RESUMEN
Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismoRESUMEN
MicroRNAs play an essential role in cell homeostasis and have been proposed as therapeutic agents. One strategy to deliver microRNAs is to genetically engineer target cells to express microRNAs of interest. However, to control dosage and timing, as well as to limit potential side-effects, microRNAs' expression should ideally be under exogenous, inducible control. Conditional expression of miRNA-based short hairpin RNAs (shRNAmirs) via gene regulatory circuits such as the Tet-system is therefore a promising strategy to control shRNAmirs' expression in research and therapy. Single vector approaches like Tet-On all-in-one designs are more compatible with potential clinical applications by providing the Tet-On system components in a single round of genetic engineering. However, all-in-one systems often come at the expense of heterogeneous and unstable expression. In this study, we aimed to understand the causes that lead to such erratic transgene expression. By using a reporter cell, we found that the degree of heterogeneity mostly correlated with reverse tetracycline transactivator (rtTA) expression levels. Moreover, the targeted integration of a potent rtTA expression cassette into a genomic safe harbor locus functionally rescued previously silenced rtTA-responsive transcription units. Overall, our results suggest that ensuring homogenous and stable rtTA expression is essential for the robust and reliable performance of future Tet-On all-in-one designs.
Asunto(s)
MicroARNs , Transactivadores , Antibacterianos , Regulación de la Expresión Génica , MicroARNs/genética , Mosaicismo , Tetraciclina/farmacología , Transactivadores/genética , Transactivadores/metabolismo , Transgenes/genéticaRESUMEN
The cardioprotective properties of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are currently being investigated in preclinical studies. Although microRNAs (miRNAs) encapsulated in EVs have been identified as one component responsible for the cardioprotective effect of MSCs, their potential off-target effects have not been sufficiently characterized. In the present study, we aimed to investigate the miRNA profile of EVs isolated from MSCs that were derived from cord blood (CB) and adipose tissue (AT). The identified miRNAs were then compared to known targets from the literature to discover possible adverse effects prior to clinical use. Our data show that while many cardioprotective miRNAs such as miR-22-3p, miR-26a-5p, miR-29c-3p, and miR-125b-5p were present in CB- and AT-MSC-derived EVs, a large number of known oncogenic and tumor suppressor miRNAs such as miR-16-5p, miR-23a-3p, and miR-191-5p were also detected. These findings highlight the importance of quality assessment for therapeutically applied EV preparations.
Asunto(s)
Tejido Adiposo/citología , Vesículas Extracelulares/genética , Sangre Fetal/citología , Perfilación de la Expresión Génica/métodos , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , Adulto , Células Cultivadas , Análisis por Conglomerados , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/citología , MicroARNs/clasificación , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Transducción de Señal/genéticaRESUMEN
For more than 20 years, tremendous efforts have been made to develop cell-based therapies for treatment of heart failure. However, the results of clinical trials using somatic, nonpluripotent stem or progenitor cells have been largely disappointing in both cardiology and cardiac surgery scenarios. Surgical groups were among the pioneers of experimental and clinical myocyte transplantation ("cellular cardiomyoplasty"), but little translational progress was made prior to the development of cellular reprogramming for creation of induced pluripotent stem cells (iPSC). Ever since, protocols have been developed which allow for the derivation of large numbers of autologous cardiomyocytes (CMs) from patient-specific iPSC, moving translational research closer toward clinical pilot trials. However, compared with somatic cell therapy, the technology required for safe and efficacious pluripotent stem cell (PSC)-based therapies is extremely complex and requires tremendous resources and close interactions between basic scientists and clinicians. This review summarizes PSC sources, strategies to derive CMs, current cardiac tissue engineering approaches, concerns regarding immunogenicity and cellular maturity, and highlights the contributions made by surgical groups.
Asunto(s)
Enfermedades Cardiovasculares/cirugía , Células Madre Embrionarias/trasplante , Miocardio/patología , Miocitos Cardíacos/trasplante , Células Madre Pluripotentes/trasplante , Regeneración , Medicina Regenerativa/métodos , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/fisiopatología , Linaje de la Célula , Reprogramación Celular , Técnicas de Reprogramación Celular , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fenotipo , Células Madre Pluripotentes/metabolismo , Recuperación de la Función , Transducción de Señal , Resultado del TratamientoRESUMEN
UNLABELLED: Reduction-oxidation (redox) signaling, the translation of an oxidative intracellular environment into a cellular response, is mediated by the reversible oxidation of specific cysteine thiols. The latter can result in disulfide formation between protein hetero- or homodimers that alter protein function until the local cellular redox environment has returned to the basal state. We have previously shown that this mechanism promotes the nuclear localization and activity of the Forkhead Box O4 (FOXO4) transcription factor. AIMS: In this study, we sought to investigate whether redox signaling differentially controls the human FOXO3 and FOXO4 paralogs. RESULTS: We present evidence that FOXO3 and FOXO4 have acquired paralog-specific cysteines throughout vertebrate evolution. Using a proteome-wide screen, we identified previously unknown redox-dependent FOXO3 interaction partners. The nuclear import receptors Importin-7 (IPO7) and Importin-8 (IPO8) form a disulfide-dependent heterodimer with FOXO3, which is required for its reactive oxygen species-induced nuclear translocation. FOXO4 does not interact with IPO7 or IPO8. INNOVATION AND CONCLUSION: IPO7 and IPO8 control the nuclear import of FOXO3, but not FOXO4, in a redox-sensitive and disulfide-dependent manner. Our findings suggest that evolutionary acquisition of cysteines has contributed to regulatory divergence of FOXO paralogs, and that phylogenetic analysis can aid in the identification of cysteines involved in redox signaling.