, and blocked the phosphorylation of AKT signaling pathways. Compared to DTX alone, FA-PLN/DTX showed superior antitumor efficacy, with no signs of toxic effects in cancer xenograft animal models. We propose that PUFA could improve the therapeutic efficacy of anticancer agents in cancer therapy. Further studies are necessary to fully understand these findings and achieve clinical translation.
Asunto(s)
Nanoestructuras , Animales , Antineoplásicos , Línea Celular Tumoral , Docetaxel , Ácidos Grasos Insaturados , Humanos , TaxoidesRESUMEN
Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2 Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis.
Asunto(s)
Citocinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedad de Parkinson/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Animales , Sitios de Unión , Células COS , Línea Celular , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Ratones , Mutación Missense , Células 3T3 NIH , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Regulator of calcineurin 1 (RCAN1; also referred as DSCR1 or MCIP1) is located in close proximity to a Down syndrome critical region of human chromosome 21. Although RCAN1 is an endogenous inhibitor of calcineurin signaling that controls lymphocyte activation, apoptosis, heart development, skeletal muscle differentiation, and cardiac function, it is not yet clear whether RCAN1 might be involved in other cellular activities. In this study, we explored the extra-functional roles of RCAN1 by searching for novel RCAN1-binding partners. Using a yeast two-hybrid assay, we found that RCAN1 (RCAN1-1S) interacts with histone deacetylase 3 (HDAC3) in mammalian cells. We also demonstrate that HDAC3 deacetylates RCAN1. In addition, HDAC3 increases RCAN1 protein stability by inhibiting its poly-ubiquitination. Furthermore, HDAC3 promotes RCAN1 nuclear translocation. These data suggest that HDAC3, a new binding regulator of RCAN1, affects the protein stability and intracellular localization of RCAN1.