Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models.

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.

Processing of the capsid proteins of the Betachrysovirus Fusarium graminearum virus-China 9 (FgV-ch9).

While the capsid of viruses in the Alphachrysovirus genus is built of subunits of a single coat protein, the capsid of viruses grouped in the Betachrysovirus genus may consist of subunits of two different proteins. For four of these betachrysoviruses, the detected molecular weights of the putative coat proteins differ from the sizes deduced from the nucleic acid sequence. The origin of these modifications remained unclear and it was hypothesized that the coat proteins undergo unspecific degradation. In our study, we show that these modifications are based on processing steps performed by unknown factors present in extracts of several eukaryotic organisms. Furthermore, we show that the C-terminal domain of P3 is fully degraded after capsid processing and particle assembly.

Functional analysis of the biochemical activity of mammalian phosphatidylinositol 5 phosphate 4-kinase enzymes.

Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) are enzymes that catalyse the phosphorylation of phosphatidylinositol 5-phosphate (PI5P) to generate PI(4,5)P2 Mammalian genomes contain three genes, PIP4K2Α, 2B and 2C and murine knockouts for these suggested important physiological roles in vivo The proteins encoded by PIP4K2A, 2B and 2C show widely varying specific activities in vitro; PIP4K2A is highly active and PIP4K2C 2000-times less active, and the relationship between this biochemical activity and in vivo function is unknown. By contrast, the Drosophila genome encodes a single PIP4K (dPIP4K) that shows high specific activity in vitro and loss of this enzyme results in reduced salivary gland cell size in vivo We find that the kinase activity of dPIP4K is essential for normal salivary gland cell size in vivo Despite their highly divergent specific activity, we find that all three mammalian PIP4K isoforms are able to enhance salivary gland cell size in the Drosophila PIP4K null mutant implying a lack of correlation between in vitro activity measurements and in vivo function. Further, the kinase activity of PIP4K2C, reported to be almost inactive in vitro, is required for in vivo function. Our findings suggest the existence of unidentified factors that regulate PIP4K enzyme activity in vivo.

WW domain-mediated regulation and activation of E3 ubiquitin ligase Suppressor of Deltex.

The Nedd4 family E3 ligases Itch and WWP1/2 play crucial roles in the regulation of cell cycle progression and apoptosis and are closely correlated with cancer development and metastasis. It has been recently shown that the ligase activities of Itch and WWP1/2 are tightly regulated, with the HECT domain sequestered intramolecularly by a linker region connecting WW2 and WW3. Here, we show that a similar autoinhibitory mechanism is utilized by the Drosophila ortholog of Itch and WWP1/2, Suppressor of Deltex (Su(dx)). We show that Su(dx) adopts an inactive steady state with the WW domain region interacting with the HECT domain. We demonstrate that both the linker and preceding WW2 are required for the efficient binding and regulation of Su(dx) HECT. Recruiting the multiple-PY motif-containing adaptor dNdfip via WW domains relieves the inhibitory state of Su(dx) and leads to substrate (e.g. Notch) ubiquitination. Our study demonstrates an evolutionarily conservative mechanism governing the regulation and activation of some Nedd4 family E3 ligases. Our results also suggest a dual regulatory mechanism for specific Notch down-regulation via dNdfip-Su(dx)-mediated Notch ubiquitination.

Activation mechanisms of the Hippo kinase signaling cascade.

First discovered two decades ago through genetic screens in Drosophila, the Hippo pathway has been shown to be conserved in metazoans and controls organ size and tissue homeostasis through regulating the balance between cell proliferation and apoptosis. Dysregulation of the Hippo pathway leads to aberrant tissue growth and tumorigenesis. Extensive studies in Drosophila and mammals have identified the core components of Hippo signaling, which form a central kinase cascade to ultimately control gene expression. Here, we review recent structural, biochemical, and cellular studies that have revealed intricate phosphorylation-dependent mechanisms in regulating the formation and activation of the core kinase complex in the Hippo pathway. These studies have established the dimerization-mediated activation of the Hippo kinase (mammalian Ste20-like 1 and 2 (MST1/2) in mammals), the dynamic scaffolding and allosteric roles of adaptor proteins in downstream kinase activation, and the importance of multisite linker autophosphorylation by Hippo and MST1/2 in fine-tuning the signaling strength and robustness of the Hippo pathway. We highlight the gaps in our knowledge in this field that will require further mechanistic studies.

Deducing the presence of proteins and proteoforms in quantitative proteomics.

The human genome harbors just 20,000 genes suggesting that the variety of possible protein products per gene plays a significant role in generating functional diversity. In bottom-up proteomics peptides are mapped back to proteins and proteoforms to describe a proteome; however, accurate quantitation of proteoforms is challenging due to incomplete protein sequence coverage and mapping ambiguities. Here, we demonstrate that a new software tool called ProteinClusterQuant (PCQ) can be used to deduce the presence of proteoforms that would have otherwise been missed, as exemplified in a proteomic comparison of two fly species, Drosophila melanogaster and D. virilis. PCQ was used to identify reduced levels of serine/threonine protein kinases PKN1 and PKN4 in CFBE41o- cells compared to HBE41o- cells and to elucidate that shorter proteoforms of full-length caspase-4 and ephrin B receptor are differentially expressed. Thus, PCQ extends current analyses in quantitative proteomics and facilitates finding differentially regulated proteins and proteoforms.

Structure-based analysis of Clot as a thioredoxin-related protein of 14 kDa in Drosophila via experimental and computational approaches.

The clot gene is required for the biosynthesis of drosopterins, the red components of Drosophila eye pigments. However, the enzymatic role of Clot in Drosophila eye pigment formation and the molecular mechanisms underlying Clot function are not fully elucidated. In this study, we cloned and characterized Clot derived from Drosophila cDNA, and results showed that Clot exhibited ∼30% sequence identity with mammalian TRP14. In addition, we reported the three-dimensional structure of Drosophila Clot based on homology modeling. Furthermore, we identified NFκB as a novel Clot substrate using the I-TASSER program. The NFκB fragment can bind near the active site of Clot. These findings predicted the novel regulatory mechanisms underlying Clot function in the pyrimidodiazepine synthesis pathway and increased the understanding of the molecular mechanisms and physiological function of Clot in Drosophila eye pigment formation.

Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability.

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical.

Highly Selective Sub-ppm Naked-Eye Detection of Hydrazine with Conjugated-1,3-Diketo Probes: Imaging Hydrazine in Drosophila Larvae.

A pair of pyrene- and anthracene-based turn-on fluorescent probes (1 and 2, respectively) reported here can be easily synthesized in a single-step process and also exhibit outstanding sensing behavior toward hydrazine over various competing nucleophilic species and environmentally relevant ions. The probes display dramatic enhancements in the emission intensity with as high as 83- and 173-fold increases in the presence of hydrazine. Nitrogenous bases, thiols, and lanthanides do not interfere in the fluorometric detection. These probes enable the detection of hydrazine with the naked eye well below sub-ppm concentrations (ca. 30 ppb) with analytical detection limits of 5.4 ppb for 1 and 7.7 ppb for 2, which are far exceeded by the accepted lower limit for hydrazine (10 ppb) set by the US EPA. Simple paper strips based on these probes could be used for the detection of hydrazine even in the gas phase. Both of the probes could selectively detect hydrazine even in pond water samples efficiently. The probes were successfully applied to visualize, for the first time, accumulation of hydrazine in live fruit-fly larvae using epifluorescence microscopy. The novel and interesting detection mechanism, proposed on the basis of spectroscopic evidence and single crystal XRD results, indicates that the detection pathway proceeds via the initial step of a five-membered ring formation upon attack of the hydrazine, followed by a dehydration step for gaining aromaticity.

Quantitative Proteomics Reveals Extensive Changes in the Ubiquitinome after Perturbation of the Proteasome by Targeted dsRNA-Mediated Subunit Knockdown in Drosophila.

The ubiquitin-proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. Here we investigate the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in Drosophila S2 cells. dsRNA-mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, with the overall impact on the ubiquitinome being the most dramatic. The abundances of ∼10% of all proteins are increased, while the abundances of the far majority of over 14 000 detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Because this is to our knowledge the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the Drosophila community.

Optimized FISH methods for visualizing RNA localization properties in Drosophila and human tissues and cultured cells.

Eukaryotic gene expression is orchestrated by a large number of regulatory steps to modulate the synthesis, maturation and fate of various families of protein-coding and non-coding RNA molecules. Defining the subcellular localization properties of an RNA molecule is thus of considerable importance for gleaning its function(s) and for elucidating post-transcriptional gene regulation pathways. For decades, fluorescent In Situ hybridization (FISH) has constituted the gold-standard technique for assessing RNA expression and distribution properties in cultured cells, tissue specimens, and whole mount organisms. Recently, several attempts aimed at advancing multiplex RNA-FISH experiments have been published. However, these procedures are both financially demanding and technically challenging, while their full potential remains unexploited. Here we describe an optimized RNA-FISH method employing the Tyramide Signal Amplification system that robustly enhances resolution and sensitivity needed for exploring RNA localization in Drosophila embryos, tissues and commonly cultured human and insect cell lines. Methodological details and key parameters are outlined for high-throughput analyses conducted in 96-well plate format.

Axin cancer mutants form nanoaggregates to rewire the Wnt signaling network.

Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode of action are poorly understood. Here we show that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. Our findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment.

Computational study of remodeling in a nucleosomal array.

Chromatin remodeling complexes utilize the energy of ATP hydrolysis to change the packing state of chromatin, e.g. by catalysing the sliding of nucleosomes along DNA. Here we present simple models to describe experimental data of changes in DNA accessibility along a synthetic, repetitive array of nucleosomes during remodeling by the ACF enzyme or its isolated ATPase subunit, ISWI. We find substantial qualitative differences between the remodeling activities of ISWI and ACF. To understand better the observed behavior for the ACF remodeler, we study more microscopic models of nucleosomal arrays.

An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors.

Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. TWIST is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of TWIST occupancy in human and Drosophila genomes reveals that TWIST, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, we found that the strict spatial configuration in the double E-box motif aligns two TWIST-E47 dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of TWIST dimerizes to mediate tetramer formation, which is functionally required for TWIST-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region.

Evolutionary divergence of Threonine Aspartase1 leads to species-specific substrate recognition.

Proteases are key regulators of life. Human Threonine Aspartase1 processes substrates, such as the mixed-lineage leukemia (MLL) protein, containing two cleavage sites, CS1 and CS2. Likewise, MLL's Drosophila ortholog trithorax is cleaved by Drosophila Threonine Aspartase1 (dTasp), suggesting a mechanistic coevolution. However, a detailed analysis of dTasp's function was missing so far. Here, active and inactive dTasp mutants allowed to compare substrate recognition and cleavage site selectivity of human and Drosophila enzymes. In contrast to the human protease, our cell-based assay revealed a preferential processing of CS2-like (QLD↓Gx[xD/Dx]) targets for dTasp, whereas cleavage of CS1-like targets (QVD↓Gx[xD/Dx]) was significantly impaired. Systematic mutagenesis of the CS2 sequence defined the motif x[FILMW]D↓Gx[xD/Dx] as the consensus cleavage sequence for dTasp. Substrate species selectivity of the enzymes was uncovered by demonstrating that dTasp cleaves Drosophila TFIIA, but not the human ortholog, suggesting evolutionary divergence of TFIIA downstream networks. Also, Drosophila USF2 was neither predicted nor cleaved by dTasp. Moreover, we found that dTasp cleavage site selectivity is independent of heterocomplex formation, as dTasp exists predominantly as an αß-monomer. Collectively, we provide novel insights into evolutionary similarities and divergence concerning Threonine Aspartase1 function in different species, which may aid to dissect and better target human Threonine Aspartase1 in malignancies.

Genomic and peptidomic analyses of the neuropeptides from the emerging pest, Drosophila suzukii.

Drosophila suzukii is a highly polyphagous invasive pest which has been recently introduced into Europe and North America, where it is causing severe economic losses through larval infestations of stone and berry fruits. The peptidome of the selected nervous tissues of adult D. suzukii was investigated as a first step in identifying potential targets for the development of novel insecticides. Through in silico analyses of the D. suzukii genome databases 28 neuropeptide families, comprising more than 70 predicted peptides were identified. Using a combination of liquid chromatography and mass spectrometry of tissue extracts, 33 predicted peptides, representing 15 different peptide families were identified by their molecular masses and a total of 17 peptide sequences were confirmed by ion fragmentation. A comparison between the peptides and precursors of D. suzukii and D. melanogaster shows they are highly conserved, with differences only identified in the amino acid sequences of the peptides encoded in the FMRFamide, hugin and ecydysis triggering hormone precursors. All other peptides predicted and identified from D. suzukii appear to be identical to those previously characterized from D. melanogaster. Adipokinetic hormone was only identified in the corpus cardiacum, other peptides present included short neuropeptide F, a pyrokinin and myosuppressin, the latter of which was the only peptide identified from the crop nerve bundle. Peptides present in extracts of the brain and/or thoracico-abdominal ganglion included allatostatins, cardioacceleratory peptide 2b, corazonin, extended FMRFamides, pyrokinins, myoinihibitory peptides, neuropeptide-like precursor 1, SIFamide, short neuropeptide F, kinin, sulfakinins and tachykinin related peptides.

Conserved metallomics in two insect families evolving separately for a hundred million years.

Μetal cofactors are required for enzymatic catalysis and structural stability of many proteins. Physiological metal requirements underpin the evolution of cellular and systemic regulatory mechanisms for metal uptake, storage and excretion. Considering the role of metal biology in animal evolution, this paper asks whether metal content is conserved between different fruit flies. A similar metal homeostasis was previously observed in Drosophilidae flies cultivated on the same larval medium. Each species accumulated in the order of 200 µg iron and zinc and approximately ten-fold less manganese and copper per gram dry weight of the adult insect. In this paper, data on the metal content in fourteen species of Tephritidae, which are major agricultural pests worldwide, are presented. These fruit flies can be polyphagous (e.g., Ceratitis capitata) or strictly monophagous (e.g., Bactrocera oleae) or oligophagous (e.g., Anastrepha grandis) and were maintained in the laboratory on five distinct diets based on olive oil, carrot, wheat bran, zucchini and molasses, respectively. The data indicate that overall metal content and distribution between the Tephritidae and Drosophilidae species was similar. Reduced metal concentration was observed in B. oleae. Feeding the polyphagous C. capitata with the diet of B. oleae resulted in a significant quantitative reduction of all metals. Thus, dietary components affect metal content in some Tephritidae. Nevertheless, although the evidence suggests some fruit fly species evolved preferences in the use or storage of particular metals, no metal concentration varied in order of magnitude between these two families of Diptera that evolved independently for over 100 million years.

Remote control of myosin and kinesin motors using light-activated gearshifting.

Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.

Mathematical modeling of sub-cellular asymmetry of fat-dachsous heterodimer for generation of planar cell polarity.

Planar Cell Polarity (PCP) is an evolutionarily conserved characteristic of animal tissues marked by coordinated polarization of cells or structures in the plane of a tissue. In insect wing epithelium, for instance, PCP is characterized by en masse orientation of hairs orthogonal to its apical-basal axis and pointing along the proximal-distal axis of the organ. Directional cue for PCP has been proposed to be generated by complex sets of interactions amongst three proteins - Fat (Ft), Dachsous (Ds) and Four-jointed (Fj). Ft and Ds are two atypical cadherins, which are phosphorylated by Fj, a Golgi kinase. Ft and Ds from adjacent cells bind heterophilically via their tandem cadherin repeats, and their binding affinities are regulated by Fj. Further, in the wing epithelium, sub-cellular levels of Ft-Ds heterodimers are seen to be elevated at the distal edges of individual cells, prefiguring their PCP. Mechanisms generating this sub-cellular asymmetry of Ft-Ds heterodimer in proximal and distal edges of cells, however, have not been resolved yet. Using a mathematical modeling approach, here we provide a framework for generation of this sub-cellular asymmetry of Ft-Ds heterodimer. First, we explain how the known interactions within Ft-Ds-Fj system translate into sub-cellular asymmetry of Ft-Ds heterodimer. Second, we show that this asymmetric localization of Ft-Ds heterodimer is lost when tissue-level gradient of Fj is flattened, or when phosphorylation of Ft by Fj is abolished, but not when tissue-level gradient of Ds is flattened or when phosphorylation of Ds is abrogated. Finally, we show that distal enrichment of Ds also amplifies Ft-Ds asymmetry. These observations reveal that gradient of Fj expression, phosphorylation of Ft by Fj and sub-cellular distal accumulation of Ds are three critical elements required for generating sub-cellular asymmetry of Ft-Ds heterodimer. Our model integrates the known experimental data and presents testable predictions for future studies.

Structure-function analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin signaling.

The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila.