Activity profiling of DUBs in <i>Salmonella</i>-infected cells by using ubiquitin-specific active-site probe.

<p><b>(A). DUB probe anatomy.</b> Ubiquitin-specific active-site probes consist of a retrieval element (tag), such as HA, which enables visualization and purification of a probe-bound DUB, as well as a ubiquitin molecule used for site recognition, and warhead (such as hereby used vinyl sulfone), which is a reactive group that interacts with DUB’s cysteine by formation of a thioester. <b>(B). Activity profiling by ubiquitin-specific active-site probe in <i>Salmonella</i>-infected macrophages.</b> HD11 macrophages were infected in duplicate for 0, 1, 2 and 18 hours with <i>Salmonella</i> Typhimurium at multiplicity of infection (MOI) of 50:1. After infection, the protein content was obtained and subjected to the enzymatic reaction with Ub-VS-HA probe. The proteins were separated by SDS-PAGE and subjected to anti-HA western blotting to visualize the active DUBs. One representative blot is shown. <b>(C, D)</b>. <b>Identification of DUBs regulated in HD11 macrophages during <i>Salmonella</i> infection by chemical proteomics.</b> HD11 macrophages were infected for 0 and 18 hours with <i>Salmonella</i> Typhimurium at MOI of 50:1. After infection, the protein content was obtained and subjected to the enzymatic reaction with the Ub-VS-HA probe. Probe-bound DUBs were immunoprecipitated by using anti-HA agarose and subjected to tryptic digestion. The peptide mixtures were then analyzed by quantitative proteomics (HPLC-MS/MS). The table shows names of the identified proteins, their accession numbers (NCBI), molecular weight, Fisher’s exact test p-values, fold change (calculated from the weighted spectral count in infected versus uninfected samples), protein identification probabilities as well as percent of protein sequence coverage [%] in individual replicas. Only the DUBs identified with high confidence are shown (C). A graph displays fold change (calculated using weighted spectral count) of DUBs and other relevant ubiquitin-binding proteins in infected versus uninfected samples (D). The abbreviations refer to the names of proteins from the table (C). <b>(E-F). Identification of UCH-L5 upregulated in infected HD11 macrophages upon infection.</b> Immunoprecipitated DUBs obtained from uninfected and infected HD11 macrophages as described in (C) were resolved on SDS-PAGE and subjected to silver staining prior to band excision and tryptic digestion of the indicated band. The identified DUB corresponded to chicken UCH-L5 (F). Accession, protein name, protein score, protein sequence coverage [%], number of identified peptides and Peptide spectrum matches (PSMs) as well as expected molecular weight are shown for the identified protein. For each one of three UCH-L5 peptides, sequence, modification, XCorr value, number of missed cleavages, delta Cn value, peptide rank, search engine rank, peptide charge, molecular weight of a precursor ion and molecular weight of the calculated singly charged peptide are shown, as well as delta mass [ppm], retention time [minute] and number of ions matched.</p

UCH-L5 regulates inflammasome activity in chicken macrophages.

<p><b>(A-B). UCH-L5 overexpression leads to reduced cell viability in infected as well as uninfected HD11 macrophages, which depends on its catalytic activity.</b> UCH-L5 was overexpressed in HD11 macrophages and empty vector was used as a control (Ctrl). 24-hours past overexpression, the cells were treated with b-AP15 (UCH-L5 inhibitor) used at indicated concentrations for 18 hours. The cell viability was measured by Presto Blue assay (A). Alternatively, UCH-L5 was overexpressed in HD11 macrophages for 2 days prior to infection with <i>Salmonella</i> Typhimurium for 1 hour. The cell viability was measured by Presto Blue assay. The p-values were calculated by using Student T test. <b>(C) UCH-L5 overexpression leads to an increase in caspase-1 activity in HD11 macrophages.</b> UCH-L5 was overexpressed in HD11 macrophages (UCH-L5) and empty vector was used as a control (ctrl). 24-hours past overexpression, the cells were subjected to <i>Salmonella</i> infection at MOI of 50:1 for 18-hours (inf) or left uninfected. The caspase-1 activity was measured by using specific inhibitor. The p-values were calculated by using Student T test. <b>(D). Overexpressed UCH-L5 is increased in infected cells.</b> The protein cell lysates from (C) were analyzed by SDS-PAGE, followed by anti-FLAG western blotting to demonstrate UCH-L5 expression; anti-GAPDH western blotting was used as a loading control. <b>(E). Caspase-1 activity in HD11 macrophages is dampened upon b-AP15 inhibitor treatment.</b> UCH-L5 was overexpressed in HD11 macrophages (UCH-L5-FLAG) and empty vector was used as a control. 24-hours past overexpression, cells were primed with 1μg/ml LPS for 4 hrs followed by treatment with 1uM b-AP15 or vehicle control (DMSO) for 15 minutes to inhibit the activity of UCH-L5. Cells were then treated with 10μM nigericin for 1 hour to induce inflammasome. The cell pellets were collected and caspase-1 activity was measured by using Z-YVAD-AFC substrate. The p-values were calculated by using Student T test. <b>(F). Exposure of cells to b-AP15 inhibitor leads to decrease in IL-1β secretion in HD11 macrophages upon inflammasome activation.</b> The HD11 cells were seeded on 6-well plates (1x10⁶ cells, 3 replicates each) and primed with LPS (1μg/ml) for 4 hours followed by treatment with 1uM b-AP15 (or vehicle control, DMSO) for 15 minutes. Cells were then treated with 10uM nigericin (or not) for 1 hour to induce inflammasome. Media were collected and used for detection of chicken IL-1β by western blot. <b>(G). Exposure of chicken HD11 macrophages to b-AP15 inhibitor leads to decrease in IL-1β secretion during <i>Salmonella</i> infection.</b> HD11 cells were treated with 1μM b-AP15 or DMSO (vehicle control) for 60 min. They were then infected (or not) with <i>Salmonella</i> Typhimurium wild-type at MOI 50:1 for 60 minutes. Media were collected for ELISA-based quantitation of chicken IL-1β (MyBioSource, Inc., USA).</p

UCH-L5 regulates inflammasome activity in human macrophages.

<p><b>(A). Exposure of THP-1 macrophages to b-AP15 inhibitor leads to decrease in IL-1β upon inflammasome activation.</b> The THP-1 cells were seeded on 6-well plates, treated with 100nM PMA for 24 hours and exposed to LPS treatment for hours, followed by treatment with 1μM b-AP15 or DMSO (vehicle control) for 60 min and treatment with nigericin for 60 minutes. Media were collected for western blotting of human IL-1β. <b>(B). Exposure of THP-1 macrophages to b-AP15 leads to decrease in IL-1β secretion upon <i>Salmonella</i> infection at different time points of infection.</b> The THP-1 cells were seeded on 24-well plates, treated with 100nM PMA for 24 hours and exposed to 1μM b-AP15 or DMSO (vehicle control) for 60 min. They were then infected with <i>Salmonella</i> Typhimurium wild-type at MOI 50:1 for indicated time points. Human IL-1β ELISA was used to quantify the amount of IL-1β I into the cell culture medium, which is shown in pg/ml. <b>(C). IL-1β secretion from <i>Salmonella</i>-infected control and NRLP3-deficient cells exposed and not exposed to b-AP15 inhibitor.</b> The THP-1 cells control cells as well as NLRP3-deficient cells (Invivogen Inc, USA) were seeded on 24-well plates, treated with 100nM PMA for 24 hours and exposed to b-AP15 or DMSO (vehicle control) for 60 min. They were then infected with <i>Salmonella</i> Typhimurium wild-type at MOI 50:1 for 1 hour. Human IL-1β ELISA was used to quantify the amount of IL-1β I into the cell culture medium, which is shown in pg/ml. <b>(D). The b-AP15 inhibitor does not affect IL-1β secretion from uninfected cells.</b> The THP-1 were treated with 100nM PMA for 24 hours and exposed to b-AP15 or DMSO (vehicle control) for 60 min. They were then infected or not with <i>Salmonella</i> Typhimurium wild-type for 30 minutes. Human IL-1β ELISA was used to quantify the amount of IL-1β I into the cell culture medium, which is shown in pg/ml. <b>(E-F). Partial knock-down of UCH-L5 in THP-1 macrophages leads to attenuation in IL-1β secretion in <i>Salmonella</i>-infected cells.</b> UCH-L5 was knocked-down in THP-1 macrophages by UCH-L5 siRNA (negative control siRNA was used as a control). After nucleofection was complete, new medium was added onto cells and cells were incubated for 24 hours prior to infection with <i>Salmonella</i> Typhimurium, MOI 50:1 for 1 hour. The cells were lysed and obtained proteins were resolved on SDS-PAGE and subjected to western blotting (Fig 4E; anti-UCH-L5, anti-GAPDH for loading control). IL-1β secretion to medium was quantified by ELISA (F). <b>(E). Model of UCH-L5’s effect on inflammasome activation in macrophages infected with <i>Salmonella</i> Typhimurium.</b></p

Classification of <i>L</i>. <i>lineolaris</i> salivary gland transcriptome based on predicted Gene ontology (GO) terms.

<p>(<b>A</b>) Biological Processes (<b>B</b>) Molecular Function (<b>C</b>) Cellular Component.</p

Phylogenetic relationships of polygalacturonase transcripts of <i>L</i>. <i>lineolaris</i> with polygalacturonases from other mirid bugs.

<p>Phylogenetic relationships of polygalacturonase transcripts of <i>L</i>. <i>lineolaris</i> with polygalacturonases from other mirid bugs.</p

Scanning electron micrograph of excised salivary glands of the tarnished plant bug <i>Lygus lineolaris</i>.

<p>Scanning electron micrograph of excised salivary glands of the tarnished plant bug <i>Lygus lineolaris</i>.</p

Venn diagram showing distribution of 15,980 <i>L</i>. <i>lineolaris</i> salivary gland proteins with BLASTP alignments to <i>Acyrthosiphon pisum</i>, <i>Drosophila melanogaster</i>, <i>Tribolium castaneum</i>, and <i>Lygus hesperus</i>.

<p>Venn diagram showing distribution of 15,980 <i>L</i>. <i>lineolaris</i> salivary gland proteins with BLASTP alignments to <i>Acyrthosiphon pisum</i>, <i>Drosophila melanogaster</i>, <i>Tribolium castaneum</i>, and <i>Lygus hesperus</i>.</p

Top 20 species exhibiting alignments (E-value ≤1.00E-05) to the <i>L</i>. <i>lineolaris</i> sialotranscriptome.

<p>Top 20 species exhibiting alignments (E-value ≤1.00E-05) to the <i>L</i>. <i>lineolaris</i> sialotranscriptome.</p

Insight into the Salivary Gland Transcriptome of <i>Lygus lineolaris</i> (Palisot de Beauvois)

<div><p>The tarnished plant bug (TPB), <i>Lygus lineolaris</i> (Palisot de Beauvois) is a polyphagous, phytophagous insect that has emerged as a major pest of cotton, alfalfa, fruits, and vegetable crops in the eastern United States and Canada. Using its piercing-sucking mouthparts, TPB employs a “lacerate and flush” feeding strategy in which saliva injected into plant tissue degrades cell wall components and lyses cells whose contents are subsequently imbibed by the TPB. It is known that a major component of TPB saliva is the polygalacturonase enzymes that degrade the pectin in the cell walls. However, not much is known about the other components of the saliva of this important pest. In this study, we explored the salivary gland transcriptome of TPB using Illumina sequencing. After <i>in silico</i> conversion of RNA sequences into corresponding polypeptides, 25,767 putative proteins were discovered. Of these, 19,540 (78.83%) showed significant similarity to known proteins in the either the NCBI nr or Uniprot databases. Gene ontology (GO) terms were assigned to 7,512 proteins, and 791 proteins in the sialotranscriptome of TPB were found to collectively map to 107 Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathways. A total of 3,653 Pfam domains were identified in 10,421 sialotranscriptome predicted proteins resulting in 12,814 Pfam annotations; some proteins had more than one Pfam domain. Functional annotation revealed a number of salivary gland proteins that potentially facilitate degradation of host plant tissues and mitigation of the host plant defense response. These transcripts/proteins and their potential roles in TPB establishment are described.</p></div

Genome-wide identification and characterization of microRNAs differentially expressed in fibers in a cotton phytochrome A1 RNAi line

<div><p>Cotton fiber is an important commodity throughout the world. Fiber property determines fiber quality and commercial values. Previous studies showed that silencing phytochrome A1 gene (<i>PHYA1</i>) by RNA interference in Upland cotton (<i>Gossypium hirsutum</i> L. cv. Coker 312) had generated <i>PHYA1</i> RNAi lines with simultaneous improvements in fiber quality (longer, stronger and finer fiber) and other key agronomic traits. Characterization of the altered molecular processes in these RNAi genotypes and its wild-type controls is a great interest to better understand the <i>PHYA1</i> RNAi phenotypes. In this study, a total of 77 conserved miRNAs belonging to 61 families were examined in a <i>PHYA1</i> RNAi line and its parental Coker 312 genotype by using multiplex sequencing. Of these miRNAs, seven (miR7503, miR7514, miR399c, miR399d, miR160, miR169b, and miR2950) were found to be differentially expressed in <i>PHYA1</i> RNAi cotton. The target genes of these differentially expressed miRNAs were involved in the metabolism and signaling pathways of phytohormones, which included Gibberellin, Auxin and Abscisic Acid. The expression of several MYB transcription factors was also affected by miRNAs in RNAi cotton. In addition, 35 novel miRNAs (novel miR1-novel miR35) were identified in fibers for the first time in this study. Target genes of vast majority of these novel miRNAs were also predicted. Of these, nine novel miRNAs (novel-miR1, 2, 16, 19, 26, 27, 28, 31 and 32) were targeted to cytochrome P450-like TATA box binding protein (TBP). The qRT-PCR confirmed expression levels of several differentially regulated miRNAs. Expression patterns of four miRNAs-targets pairs were also examined via RNA deep sequencing. Together, the results imply that the regulation of miRNA expression might confer to the phenotype of the <i>PHYA1</i> RNAi line(s) with improved fiber quality.</p></div