RESUMEN
Maintaining metabolic homeostasis requires coordinated nutrient utilization between intracellular organelles and across multiple organ systems. Many organs rely heavily on mitochondria to generate (ATP) from glucose, or stored glycogen. Proteins required for ATP generation are encoded in both nuclear and mitochondrial DNA (mtDNA). We show that motoneuron to muscle signaling by the TGFß/Activin family member Actß positively regulates glycogen levels during Drosophila development. Remarkably, we find that levels of stored glycogen are unaffected by altering cytoplasmic glucose catabolism. Instead, Actß loss reduces levels of mtDNA and nuclearly encoded genes required for mtDNA replication, transcription and translation. Direct RNAi mediated knockdown of these same nuclearly encoded mtDNA expression factors also results in decreased glycogen stores. Lastly, we find that expressing an activated form of the type I receptor Baboon in muscle restores both glycogen and mtDNA levels in actß mutants, thereby confirming a direct link between Actß signaling, glycogen homeostasis and mtDNA expression.
RESUMEN
Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. In this report, we show that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. Our research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia , Autofagia , Proteínas de Drosophila , Drosophila melanogaster , Longevidad , Animales , Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Longevidad/genética , Interferencia de ARNRESUMEN
Coiled-coil protein structural motifs have proven amenable to the design of structurally well-defined biomaterials. Mesoscale structural properties can be fairly well predicted based on rules governing the chemical interactions between the helices that define this structural motif. We explore the role of the hydrophobic core residues on the self-assembly of a coiled-coil polymer through a mutational analysis coupled with a salting-out procedure. Because the resultant polymers remain in solution, a thermodynamic approach is applied to characterize the polymer assembly using conventional equations from polymer theory to extract nucleation and elongation parameters. The stabilities and lengths of the polymers are measured using circular dichroism spectropolarimetry, sizing methods including dynamic light scattering and analytical ultracentrifugation, and atomic force microscopy to assess mesoscale morphology. Upon mutating isoleucines at two core positions to serines, we find that polymer stability is decreased while the degree of polymerization is about the same. Differences in results from circular dichroism and dynamic light scattering experiments suggest the presence of a stable intermediate state, and a scheme is proposed for how this intermediate might relate to the monomer and polymer states.
Asunto(s)
Materiales Biocompatibles/química , Péptidos/química , Proteínas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
BRCC36 is a JAMM (JAB1/MPN/Mov34 metalloenzyme) domain, lysine 63-ubiquitin (K63-Ub)-specific deubiquitinating enzyme (DUB) and a member of two protein complexes: the DNA damage-responsive BRCA1-RAP80 complex, and the cytoplasmic BRCC36 isopeptidase complex (BRISC). The presence of several identical constituents in both complexes suggests common regulatory mechanisms and potential competition between K63-Ub-related signaling in cytoplasmic and nuclear compartments. Surprisingly, we discover that BRCC36 DUB activity requires different interactions within the context of each complex. Abraxas and BRCC45 were essential for BRCC36 DUB activity within the RAP80 complex, whereas KIAA0157/Abro was the only interaction required for DUB activity within the BRISC. Poh1 also required protein interactions for activity, suggesting a common regulatory mechanism for JAMM domain DUBs. Finally, BRISC deficiency enhanced formation of the BRCA1-RAP80 complex in vivo, increasing BRCA1 levels at DNA double strand breaks. These findings reveal that JAMM domain DUB activity and K63-Ub levels are regulated by multiple mechanisms within the cell.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Citoplasma/enzimología , Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas Portadoras/genética , Núcleo Celular/genética , Citoplasma/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Enzimas Desubicuitinizantes , Endopeptidasas/genética , Células HeLa , Chaperonas de Histonas , Humanos , Proteínas de la Membrana/genética , Complejos Multienzimáticos/genética , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.
Asunto(s)
Rotura Cromosómica , Segregación Cromosómica , Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Drosophila melanogaster/metabolismo , Transducción de Señal , Animales , Roturas del ADN de Doble Cadena , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Pruebas Genéticas , Micronúcleo Germinal/metabolismo , Mitosis , Mutación/genética , Ubiquitinación , ADN Polimerasa thetaRESUMEN
Maintaining energy homeostasis is critical for ensuring proper growth and maximizing survival potential of all organisms. Here we review the role of somatic muscle in regulating energy homeostasis in insects. The muscle is not only a large consumer of energy, it also plays a crucial role in regulating metabolic signaling pathways and energy stores of the organism. We examine the metabolic pathways required to supply the muscle with energy, as well as muscle-derived signals that regulate metabolic energy homeostasis.
RESUMEN
Conserved DNA-damage responses (DDRs) sense genome damage and prevent mitosis of broken chromosomes. How cells lacking DDRs cope with broken chromosomes during mitosis is poorly understood. DDRs are frequently inactivated in cells with extra genomes (polyploidy), suggesting that study of polyploidy can reveal how cells with impaired DDRs/genome damage continue dividing. Here, we show that continued division and normal organ development occurs in polyploid, DDR-impaired Drosophila papillar cells. As papillar cells become polyploid, they naturally accumulate broken acentric chromosomes but do not apoptose/arrest the cell cycle. To survive mitosis with acentric chromosomes, papillar cells require Fanconi anemia proteins FANCD2 and FANCI, as well as Blm helicase, but not canonical DDR signaling. FANCD2 acts independently of previous S phases to promote alignment and segregation of acentric DNA produced by double-strand breaks, thus avoiding micronuclei and organ malformation. Because polyploidy and impaired DDRs can promote cancer, our findings provide insight into disease-relevant DNA-damage tolerance mechanisms.