Wolbachia Endosymbionts Modify Drosophila Ovary Protein Levels in a Context-Dependent Manner

ABSTRACT Endosymbiosis is a unique form of interaction between organisms, with one organism dwelling inside the other. One of the most widespread endosymbionts is Wolbachia pipientis, a maternally transmitted bacterium carried by insects, crustaceans, mites, and filarial nematodes. Although candidate proteins that contribute to maternal transmission have been identified, the molecular basis for maternal Wolbachia transmission remains largely unknown. To investigate transmission-related processes in response to Wolbachia infection, ovarian proteomes were analyzed from Wolbachia-infected Drosophila melanogaster and D. simulans. Endogenous and variant host-strain combinations were investigated. Significant and differentially abundant ovarian proteins were detected, indicating substantial regulatory changes in response to Wolbachia. Variant Wolbachia strains were associated with a broader impact on the ovary proteome than endogenous Wolbachia strains. The D. melanogaster ovarian environment also exhibited a higher level of diversity of proteomic responses to Wolbachia than D. simulans. Overall, many Wolbachia-responsive ovarian proteins detected in this study were consistent with expectations from the experimental literature. This suggests that context-specific changes in protein abundance contribute to Wolbachia manipulation of transmission-related mechanisms in oogenesis. IMPORTANCE Millions of insect species naturally carry bacterial endosymbionts called Wolbachia. Wolbachia bacteria are transmitted by females to their offspring through a robust egg-loading mechanism. The molecular basis for Wolbachia transmission remains poorly understood at this time, however. This proteomic study identified specific fruit fly ovarian proteins as being upregulated or downregulated in response to Wolbachia infection. The majority of these protein responses correlated specifically with the type of host and Wolbachia strain involved. This work corroborates previously identified factors and mechanisms while also framing the broader context of ovarian manipulation by Wolbachia

New Coordinative Compounds with 4-(4’-pyridyl)pyridinium Disubstituted Monoylides

The complexes with manganese(II), iron(II), cobalt(II), nickel(II) and copper(II) of 2-(4, 4’-bipyridin-1-ium-1-yl)-1-(4-bromophenylamino)-3-(4-methoxyphenyl)-3-oxo-1-thioxopropan-2-ide (ylide, Y) were synthesized and characterized. The obtained compounds with 1 : 2 metal/ligand ratios have been characterized by FTIR, UV Vis spectroscopy, ESI MS spectrometry, molecular conductance, magnetic measurements and thermal analysis. The ylide ligand forms chelates with metallic (II) ions through their amide nitrogen and oxygen atoms

AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

The Hedgehog (Hh) pathway regulates cell differen- tiation and proliferation during development by controlling the Gli transcription factors. Cell fate de- cisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP- activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthe- sis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhib- iting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcrip- tional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency

Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

<div><p>Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption.</p></div

ISE6 proteins associated with the TCA cycle and glutaminolysis.

<p>ISE6 glutaminolysis and mTOR signaling proteins altered with LGTV infection are shown. GDH denotes glutamate dehydrogenase enzymes and Glase denotes glutaminase enzymes. ISCWxxxxxx denotes corresponding VB accession ID for corresponding <i>I</i>. <i>scapularis</i> protein.</p

Citrate cycle showing ISE6 proteins that exhibited increased/decreased expression following treatment to LGTV and UV-LGTV.

<p>The enzymes are indicated with KEGG abbreviated nomenclature and the corresponding substrates are shown in circles. * denotes proteins identified in this study. Dotted lines denote indirect involvement with production. The increased expression of malate dehydrogenase (MDH2) is unique to LGTV-treated cells while increase in the expression of acetyl-CoA acetyltransferase 1 (ACAT1), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH4A1), glutamate dehydrogenase (GLUD1), and fumarylacetoacetase (FAH) is common to cell samples following LGTV infection and UV-LGTV treatment. Decreased expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was observed in LGTV-treated cells and decreased expression of fumarate hydratase (FH), aldolase A/B/C fructose-bisphosphate (ALDOA/B/C), and aldehyde dehydrogenase 2/1B1/3A2 family protein (ALDH2/1B1/3A2) was observed in both LGTV-infected and UV-LGTV—treated cells. ATP citrate lyase (ACLY), aconitase (ACO), isocitrate dehydrogenase 2/3a (IDH2/3a), oxoglutarate/alpha-ketoglutarate dehydrogenase complex (OGDH/DLST), succinyl-CoA synthetase alpha/beta subunit (LSC1/2), succinate dehydrogenase flavoprotein subunit (SDHA), pyruvate kinase (PK), enolase 1/2/3 (ENO1/2/3), and aldehyde dehydrogenase 7A1 family protein (ALDH7A1).</p

ISE6 proteins with increased expression associate with pathways for protein production, transport, assembly, and proteolysis.

<p>The ISCW accession numbers corresponding to proteins identified with increased expression in LGTV-infected and UV-LGTV-treated cells are shown (KEGG abbreviated nomenclature provided as well if available in parentheses). The schematic presents potential mechanisms for LGTV-induced perturbation and increase in cell protein expression. See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004180#pntd.0004180.s010" target="_blank">S5 Table</a> for information on proteins and KEGG protein classes. Underlined proteins denote ISE6 proteins exhibiting increased expression following flavivirus infection not identified before. “Fld, Srt, & Deg” is the folding, sorting, and degradation KEGG pathway. <b>**</b> denotes ortholog in human [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004180#pntd.0004180.ref019" target="_blank">19</a>]; + denotes human ortholog showing increased expression following HCV infection;—denotes human ortholog showing decreased expression following HCV infection. Gray boxes denote cellular protein functions. White boxes denote KEGG pathways.</p

<i>In vitro</i> assays impact of (A) Trichostatin A and (B) Oligomycin A on LGTV replication.

<p>Trichostatin (TSA) and Oligomycin A (OligoA) concentrations on the x-axis, percent cell viability on the right y-axis, and virus release in pfu/mL on the left y-axis. Compound assays out of Vero cells are shown in the left panels and out of ISE6 cells are shown in the right panels. Release of LGTV was assessed by viral titer (pfu/ml) using plaque assays in BHK15 cells. Cell viability was determined with alamarBlue reagent and fluorescent assay and percentage was normalized against solvent only control in both LGTV-infected and mock-treated ISE6/Vero cells. * denotes p<0.05 and *** denotes p<0.001. Standard error shown in error bars with five technical replicates. Two biological replicate experiments were completed.</p

ISE6 proteins identified in KEGG pathways with differential expression following LGTV, UV-LGTV and mock treatment.

<p>(A) Total number of ISE6 cell proteins categorized by treatment and change in expression (increase/decrease/no change). Total number of proteins showing (B) increased expression, (C) decreased expression, and (D) no change in expression following LGTV infection and UV-LGTV treatment as compared to mock-treated cells and in LGTV-infected cells as compared to UV-LGTV-treated cells. Proteins were categorized by the KEGG classes for cellular function: metabolism, genetic information processing (GIP), environmental information processing (EIP), cellular processes (CP), and organismal systems (OS).</p

Quantification of LGTV infection in <i>I</i>. <i>scapularis</i> ISE6 cells via immunofluorescence and plaque assay.

<p>(A) Immunofluorescent detection of virus in ISE6 cells at 3, 9, 24 and 48 hours post infection (hpi) with LGTV MOIs of 7, 13 and 26. LGTV NS3 nonstructural protein (green), DAPI-stained nuclei (blue). (B) Percentage of infected ISE6 cells at 3, 9, 24 and 48 hpi following treatment with LGTV at MOIs of 7, 13 and 26 as determined from quantifying ISE6 cells with immunofluorescent LGTV NS3 expression. (C) Timecourse experiment showing amount of infectious LGTV (Log pfu/mL) released from ISE6 cells initially infected with LGTV MOI = 10 (n = 3). Titration was performed using both plaque assays and immunofluorescent focus forming assays in BHK15 cells. The red box corresponds to the time of peak release of infectious virus. (D) Plaque assays in BHK15 cells showing the reduction in infectious viral titer (pfu/mL) following treatment with UV-irradiated LGTV viral stocks for up to 300 seconds (n = 2).</p