Mass Spectrometry-Based Analysis of Rat Liver and Hepatocellular Carcinoma Morris Hepatoma 7777 Plasma Membrane Proteome

The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide “tube gel” followed by in-gel digestion of “tube gel” pieces significantly improved detection by electrospray ionization–liquid chromatography–tandem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues

Transcriptome analysis of <i>Phytophthora litchii</i> reveals pathogenicity arsenals and confirms taxonomic status

<div><p>Litchi downy blight, caused by <i>Peronophythora litchii</i>, is one of the major diseases of litchi and has caused severe economic losses. <i>P</i>. <i>litchii</i> has the unique ability to produce downy mildew like sporangiophores under artificial culture. The pathogen had been placed in a new family Peronophytophthoraceae by some authors. In this study, the whole transcriptome of <i>P</i>. <i>litchii</i> from mycelia, sporangia, and zoospores was sequenced for the first time. A set of 23637 transcripts with an average length of 1284 bp was assembled. Using six open reading frame (ORF) predictors, 19267 representative ORFs were identified and were annotated by searching against several public databases. There were 4666 conserved gene families and various sets of lineage-specific genes among <i>P</i>. <i>litchii</i> and other four closely related oomycetes. In silico analyses revealed 490 pathogen-related proteins including 128 RXLR and 22 CRN effector candidates. Based on the phylogenetic analysis of 164 single copy orthologs from 22 species, it is validated that <i>P</i>. <i>litchii</i> is in the genus <i>Phytophthora</i>. Our work provides valuable data to elucidate the pathogenicity basis and ascertain the taxonomic status of <i>P</i>. <i>litchii</i>.</p></div

Gene families of <i>P</i>. <i>litchii</i> potentially implicated in plant pathogenesis.

<p>Gene families of <i>P</i>. <i>litchii</i> potentially implicated in plant pathogenesis.</p

Overview of functional annotation of the <i>P</i>. <i>litchii</i> transcriptome.

<p>Overview of functional annotation of the <i>P</i>. <i>litchii</i> transcriptome.</p

Phylogenetic tree of 22 oomycete species.

<p>Phylogenetic tree infered from the 164 single-copy proteins dataset by RAxML under the GAMMALGF model. ML bootstrap support (lower value) and genetic distance (upper value) were estimated under the uniform model.</p

Summary of transcriptome sequencing and assembly for <i>P</i>. <i>litchii</i>.

<p>Summary of transcriptome sequencing and assembly for <i>P</i>. <i>litchii</i>.</p

Five-way-Venn-diagram of unique and shared gene families among oomycete species.

<p>Homologous proteins in <i>P</i>. <i>litchii</i>, <i>H</i>. <i>arabidopsidis</i>, <i>P</i>. <i>halstedii</i>, <i>P</i>. <i>infestans</i>, and <i>P</i>. <i>sojae</i> were clustered into gene families using OrthoMCL. Numbers in each section refer to number of gene families (not genes). Overlapping regions denote groups with at least one protein of all species that are part of the intersection. The number under the organism name refer to the total number of clusters.</p

MXene Enhanced Photoactivity of Bi₂O₃/Bi₂S₃ Heterojunction with G‑wire Superstructure for Photoelectrochemical Detection of TET1 Protein

Ten-eleven translocation 1 (TET1) protein has the potential to accelerate the oxygenation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC); then the −CH2OH of 5hmC can further covalently react with −SH catalyzed by M.HhaI methyltransferase. A brand-new photoelectrochemical (PEC) detection technique for the TET1 protein was created in light of this. For this objective, the Bi2O3/Bi2S3 heterojunction was first prepared by a one-pot hydrothermal method and served for photosensitive materials. For further enhancing the photoactivity, Bi2O3/Bi2S3 was blended with MXene to form an energy band-matched structure, thus improving the migration kinetics of photogenerated carriers. For achieving a high sensitivity of detection, a DNA Walker incorporated with the nicking endonuclease (Nb.BbvCI enzyme)-assisted signal amplification strategy was presented to output exponential G-quadruplex fragments. Self-assembly of the free G-quadruplex sequence into a G-wire superstructure with the assistance of Mg2+ provided more loading sites for MB and amplified the PEC signal. The linear range of the biosensor was 0.1–10 μg/mL with a detection limit of 0.024 μg/mL (S/N = 3) for TET1 protein under optimal experimental conditions. The suitability of the proposed method was evaluated by inhibitor screening experiments and the influence of environmental degradation on the activity of TET1 protein

Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

<div><p>The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.</p> </div

Erk positive feedback on Lck, ZAP70, and CD3 ITAMs.

<p>Phosphorylation kinetics of Lck, ZAP70, and CD3 ITAMs from SLP-76 reconstituted (WT) and deficient (ΔSLP76) cells are presented for 8 timepoints. The differences of phosphorylations between WT and ΔSLP76 cells (ΔSLP76–WT) are also presented to show the trend of phosphorylation changes. Results represent the means of three replicate experiments (error bars indicate SD). “*” represents timepoints with significant changes (less than 2% false discovery rate) in phosphorylation abundance between WT and ΔSLP76 cells.</p