Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms.

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

The indispensable contribution of s38 protein to ovarian-eggshell morphogenesis in Drosophila melanogaster.

Drosophila chorion represents a remarkable model system for the in vivo study of complex extracellular-matrix architectures. For its organization and structure, s38 protein is considered as a component of major importance, since it is synthesized and secreted during early choriogenesis. However, there is no evidence that proves its essential, or redundant, role in chorion biogenesis. Hence, we show that targeted downregulation of s38 protein, specifically in the ovarian follicle-cell compartment, via employment of an RNAi-mediated strategy, causes generation of diverse dysmorphic phenotypes, regarding eggshell's regionally and radially specialized structures. Downregulation of s38 protein severely impairs fly's fertility and is unable to be compensated by the s36 homologous family member, thus unveiling s38 protein's essential contribution to chorion's assembly and function. Altogether, s38 acts as a key skeletal protein being critically implicated in the patterning establishment of a highly structured tripartite endochorion. Furthermore, it seems that s38 loss may sensitize choriogenesis to stochastic variation in its coordination and timing.

Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis.

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In our study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion's regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion's fragility but also induces severe structural irregularities on chorion's surface and entirely impairs fly's fertility. Moreover, we herein unveil a novel function of s36 chorionic protein in the regulation of number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress.

Global Proteomic Profiling of Drosophila Ovary: A High-resolution, Unbiased, Accurate and Multifaceted Analysis.

BACKGROUND: Drosophila melanogaster ovary serves as an attractive model system for the investigation of the cell cycle, death, signaling, migration, differentiation, development and stemness. By employing the 3750/+ heterozygote fly strain that carries specific functions in the follicle cell compartment, and a reliable control in GAL4/UAS-based transgenic technology, we herein characterized the protein-expression profiling of D. melanogaster ovary by applying high-resolution proteomic tools and bioinformatics programs. MATERIALS AND METHODS: Whole-cell total protein extracts derived from 3750/+ fly ovaries were prepared under highly denaturing conditions and after tryptic digestion, their cognate peptides were processed to liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis in a high-resolution LTQ Orbitrap Elite instrument. Obtained protein data were analyzed through use of UniProt, DAVID, KEGG and PANTHER bioinformatics platforms. RESULTS: The 7,583 unique peptides identified show that fly ovary contains at least 2,103 single proteins, which are distributed to all egg chamber compartments, in cytoplasm, membrane and nucleus, compartmentalized into major cellular organelles, and categorized into critical macromolecular assemblies. Among the recognized specific functions, nucleic acid binding, hydrolase, oxidoreductase, transporter and vesicle-mediated trafficking activities were the most prevalent. Determinants implicated in cellular metabolism and gene expression are represented by ~41% and ~17% of the ovarian proteome, respectively. Surprisingly, several proteins were found engaged in aging, immune response and neurogenesis. All major signaling pathways were detected, while apoptotic and non-apoptotic cell death programs were also identified. Remarkably, proteins involved in tumor formation, neurodegenerative and inflammatory diseases were also recognized. The successful remodeling of the proteasome and nearly complete molecular reconstruction of the citrate cycle and fatty acid degradation pathways demonstrate the efficacy, accuracy and fidelity of our combined proteomics/bioinformatics approach. CONCLUSION: Global proteomic characterization of D. melanogaster ovary allows the discovery of novel regulators and pathways, and provides a systemic view of networks that govern ovarian pathophysiology and embryonic development in fly species as well in humans.