scholarly journals The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes

2021 ◽  
Author(s):  
Elena Porcellato ◽  
Juan Carlos Gonzalez ◽  
Constantin Ahlmann-Eltze ◽  
Mahmoud Ali Elsakka ◽  
Itamar Shapira ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Protein Modification ◽  
Protein Localization ◽  
Ectopic Expression ◽  
Negative Control ◽  
Neuronal System ◽  
Post Translational Modification ◽  
Systematic Analysis ◽  
Interaction Partners ◽  
Client Proteins

ABSTRACTProtein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modification in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster.Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24- mutant, as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic.In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.

scholarly journals Needs and Targets for the multi sex combs Gene Product in Drosophila melanogaster

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1823-1838 ◽  
Author(s):  
Olivier Saget ◽  
Françoise Forquignon ◽  
Pedro Santamaria ◽  
Neel B Randsholt
Keyword(s):  
Drosophila Melanogaster ◽  
Ectopic Expression ◽  
Polycomb Group ◽  
Homeotic Genes ◽  
Maternal Control ◽  
Gap Gene ◽  
Sex Combs ◽  
Normal Expression ◽  
Selector Genes ◽  
Mutant Phenotypes

Abstract We have analyzed the requirements for the multi sex combs (mxc) gene during development to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila melanogaster. mxc is allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm (l(1)mbn). We show that the mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc has also a strong maternal effect. We find that hypomorphic mxc mutations enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. We also demonstrate that the mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. Our analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression previously reported for some PcG genes and suggests that the mechanism of this early PcG function could be different from the PcG-mediated regulation of homeotic selector genes later in development. We discuss these data in view of the numerous functions of PcG genes during development.

scholarly journals Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1519 ◽  
Author(s):  
Jessica R. Blount ◽  
Kozeta Libohova ◽  
Gustavo M. Silva ◽  
Sokol V. Todi
Keyword(s):  
Drosophila Melanogaster ◽  
Fruit Fly ◽  
Post Translational Modification ◽  
Ubiquitin Chain ◽  
Interaction Site ◽  
Cellular Processes ◽  
Protein Adduct ◽  
Linear Chains ◽  
Linear Poly ◽  
Free Ubiquitin

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.

scholarly journals Effects of O-GlcNAcylation on functional mitochondrial transfer from astrocytes

2020 ◽  
pp. 0271678X2096958 ◽  
Author(s):  
Ji-Hyun Park ◽  
Yoshihiko Nakamura ◽  
Wenlu Li ◽  
Gen Hamanaka ◽  
Ken Arai ◽  
...  
Keyword(s):  
Protein Modification ◽  
Mitochondrial Protein ◽  
Brefeldin A ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Mitochondrial Transfer ◽  
The Central Nervous System ◽  
Critical Process

Mitochondria may be transferred from cell to cell in the central nervous system and this process may help defend neurons against injury and disease. But how mitochondria maintain their functionality during the process of release into extracellular space remains unknown. Here, we report that mitochondrial protein O-GlcNAcylation is a critical process to support extracellular mitochondrial functionality. Activation of CD38-cADPR signaling in astrocytes robustly induced protein O-GlcNAcylation in mitochondria, while oxygen-glucose deprivation and reoxygenation showed transient and mild protein modification. Blocking the endoplasmic reticulum – Golgi trafficking with Brefeldin A or slc35B4 siRNA reduced O-GlcNAcylation, and resulted in the secretion of mitochondria with decreased membrane potential and mtDNA. Finally, loss-of-function studies verified that O-GlcNAc-modified mitochondria demonstrated higher levels of neuroprotection after astrocyte-to-neuron mitochondrial transfer. Collectively, these findings suggest that post-translational modification by O-GlcNAc may be required for supporting the functionality and neuroprotective properties of mitochondria released from astrocytes.

scholarly journals Drosophila Zic family member odd-paired is needed for adult post-ecdysis maturation

Open Biology ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 190245
Author(s):  
Eléanor Simon ◽  
Sergio Fernández de la Puebla ◽  
Isabel Guerrero
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Transcription Factor ◽  
Drosophila Melanogaster ◽  
Family Member ◽  
Ectopic Expression ◽  
Functional Conservation ◽  
The Central Nervous System ◽  
Behavioural Sequence

Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed by maturation of the new cuticle. In Drosophila melanogaster , the last ecdysis occurs at eclosion from the pupal case, with a post-eclosion behavioural sequence that leads to wing extension, cuticle stretching and tanning. These events are highly stereotyped and are controlled by a subset of crustacean cardioactive peptide (CCAP) neurons through the expression of the neuropeptide Bursicon (Burs). We have studied the role of the transcription factor Odd-paired (Opa) during the post-eclosion period. We report that opa is expressed in the CCAP neurons of the central nervous system during various steps of the ecdysis process and in peripheral CCAP neurons innerving the larval muscles involved in adult ecdysis. We show that its downregulation alters Burs expression in the CCAP neurons. Ectopic expression of Opa, or the vertebrate homologue Zic2 , in the CCAP neurons also affects Burs expression, indicating an evolutionary functional conservation. Finally, our results show that, independently of its role in Burs regulation, Opa prevents death of CCAP neurons during larval development.

A genetic and molecular analysis of an invectedDominant mutation in Drosophila melanogaster

Genome ◽  
1998 ◽  
Vol 41 (3) ◽  
pp. 381-390 ◽  
Author(s):  
A J Simmonds ◽  
J B Bell
Keyword(s):  
Drosophila Melanogaster ◽  
Imaginal Disc ◽  
Transcriptional Start Site ◽  
Ectopic Expression ◽  
Careful Analysis ◽  
Wing Imaginal Disc ◽  
Coding Region ◽  
Specific Expression ◽  
Additional Lesion ◽  
Normal Expression

The invected gene of Drosophila melanogaster is a homeobox-containing gene that is closely related to engrailed. A dominant gain of function allele, invectedDominant, was derived from mutagenesis of a dominant allele of vestigial, In(2R)vgW. A careful analysis of the phenotype of invectedDominant shows that it is associated with a transformation of the anterior compartment of the wing to a posterior fate. This transformation is normally limited to the wing blade itself and does not involve the remaining tissues derived from the wing imaginal disc, including the wing hinge and dorsal thorax of the fly. The ectopic expression of invected protein associated with invectedDominant correlates spatially with the normal expression pattern of vestigial in the wing imaginal disc, suggesting that control elements of vestigial are driving ectopic invected expression. This was confirmed by sequence analysis that shows that the dominant vestigial activity was eliminated by a deletion that removes the 3' portion of the vestigial coding region. This leaves a gene fusion wherein the vestigial enhancer elements are still juxtaposed immediately 5' to the invected transcriptional start site, but with the vg sequences harboring an additional lesion. Unlike recessive invected alleles, the invectedDominant allele produces an observable phenotype, and as such, should prove useful in determining the role of invected in patterning the wing imaginal disc. Genetic analysis has shown that mutations of polyhomeotic, a gene involved in regulating engrailed expression, cause a reproducible alteration in the invectedDominant phenotype. Finally, the invectedDominant allele should prove valuable for identifying and characterizing genes that are activated within the posterior compartment. A screen using various lacZ lines that are asymmetrically expressed in an anterior-posterior manner in the wing imaginal disc isolated one line that shows posterior-specific expression within the transformed anterior compartment.Key words: Drosophila, development, dominant mutation, ectopic, wings.

scholarly journals Miro, a Rho GTPase genetically interacts with Alzheimer's disease-associated genes (Tau, Aβ42 and Appl) in Drosophila melanogaster

Biology Open ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. bio049569 ◽  
Author(s):  
Komal Panchal ◽  
Anand Krishna Tiwari
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Drosophila Melanogaster ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Genetic Interaction ◽  
Mitochondrial Dynamics ◽  
Ectopic Expression ◽  
Mitochondrial Outer Membrane ◽  
Disease Associated Genes

ABSTRACTMiro (mitochondrial Rho GTPases), a mitochondrial outer membrane protein, facilitates mitochondrial axonal transport along the microtubules to facilitate neuronal function. It plays an important role in regulating mitochondrial dynamics (fusion and fission) and cellular energy generation. Thus, Miro might be associated with the key pathologies of several neurodegenerative diseases (NDs) including Alzheimer's disease (AD). In the present manuscript, we have demonstrated the possible genetic interaction between Miro and AD-related genes such as Tau, Aβ42 and Appl in Drosophila melanogaster. Ectopic expression of Tau, Aβ42 and Appl induced a rough eye phenotype, defects in phototaxis and climbing activity, and shortened lifespan in the flies. In our study, we have observed that overexpression of Miro improves the rough eye phenotype, behavioral activities (climbing and phototaxis) and ATP level in AD model flies. Further, the improvement examined in AD-related phenotypes was correlated with decreased oxidative stress, cell death and neurodegeneration in Miro overexpressing AD model flies. Thus, the obtained results suggested that Miro genetically interacts with AD-related genes in Drosophila and has the potential to be used as a therapeutic target for the design of therapeutic strategies for NDs.This article has an associated First Person interview with the first author of the paper.

scholarly journals FlyRNAi.org—the database of the Drosophila RNAi screening center and transgenic RNAi project: 2021 update

2020 ◽  
Vol 49 (D1) ◽  
pp. D908-D915
Author(s):  
Yanhui Hu ◽  
Aram Comjean ◽  
Jonathan Rodiger ◽  
Yifang Liu ◽  
Yue Gao ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Functional Genomics ◽  
Protein Interactions ◽  
Protein Modification ◽  
Genetic Model ◽  
Online Resources ◽  
Model Species ◽  
Rnai Screening ◽  
Transcriptomics Data

Abstract The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) provides a suite of online resources that facilitate functional genomics studies with a special emphasis on Drosophila melanogaster. Currently, the database provides: gene-centric resources that facilitate ortholog mapping and mining of information about orthologs in common genetic model species; reagent-centric resources that help researchers identify RNAi and CRISPR sgRNA reagents or designs; and data-centric resources that facilitate visualization and mining of transcriptomics data, protein modification data, protein interactions, and more. Here, we discuss updated and new features that help biological and biomedical researchers efficiently identify, visualize, analyze, and integrate information and data for Drosophila and other species. Together, these resources facilitate multiple steps in functional genomics workflows, from building gene and reagent lists to management, analysis, and integration of data.

scholarly journals Systematic Analysis of Gibberellin Pathway Components in Medicago truncatula Reveals the Potential Application of Gibberellin in Biomass Improvement

2020 ◽  
Vol 21 (19) ◽  
pp. 7180
Author(s):  
Hongfeng Wang ◽  
Hongjiao Jiang ◽  
Yiteng Xu ◽  
Yan Wang ◽  
Lin Zhu ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Feedback Regulation ◽  
Specific Expression ◽  
Systematic Analysis ◽  
Model Legume ◽  
Genome Wide ◽  
A Genome

Gibberellins (GAs), a class of phytohormones, act as an essential natural regulator of plant growth and development. Many studies have shown that GA is related to rhizobial infection and nodule organogenesis in legume species. However, thus far, GA metabolism and signaling components are largely unknown in the model legume Medicago truncatula. In this study, a genome-wide analysis of GA metabolism and signaling genes was carried out. In total 29 components, including 8 MtGA20ox genes, 2 MtGA3ox genes, 13 MtGA2ox genes, 3 MtGID1 genes, and 3 MtDELLA genes were identified in M. truncatula genome. Expression profiles revealed that most members of MtGAox, MtGID1, and MtDELLA showed tissue-specific expression patterns. In addition, the GA biosynthesis and deactivation genes displayed a feedback regulation on GA treatment, respectively. Yeast two-hybrid assays showed that all the three MtGID1s interacted with MtDELLA1 and MtDELLA2, suggesting that the MtGID1s are functional GA receptors. More importantly, M. truncatula exhibited increased plant height and biomass by ectopic expression of the MtGA20ox1, suggesting that enhanced GA response has the potential for forage improvement.

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