RESUMEN
The basement membrane (BM) constitutes a specialised form of the extracellular matrix (ECM) and plays important roles in many biological processes, such as cell migration, organ and tissue integrity, cell polarity, and the formation of metastases. In metazoans, a canonical BM is formed by only a few conserved structural core proteins: Laminin, Collagen IV, Nidogen and Perlecan. Depending on the tissue's function and mechanical load, additional matrix proteins interact with, or are incorporated into the BM, resulting in tissue-specific mechanical properties, such as higher stiffness or elasticity, or special resistance to mechanical stress or harmful environmental conditions. In flies, the collagen IV-like protein Pericardin forms an integral constituent of matrices around the heart and tension sensors (chordotonal organs) of the peripheral nervous system. The function and integrity of both organ systems strongly relies on the appropriate establishment of a Pericardin (Prc) matrix and the function of its adapter protein-Lonely heart (Loh). In this review, we provide an overview of the four collagens present in flies, and will discuss our recent work on the formation and function of Pericardin-containing matrices, the role of the adapter protein Lonely heart and the necessity of specialised ECM molecules in tissue architecture and function.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Proteínas de Drosophila/análisis , Matriz Extracelular/química , Laminina/análisis , Laminina/metabolismo , Colágeno Tipo IV/metabolismoRESUMEN
The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/metabolismo , Proteínas con Dominio LIM/fisiología , Animales , Citoesqueleto/química , Citoesqueleto/fisiología , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Proteínas con Dominio LIM/análisis , Proteínas con Dominio LIM/genética , Proteínas con Homeodominio LIM/análisis , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/fisiología , Transducción de Señal , Factores de Transcripción/metabolismo , Alas de Animales/crecimiento & desarrollo , alfa Catenina/metabolismoRESUMEN
The Drosophila genome codes for two decapping proteins, DCP1 and DCP2, out of which DCP2 is the active decapping enzyme. The present endeavour explores the endogenous promoter firing, transcript and protein expression of DCP2 in Drosophila wherein, besides a ubiquitous expression across development, we identify an active expression paradigm during dorsal closure and a plausible moonlighting expression in the Corazonin neurons of the larval brain. We also demonstrate that the ablation of DCP2 leads to embryonic lethality and defects in vital morphogenetic processes whereas a knockdown of DCP2 in the Corazonin neurons reduces the sensitivity to ethanol in adults, thereby ascribing novel regulatory roles to DCP2. Our findings unravel novel putative roles for DCP2 and identify it as a candidate for studies on the regulated interplay of essential molecules during early development in Drosophila, nay the living world.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Larva/genética , Larva/crecimiento & desarrollo , Neuronas/citología , Neuronas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
Dietary restriction (DR) extends healthy lifespan in diverse species. Age and nutrient-related changes in the abundance of microRNAs (miRNAs) and their processing factors have been linked to organismal longevity. However, the mechanisms by which they modulate lifespan and the tissue-specific role of miRNA-mediated networks in DR-dependent enhancement of lifespan remains largely unexplored. We show that two neuronally enriched and highly conserved microRNAs, miR-125 and let-7 mediate the DR response in Drosophila melanogaster. Functional characterization of miR-125 demonstrates its role in neurons while its target chinmo acts both in neurons and the fat body to modulate fat metabolism and longevity. Proteomic analysis revealed that Chinmo exerts its DR effects by regulating the expression of FATP, CG2017, CG9577, CG17554, CG5009, CG8778, CG9527, and FASN1. Our findings identify miR-125 as a conserved effector of the DR pathway and open the avenue for this small RNA molecule and its downstream effectors to be considered as potential drug candidates for the treatment of late-onset diseases and biomarkers for healthy aging in humans.
Asunto(s)
Restricción Calórica , Proteínas de Drosophila/metabolismo , Longevidad/fisiología , MicroARNs/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Línea Celular , Drosophila , Proteínas de Drosophila/análisis , Proteínas de Drosophila/química , Embrión no Mamífero , Femenino , Transducción de Señal/fisiologíaRESUMEN
Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. Here, we show that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients.
Asunto(s)
Centrosoma/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Proteínas de Drosophila/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Proteínas de Transporte Vesicular/fisiología , Animales , Línea Celular , Centrosoma/química , Síndrome de Chediak-Higashi , Gránulos Citoplasmáticos/química , Drosophila/química , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/análisis , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Femenino , Humanos , Lisosomas , Proteínas Asociadas a Microtúbulos/genética , Mutación , Oocitos/química , Huso Acromático/química , Proteínas de Transporte Vesicular/análisis , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
In the best studied cases (Aplysia feeding, crustacean stomatogastric system), peptidergic modulation is mediated by large numbers of peptides. Furthermore, in Aplysia, excitatory motor neurons release the peptides, obligatorily coupling target activation and modulator release. Vertebrate nervous systems typically contain about a hundred peptide modulators. These data have created a belief that modulation is, in general, complex. The stick insect leg is a well-studied locomotory model system, and the complete stick insect neuropeptide inventory was recently described. We used multiple techniques to comprehensively examine stick insect leg peptidergic modulation. Single-cell mass spectrometry (MS) and immunohistochemistry showed that myoinhibitory peptide (MIP) is the only neuronal (as opposed to hemolymph-borne) peptide modulator of all leg muscles. Leg muscle excitatory motor neurons contained no neuropeptides. Only the common inhibitor (CI) and dorsal unpaired median (DUM) neuron groups, each neuron of which innervates a group of functionally-related leg muscles, contained MIP. We described MIP transport to, and receptor presence in, one leg muscle, the extensor tibiae (ExtTi). MIP application reduced ExtTi slow fiber force and shortening by about half, increasing the muscle's ability to contract and relax rapidly. These data show neuromodulation does not need to be complex. Excitation and modulation do not need to be obligatorily coupled (Aplysia feeding). Modulation does not need to involve large numbers of peptides, with the attendant possibility of combinatorial explosion (stomatogastric system). Modulation can be simple, mediated by dedicated regulatory neurons, each innervating a single group of functionally-related targets, and all using the same neuropeptide.SIGNIFICANCE STATEMENT Vertebrate and invertebrate nervous systems contain large numbers (around a hundred in human brain) of peptide neurotransmitters. In prior work, neuropeptide modulation has been complex, either obligatorily coupling postsynaptic excitation and modulation, or large numbers of peptides modulating individual neural networks. The complete stick insect neuropeptide inventory was recently described. We comprehensively describe here peptidergic modulation in the stick insect leg. Surprisingly, out of the large number of potential peptide transmitters, only myoinhibitory peptide (MIP) was present in neurons innervating leg muscles. Furthermore, the peptide was present only in dedicated regulatory neurons, not in leg excitatory motor neurons. Peptidergic modulation can thus be simple, neither obligatorily coupling target activation and modulation nor involving so many peptides that combinatorial explosion can occur.
Asunto(s)
Proteínas de Drosophila/metabolismo , Ganglios de Invertebrados/metabolismo , Proteínas de Insectos/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Femenino , Ganglios de Invertebrados/química , Proteínas de Insectos/análisis , Proteínas de Insectos/genética , Insectos , Músculo Esquelético/químicaRESUMEN
Drosophila flight muscles are highly enriched with mitochondria and have emerged as a powerful genetic system for studying how oxidative phosphorylation (OXPHOS) complexes are assembled. Here, we describe a series of protocols for analyzing the integrity of OXPHOS complexes in Drosophila via blue native polyacrylamide gel electrophoresis (BN PAGE). We have also included protocols for the additional steps that are typically performed after OXPHOS complexes are separated by BN PAGE, such as Coomassie staining, silver staining, and in-gel OXPHOS activities. For complete details on the use and execution of this protocol, please refer to Murari et al. (2020).
Asunto(s)
Proteínas de Drosophila , Drosophila , Vuelo Animal/fisiología , Proteínas Musculares , Músculos , Animales , Drosophila/química , Drosophila/fisiología , Proteínas de Drosophila/análisis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Femenino , Masculino , Proteínas Musculares/análisis , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculos/química , Músculos/metabolismo , Electroforesis en Gel de Poliacrilamida Nativa , Fosforilación OxidativaRESUMEN
The Roundabout (Robo) family of axon guidance receptors has a conserved ectodomain arrangement of five immunoglobulin-like (Ig) domains plus three fibronectin type III (Fn) repeats. Based on the strong evolutionary conservation of this domain structure among Robo receptors, as well as in vitro structural and domain-domain interaction studies of Robo family members, this ectodomain arrangement is predicted to be important for Robo receptor signaling in response to Slit ligands. Here, we define the minimal ectodomain structure required for Slit binding and midline repulsive signaling in vivo by Drosophila Robo1. We find that the majority of the Robo1 ectodomain is dispensable for both Slit binding and repulsive signaling. We show that a significant level of midline repulsive signaling activity is retained when all Robo1 ectodomain elements apart from Ig1 are deleted, and that the combination of Ig1 plus one additional ectodomain element (Ig2, Ig5, or Fn3) is sufficient to restore midline repulsion to wild type levels. Further, we find that deleting four out of five Robo1 Ig domains (ΔIg2-5) does not affect negative regulation of Robo1 by Commissureless (Comm) or Robo2, while variants lacking all three fibronectin repeats (ΔFn1-3 and ΔIg2-Fn3) are insensitive to regulation by both Comm and Robo2, signifying a novel regulatory role for Robo1's Fn repeats. Our results provide an in vivo perspective on the importance of the conserved 5+3 ectodomain structure of Robo receptors, and suggest that specific biochemical properties and/or ectodomain structural conformations observed in vitro for domains other than Ig1 may have limited significance for in vivo signaling in the context of midline repulsion.
Asunto(s)
Drosophila/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal , Animales , Sitios de Unión , Drosophila/citología , Proteínas de Drosophila/análisis , Proteínas de Drosophila/metabolismo , Proteínas del Tejido Nervioso/análisis , Unión Proteica , Dominios Proteicos , Mapas de Interacción de Proteínas , Receptores Inmunológicos/análisis , Proteínas RoundaboutRESUMEN
The larvae of Drosophila melanogaster grow rapidly through use of a highly truncated cell cycle in which mitosis is entirely eliminated. The Drosophila homolog of the protooncogene transcription factor Myc plays a major role in promoting this endopolyploid (EP) growth. We have previously determined that the gene jim lovell (lov), which encodes a member of the BTB/POZ (Bric-a-brac, Tramtrack, Broad/Pox virus zinc finger) domain family of transcription factors, is also required for EP growth in one larval tissue, the trachea. Here we show that lov promotes EP growth in three further tissues indicating a fundamental role in this process. However, epistasis experiments revealed heterogeneity in lov's action in these tissues. Whereas in the tracheae and salivary glands lov acts downstream of Myc, in the fat body, reduced expression of lov does not impede the action of Myc, indicating an upstream action for the gene. We show here that lov's regulation of the gene uninflatable (uif) in the tracheae is a component of this difference. uif is required for tracheal EP growth downstream of Myc and lov but has no equivalent role in the fat body. Although Uif is a transmembrane component of the plasma membrane in the tracheae, its action downstream of Myc suggests an intracellular role for the protein in the tracheae. In addition to regulating uif expression in some tissues we also show that lov locates to the nucleolus, indicating it can function in both polymerase I and polymerase II transcriptional events. Our major finding is that tissue-specific mechanisms can interact with universal growth promotion by Myc to generate the individual endopolyploid organs of the larvae.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Epistasis Genética , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Poliploidía , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
Identifying moving synaptic vesicle complexes and isolating specific proteins present within such complexes in vivo is challenging. Here we detail a protocol that we have developed that is designed to simultaneously visualize the axonal transport of two fluorescently tagged synaptic vesicle proteins in living Drosophila larval segmental nerves in real time. Using a beam-splitter and split view software, larvae expressing GFP-tagged Synaptobrevin (Syb) and mRFP-tagged Rab4-GTPase or YFP-tagged Amyloid Precursor protein (APP) and mRFP-tagged Rab4-GTPase are imaged simultaneously using separate wavelengths. Merged kymographs from the two wavelengths are evaluated for colocalization analysis. Vesicle velocity analysis can also be done. Such analysis enables us to visualize the motility behaviors of two synaptic proteins present on a single vesicle complex and identify candidate proteins moving on synaptic vesicles in vivo, under physiological conditions.
Asunto(s)
Transporte Axonal , Drosophila melanogaster/metabolismo , Microscopía Intravital/métodos , Microscopía Fluorescente/métodos , Vesículas Sinápticas/ultraestructura , Precursor de Proteína beta-Amiloide/análisis , Precursor de Proteína beta-Amiloide/genética , Animales , Axones/metabolismo , Sistemas de Computación , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Colorantes Fluorescentes/análisis , GTP Fosfohidrolasas/análisis , GTP Fosfohidrolasas/genética , Quimografía , Larva , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas R-SNARE/análisis , Proteínas R-SNARE/genética , Programas Informáticos , Vesículas Sinápticas/fisiologíaRESUMEN
The small interfering RNA (siRNA) pathway of Drosophila melanogaster, mainly characterized by the activity of the enzymes Dicer 2 (Dcr-2) and Argonaute 2 (Ago-2), has been described as the major antiviral immune response. Several lines of evidence demonstrated its pivotal role in conferring resistance against viral infections at cellular and systemic level. However, only few studies have addressed the regulation and induction of this system upon infection and knowledge on stability and turnover of the siRNA pathway core components transcripts and proteins remains scarce. In the current work, we explore whether the siRNA pathway is regulated following viral infection in D. melanogaster. After infecting different fly strains with two different viruses and modes of infection, we observed changes in Dcr-2 and Ago-2 protein concentrations that were not related with changes in gene expression. This response was observed either upon viral infection or upon stress-related experimental procedure, indicating a bivalent function of the siRNA system operating as a general gene regulation rather than a specific antiviral system.
Asunto(s)
Proteínas Argonautas/análisis , Proteínas de Drosophila/análisis , Drosophila melanogaster/inmunología , ARN Helicasas/análisis , Ribonucleasa III/análisis , Virosis/inmunología , Animales , Proteínas Argonautas/genética , Susceptibilidad a Enfermedades , Proteínas de Drosophila/genética , Femenino , ARN Helicasas/genética , ARN Interferente Pequeño/fisiología , Ribonucleasa III/genética , Estrés Fisiológico , Virosis/metabolismoRESUMEN
Protein expression levels are regulated through both translation and degradation mechanisms. Levels of degradation intermediates, that is, partially degraded proteins, cannot be distinguished from those of intact proteins by global proteomics analysis, which quantify total protein abundance levels. This study aimed to develop a tool for assessing the aspects of degradation regulation via proteolytic processing through a new multiplexed N-terminomics method involving selective isobaric labeling of protein N-termini and immunoaffinity capture of the labeled N-terminal peptides. Our method allows for not only identification of proteolytic cleavage sites, but also highly multiplexed quantification of proteolytic processing. We profiled a number of potential cleavage sites by signal peptidase and provided experimental confirmation of predicted cleavage sites of signal peptide. Furthermore, the present method uniquely represents the landscape of proteomic proteolytic processing rate during early embryogenesis in Drosophila melanogaster, revealing the underlying mechanism of stringent decay regulation of zygotically expressed proteins during early stages of embryogenesis.
Asunto(s)
Proteínas de Drosophila/análisis , Péptidos/análisis , Animales , Drosophila melanogaster/embriología , Desarrollo Embrionario , Proteolisis , ProteómicaRESUMEN
Rhesus glycoproteins (Rh50) have been shown to be ammonia transporters in many species from bacteria to human. They are involved in various physiological processes including acid excretion and pH regulation. Rh50 proteins can also provide a structural link between the cytoskeleton and the plasma membranes that maintain cellular integrity. Although ammonia plays essential roles in the nervous system, in particular at glutamatergic synapses, a potential role for Rh50 proteins at synapses has not yet been investigated. To better understand the function of these proteins in vivo, we studied the unique Rh50 gene of Drosophila melanogaster, which encodes two isoforms, Rh50A and Rh50BC. We found that Drosophila Rh50A is expressed in larval muscles and enriched in the postsynaptic regions of the glutamatergic neuromuscular junctions. Rh50 inactivation by RNA interference selectively in muscle cells caused muscular atrophy in larval stages and pupal lethality. Interestingly, Rh50-deficiency in muscles specifically increased glutamate receptor subunit IIA (GluRIIA) level and the frequency of spontaneous excitatory postsynaptic potentials. Our work therefore highlights a new role for Rh50 proteins in the maintenance of Drosophila muscle architecture and synaptic physiology, which could be conserved in other species.
Asunto(s)
Compuestos de Amonio/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Larva/metabolismo , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Unión Neuromuscular/metabolismo , Compuestos de Amonio/análisis , Animales , Animales Modificados Genéticamente , Proteínas Sanguíneas/análisis , Proteínas de Drosophila/análisis , Drosophila melanogaster , Larva/crecimiento & desarrollo , Glicoproteínas de Membrana/análisis , Músculo Esquelético/química , Músculo Esquelético/crecimiento & desarrollo , Unión Neuromuscular/química , Unión Neuromuscular/crecimiento & desarrolloRESUMEN
The insect gustatory system senses taste information from environmental food substrates and processes it to control feeding behaviors. Drosophila melanogaster has been a powerful genetic model for investigating how various chemical cues are detected at the molecular and cellular levels. In addition to an understanding of how tastants belonging to five historically described taste modalities (sweet, bitter, acid, salt, and amino acid) are sensed, recent findings have identified taste neurons and receptors that recognize tastants of non-canonical modalities, including fatty acids, carbonated water, polyamines, H2O2, bacterial lipopolysaccharide (LPS), ammonia, and calcium. Analyses of response profiles of taste neurons expressing different suites of chemosensory receptors have allowed exploration of taste coding mechanisms in primary sensory neurons. In this review, we present the current knowledge of the molecular and cellular basis of taste detection of various categories of tastants. We also summarize evidence for organotopic and multimodal functions of the taste system. Functional characterization of peripheral taste neurons in different organs has greatly increased our understanding of how insect behavior is regulated by the gustatory system, which may inform development of novel insect pest control strategies.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Receptores de Superficie Celular/metabolismo , Células Receptoras Sensoriales/metabolismo , Gusto , Animales , Drosophila/anatomía & histología , Drosophila/citología , Drosophila/genética , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Expresión Génica , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Células Receptoras Sensoriales/citologíaRESUMEN
The Hippo signaling pathway is an evolutionarily conserved regulator that plays important roles in organ size control, homeostasis, and tumorigenesis. As the key effector of the Hippo pathway, Yorkie (Yki) binds to transcription factor Scalloped (Sd) and promotes the expression of target genes, leading to cell proliferation and inhibition of apoptosis. Thus, it is of great significance to understand the regulatory mechanism for Yki protein turnover. Here, we provide evidence that the deubiquitinating enzyme ubiquitin-specific protease 10 (Usp10) binds Yki to counteract Yki ubiquitination and stabilize Yki protein in Drosophila S2 cells. The results in Drosophila wing discs indicate that silence of Usp10 decreases the transcription of target genes of the Hippo pathway by reducing Yki protein. In vivo functional analysis ulteriorly showed that Usp10 upregulates the Yki activity in Drosophila eyes. These findings uncover Usp10 as a novel Hippo pathway modulator and provide a new insight into the regulation of Yki protein stability and activity.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Animales , Línea Celular , Citoplasma/metabolismo , Proteínas de Drosophila/análisis , Drosophila melanogaster/citología , Proteínas Nucleares/análisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Transactivadores/análisis , Proteasas Ubiquitina-Específicas , Ubiquitinación , Proteínas Señalizadoras YAPRESUMEN
Osteoarthritis (OA) is predominantly characterized by the progressive degradation of articular cartilage, the connective tissue produced by chondrocytes, due to an imbalance between anabolic and catabolic processes. In addition, physical activity (PA) is recognized as an important tool for counteracting OA. To evaluate PA effects on the chondrocyte lineage, we analyzed the expression of SOX9, COL2A1, and COMP in circulating progenitor cells following a half marathon (HM) performance. Therefore, we studied in-depth the involvement of metabolites affecting chondrocyte lineage, and we compared the metabolomic profile associated with PA by analyzing runners' sera before and after HM performance. Interestingly, this study highlighted that metabolites involved in vitamin B6 salvage, such as pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate, were highly modulated. To evaluate the effects of vitamin B6 in cartilage cells, we treated differentiated mesenchymal stem cells and the SW1353 chondrosarcoma cell line with vitamin B6 in the presence of IL1ß, the inflammatory cytokine involved in OA. Our study describes, for the first time, the modulation of the vitamin B6 salvage pathway following PA and suggests a protective role of PA in OA through modulation of this pathway.
Asunto(s)
Cartílago/metabolismo , Condrocitos/metabolismo , Ejercicio Físico/fisiología , Adulto , Atletas , Cartílago/fisiología , Proteína de la Matriz Oligomérica del Cartílago/análisis , Proteína de la Matriz Oligomérica del Cartílago/sangre , Cartílago Articular/metabolismo , Cartílago Articular/fisiología , Línea Celular , Células Cultivadas , Condrocitos/fisiología , Colágeno Tipo II/análisis , Colágeno Tipo II/sangre , Proteínas de Drosophila/análisis , Proteínas de Drosophila/sangre , Femenino , Humanos , Interleucina-1beta , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Metabolómica/métodos , Persona de Mediana Edad , Osteoartritis/metabolismo , Osteoartritis/fisiopatología , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/fisiopatología , Factor de Transcripción SOX9/análisis , Factor de Transcripción SOX9/sangre , Vitamina B 6/metabolismoRESUMEN
Insight into the structure-function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl-/H+ exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MS-compatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence; however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.
Asunto(s)
Antiportadores/análisis , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/análisis , Proteínas de Drosophila/análisis , Proteínas de Escherichia coli/análisis , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Fragmentos de Péptidos/análisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática/análisis , Secuencia de Aminoácidos , Animales , Antiportadores/química , Aquifex , Proteasas de Ácido Aspártico/química , Bacterias , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Drosophila/química , Drosophila melanogaster , Escherichia coli , Proteínas de Escherichia coli/química , Humanos , Modelos Moleculares , Pepsina A/química , Proteolisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Relación Estructura-Actividad , Porcinos , Urea/químicaRESUMEN
Columnar structure is a basic unit of the brain, but the mechanism underlying its development remains largely unknown. The medulla, the largest ganglion of the Drosophila melanogaster visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. In this study, using N-cadherin (Ncad) as a marker, we reveal the donut-like columnar structures along the 2D layer in the larval medulla that evolves to form three distinct layers in pupal development. Column formation is initiated by three core neurons, R8, R7, and Mi1, which establish distinct concentric domains within a column. We demonstrate that Ncad-dependent relative adhesiveness of the core columnar neurons regulates their relative location within a column along a 2D layer in the larval medulla according to the differential adhesion hypothesis. We also propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns.SIGNIFICANCE STATEMENT The columnar structure is a basic unit of the brain, but its developmental mechanism remains unknown. The medulla, the largest ganglion of the fly visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. We reveal that column formation is initiated by three core neurons that establish distinct concentric domains within a column. We demonstrate the in vivo evidence of N-cadherin-dependent differential adhesion among the core columnar neurons within a column along a 2D layer in the larval medulla. The 2D larval columns evolve to form three distinct layers in the pupal medulla. We propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns.
Asunto(s)
Cadherinas/análisis , Proteínas de Drosophila/análisis , Bulbo Raquídeo/química , Bulbo Raquídeo/citología , Neuronas/química , Animales , Animales Modificados Genéticamente , Cadherinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Femenino , Masculino , Bulbo Raquídeo/metabolismo , Neuronas/metabolismoRESUMEN
Allelic expression from each parent-of-origin is important as a backup and to ensure that enough protein products of a gene are produced. Thus far, it is not known how each cell throughout a tissue differs in parental allele expression at the level of protein synthesis. Here, we measure the expression of the Ribosomal protein L13a (Rpl13a) from both parental alleles simultaneously in single cells in the living animal. We use genome-edited Drosophila that have a quantitative reporter of protein synthesis inserted into the endogenous Rpl13a locus. We find that individual cells can have large (>10-fold) differences in protein expression between the two parental alleles. Cells can produce protein from only one allele oftentimes, and time-lapse imaging of protein production from each parental allele in each cell showed that the imbalance in expression from one parental allele over the other can invert over time. We also identify the histone methyltransferase EHMT to be involved in the protein synthesis dynamics within cells.