Genome-Wide Identification and Analysis of the Maize Serine Peptidase S8 Family Genes in Response to Drought at Seedling Stage.

Subtilisin-like proteases (subtilases) are found in almost all plant species and are involved in regulating various biotic and abiotic stresses. Although the literature on subtilases in different plant species is vast, the gene function of the serine peptidase S8 family and its maize subfamily is still unknown. Here, a bioinformatics analysis of this gene family was conducted by describing gene structure, conserved motifs, phylogenetic relationships, chromosomal distributions, gene duplications, and promoter cis-elements. In total, we identified 18 ZmSPS8 genes in maize, distributed on 7 chromosomes, and half of them were hydrophilic. Most of these proteins were located at the cell wall and had similar secondary and tertiary structures. Prediction of cis-regulatory elements in promoters illustrated that they were mainly associated with hormones and abiotic stress. Maize inbred lines B73, Zheng58, and Qi319 were used to analyze the spatial-temporal expression patterns of ZmSPS8 genes under drought treatment. Seedling drought results showed that Qi319 had the highest percent survival after 14 d of withholding irrigation, while B73 was the lowest. Leaf relative water content (LRWC) declined more rapidly in B73 and to lower values, and the nitrotetrazolium blue chloride (NBT) contents of leaves were higher in Qi319 than in the other inbreds. The qPCR results indicated that 6 serine peptidase S8 family genes were positively or negatively correlated with plant tolerance to drought stress. Our study provides a detailed analysis of the ZmSPS8s in the maize genome and finds a link between drought tolerance and the family gene expression, which was established by using different maize inbred lines.

Glycoprotein 96 in Peritoneal Dialysis Effluent-Derived Extracellular Vesicles: A Tool for Evaluating Peritoneal Transport Properties and Inflammatory Status.

Background: Extracellular vesicles (EVs) from peritoneal dialysis effluent (PDE), containing molecules such as proteins and microRNAs (miRNAs), may be potential biological markers to monitor peritoneal function or injury. Peritoneal inflammation is an important determinant of peritoneal solute transport rate (PSTR). Thus, the aim of this study is to determine whether the specific proteins capable of evaluating the PSTR could be found in PDE-EVs, and explore the underlying mechanism for the association between PSTR and peritoneal inflammation. Methods: Sixty patients undergoing peritoneal dialysis (PD) were divided into two groups: high/high average transport (H/A) group (PET >0.65) and low/low average transport (L/A) group (PET <0.65). EVs derived from PDE (PDE-EVs) were isolated by ultracentrifugation. Proteomic analysis was performed to explore the differentially expressed proteins and identify the potential biomarkers in PDE-EVs from the two groups, and we focused on glycoprotein 96 (GP96) as it could be involved in the inflammatory process. The expression of GP96 in PDE-EVs and inflammatory cytokines was quantified by real-time PCR and enzyme-linked immunosorbent assay. The infiltration of macrophages and neutrophils into the peritoneum was detected using immunohistochemistry in a PD rat model. Results: The expression of PDE-EVs-GP96 was significantly higher in the H/A group, and was positively correlated with the PSTR and the level of the inflammatory factor interleukin (IL)-6. GP96-enriched EVs enhanced the secretion of proinflammatory cytokines IL-1ß, IL-6, tumor necrosis factor (TNF)-α, and IL-8 in macrophages, which was reversed by a pharmacological GP96-specific inhibitor (PU-WS13). The GP96 inhibitor also reduced local peritoneal inflammation by decreasing the infiltration of inflammatory cells and levels of proinflammatory cytokines (IL-6 and TNF-α) and chemokines (CCL2, CXCL1, and CXCL2) in a PD rat model. Conclusions: PDE-EVs-GP96 is a new promising tool to evaluate the status of peritoneal inflammation and PSTR, and the mechanism may be related to affecting the inflammatory properties of macrophages.

BKCa channels regulate the immunomodulatory properties of WJ-MSCs by affecting the exosome protein profiles during the inflammatory response.

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from the human umbilical cord have been studied extensively due to their immunomodulatory functions. Large-conductance Ca2+-activated K+ (BKCa channels) channels are involved in many inflammatory responses, but their involvement in the anti-inflammatory activity of WJ-MSCs is unknown. The underlying molecular mechanism, through which BKCa channels mediate the immunomodulation of WJ-MSC, which may include changes in exosomes proteomics, has not yet been clarified. METHODS: Alizarin staining, Oil Red O staining, and flow cytometry were used to identify WJ-MSCs, which were isolated from human umbilical cord Wharton's jelly. BKCa channels were detected in WJ-MSCs using western blotting, real-time polymerase chain reaction (real-time PCR), and electrophysiology, and cytokine expression was examined using real-time PCR and enzyme-linked immunosorbent assays (ELISAs). Exosomes were characterized using transmission electron microscopy and nanoparticle tracking analysis. Proteomics analysis was performed to explore exosomal proteomic profiles. RESULTS: The cells derived from human umbilical cord Wharton's jelly were identified as MSCs. BKCa channels were detected in the isolated WJ-MSCs, and the expression of these channels increased after lipopolysaccharide (LPS) stimulation. BKCa channels blockade in LPS-treated WJ-MSCs induced apoptosis and decreased interleukin-6 (IL-6) expression. Furthermore, THP-1 cells (human monocytic cell line) stimulated with LPS/interferon gamma (IFN-γ) produced more anti-inflammatory cytokines after treatment with exosomes derived from BKCa channel-knockdown WJ-MSCs (si-exo). We also observed altered expression of mitochondrial ATP synthase alpha subunit (ATP5A1), filamin B, and other proteins in si-exo, which might increase the anti-inflammatory activity of macrophages. CONCLUSIONS: Our study described the functional expression of BKCa channels in WJ-MSCs, and BKCa channels regulated the immunomodulatory properties of WJ-MSCs by affecting the exosomal protein profiles during the inflammatory response.

Proteomic approach to reveal the regulatory function of aconitase AcnA in oxidative stress response in the antibiotic producer Streptomyces viridochromogenes Tü494.

The aconitase AcnA from the phosphinothricin tripeptide producing strain Streptomyces viridochromogenes Tü494 is a bifunctional protein: under iron-sufficiency conditions AcnA functions as an enzyme of the tricarboxylic acid cycle, whereas under iron depletion it is a regulator of iron metabolism and oxidative stress response. As a member of the family of iron regulatory proteins (IRP), AcnA binds to characteristic iron responsive element (IRE) binding motifs and post-transcriptionally controls the expression of respective target genes. A S. viridochromogenes aconitase mutant (MacnA) has previously been shown to be highly sensitive to oxidative stress. In the present paper, we performed a comparative proteomic approach with the S. viridochromogenes wild-type and the MacnA mutant strain under oxidative stress conditions to identify proteins that are under control of the AcnA-mediated regulation. We identified up to 90 differentially expressed proteins in both strains. In silico analysis of the corresponding gene sequences revealed the presence of IRE motifs on some of the respective target mRNAs. From this proteome study we have in vivo evidences for a direct AcnA-mediated regulation upon oxidative stress.

Quantitative proteomics reveal up-regulated protein expression of the SET complex associated with hepatocellular carcinoma.

We combined culture-derived isotope tags (CDITs) with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) to extensively survey abnormal protein expression associated with hepatocellular carcinoma (HCC) in clinical tissues. This approach yielded an in-depth quantitated proteome of 1360 proteins. Importantly, 267 proteins were significantly regulated with a fold-change of at least 1.5. The proteins up-regulated in HCC tissues are involved in regulatory processes, such as the granzyme A-mediated apoptosis pathway (The GzmA pathway). The SET complex, a central component in the GzmA pathway, was significantly up-regulated in HCC tissue. The elevated expressions of all of the SET complex components were validated by Western blotting. Among them, ANP32A and APEX1 were further investigated by immunohistochemistry staining using tissue microarrays (59 cases), confirming their overexpression in tumors. The up-regulation and nuclear accumulations of APEX1 was associated not only with HCC malignancy but also with HCC differentiation in 96 clinical HCC cases. Our work provided a systematic and quantitative analysis and demonstrated key changes in clinical HCC tissues. These proteomic signatures could help to unveil the underlying mechanisms of hepatocarcinogenesis and may be useful for the discovery of candidate biomarkers.

ESNOQ, proteomic quantification of endogenous S-nitrosation.

S-nitrosation is a post-translational protein modification and is one of the most important mechanisms of NO signaling. Endogenous S-nitrosothiol (SNO) quantification is a challenge for detailed functional studies. Here we developed an ESNOQ (Endogenous SNO Quantification) method which combines the stable isotope labeling by amino acids in cell culture (SILAC) technique with the detergent-free biotin-switch assay and LC-MS/MS. After confirming the accuracy of quantification in this method, we obtained an endogenous S-nitrosation proteome for LPS/IFN-gamma induced RAW264.7 cells. 27 S-nitrosated protein targets were confirmed and using our method we were able to obtain quantitative information on the level of S-nitrosation on each modified Cys. With this quantitative information, over 15 more S-nitrosated targets were identified than in previous studies. Based on the quantification results, we found that the S-nitrosation levels of different cysteines varied within one protein, providing direct evidence for differences in the sensitivity of cysteine residues to reactive nitrosative stress and that S-nitrosation is a site-specific modification. Gene ontology clustering shows that S-nitrosation targets in the LPS/IFN-gamma induced RAW264.7 cell model were functionally enriched in protein translation and glycolysis, suggesting that S-nitrosation may function by regulating multiple pathways. The ESNOQ method described here thus provides a solution for quantification of multiple endogenous S-nitrosation events, and makes it possible to elucidate the network of relationships between endogenous S-nitrosation targets involved in different cellular processes.

Shotgun proteomics analysis of hibernating arctic ground squirrels.

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.

Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients.

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.

Proteomic analysis of SARS associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectroemtric analysis.

The proteomes of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and its infected Vero E6 cells were detected in the present study. The cytosol and nucleus fractions of virus-infected cells as well as the crude virions were analyzed either by one-dimensional electrophoresis followed by ESI-MS/MS identification or by shotgun strategy with two-dimensional liquid chromatography-ESI-MS/MS. For the first time, all of the four predicted structural proteins of SARS-CoV were identified, including S (Spike), M (Membrane), N (Nucleocapsid), and E (Envolope) proteins. In addition, a novel phosphorylated site of M protein was observed. The combination of these gel-base and non-gel methods provides fast and complimentary approaches to SARS-CoV proteome and can be widely used in the analysis of other viruses.