RESUMEN
Membrane dynamics are important to the integrity and function of mitochondria. Defective mitochondrial fusion underlies the pathogenesis of multiple diseases. The ability to target fusion highlights the potential to fight life-threatening conditions. Here we report a small molecule agonist, S89, that specifically promotes mitochondrial fusion by targeting endogenous MFN1. S89 interacts directly with a loop region in the helix bundle 2 domain of MFN1 to stimulate GTP hydrolysis and vesicle fusion. GTP loading or competition by S89 dislodges the loop from the GTPase domain and unlocks the molecule. S89 restores mitochondrial and cellular defects caused by mitochondrial DNA mutations, oxidative stress inducer paraquat, ferroptosis inducer RSL3 or CMT2A-causing mutations by boosting endogenous MFN1. Strikingly, S89 effectively eliminates ischemia/reperfusion (I/R)-induced mitochondrial damage and protects mouse heart from I/R injury. These results reveal the priming mechanism for MFNs and provide a therapeutic strategy for mitochondrial diseases when additional mitochondrial fusion is beneficial.
Asunto(s)
Dinámicas Mitocondriales , Proteínas de Transporte de Membrana Mitocondrial , Ratones , Animales , Proteínas de Transporte de Membrana Mitocondrial/análisis , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Mitocondrias , Hidrólisis , Guanosina Trifosfato/análisis , Guanosina Trifosfato/farmacología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/farmacologíaRESUMEN
Abundant APOBEC3 (A3) deaminase-mediated mutations can dominate the mutational landscape ('mutator phenotype') of some cancers, however, the basis of this sporadic vulnerability is unknown. We show here that elevated expression of the bifunctional DNA glycosylase, NEIL2, sensitizes breast cancer cells to A3B-mediated mutations and double-strand breaks (DSBs) by perturbing canonical base excision repair (BER). NEIL2 usurps the canonical lyase, APE1, at abasic sites in a purified BER system, rendering them poor substrates for polymerase ß. However, the nicked NEIL2 product can serve as an entry site for Exo1 in vitro to generate single-stranded DNA, which would be susceptible to both A3B and DSBs. As NEIL2 or Exo1 depletion mitigates the DNA damage caused by A3B expression, we suggest that aberrant NEIL2 expression can explain certain instances of A3B-mediated mutations.
Asunto(s)
Desaminasas APOBEC/genética , Neoplasias de la Mama/metabolismo , Citidina Desaminasa/metabolismo , Reparación del ADN , Antígenos de Histocompatibilidad Menor/genética , Mutación , Desaminasas APOBEC/metabolismo , Línea Celular Tumoral , Citidina Desaminasa/genética , Humanos , Antígenos de Histocompatibilidad Menor/metabolismo , Transcripción GenéticaRESUMEN
The ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.
RESUMEN
Silkworm posterior silkgland is a model for studying intracellular trafficking. Here, using this model, we identify several potential cargo proteins of BmKinesin-1 and focus on one candidate, BmCREC. BmCREC (also known as Bombyx mori DNA supercoiling factor, BmSCF) was previously proposed to supercoil DNA in the nucleus. However, we show here that BmCREC is localized in the ER lumen. Its C-terminal tetrapeptide HDEF is recognized by the KDEL receptor, and subsequently it is retrogradely transported by coat protein I (COPI) vesicles to the ER. Lacking the HDEF tetrapeptide of BmCREC or knocking down COPI subunits results in decreased ER retention and simultaneously increased secretion of BmCREC. Furthermore, we find that BmCREC knockdown markedly disrupts the morphology of the ER and Golgi apparatus and leads to a defect of posterior silkgland tube expansion. Together, our results clarify the ER retention mechanism of BmCREC and reveal that BmCREC is indispensable for maintaining ER/Golgi morphology.
Asunto(s)
ADN Superhelicoidal/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Insectos/fisiología , Secuencias de Aminoácidos , Animales , Bombyx , Núcleo Celular/metabolismo , Proteína Coat de Complejo I/metabolismo , Citoplasma/metabolismo , ADN Superhelicoidal/química , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Insectos/genética , Cinesinas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , TransfecciónRESUMEN
Calumenin isoforms 1 and 2 (calu-1/2), encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20-46) and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Unión al Calcio/genética , Línea Celular , Dineínas/genética , Dineínas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Cinesinas/genética , Cinesinas/metabolismo , Microscopía Fluorescente , Unión Proteica/genética , Unión Proteica/fisiología , Isoformas de Proteínas/genética , Estabilidad Proteica , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Interferencia de ARNRESUMEN
Tau proteins are major microtubule-associated proteins (MAPs), which promote polymerization of tubulin and determine spacings between microtubules in axons of both the central and peripheral nervous systems (CNS and PNS). Here, we cloned and identified a tau-like protein BmTau from silkworm, Bombyx mori (GenBank accession number FJ904935). The coding sequence of BmTau is 723 bases long and encodes an approximate 30 kDa protein. In the C-terminus of BmTau are contained four predicted microtubule-binding domains, which share strong sequence homology to its ortholog in Drosophila melanoganster. Relative real-time PCR analysis showed ubiquitous expression of BmTau in both neurons and non-neural cells, with its mRNA abundantly expressing in brain but significantly less detected in trachea, fat body, and silkgland. Furthermore, immunocytochemical studies in BmN cells transfected with EGFP-BmTau indicated that BmTau functioned as microtubule bundling protein as its orthologues.
Asunto(s)
Bombyx/metabolismo , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Cartilla de ADN , ADN Complementario , Células HeLa , Humanos , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido , Proteínas tau/genéticaRESUMEN
BACKGROUND: Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation. METHODOLOGY/PRINCIPAL FINDINGS: Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α, ß, ß', δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, ß'- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion. CONCLUSIONS/SIGNIFICANCE: The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.
Asunto(s)
Proteínas de Insectos/fisiología , Animales , Bombyx , Clonación Molecular , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Fracciones Subcelulares/metabolismoRESUMEN
Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland (PSG), specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesin-driven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins (KLPs), BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein (RNP) complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.