iTRAQ-based Quantitative Proteomic Analysis Research Articles

NudCL2 is required for cytokinesis by stabilizing RCC2 with Hsp90 at the midbody.

Cytokinesis is required for faithful division of cytoplasmic components and duplicated nuclei into two daughter cells. Midbody, a protein-dense organelle that forms at the intercellular bridge, is indispensable for successful cytokinesis. However, the regulatory mechanism of cytokinesis at the midbody still remains elusive. Here, we unveil a critical role for NudC-like protein 2 (NudCL2), a co-chaperone of heat shock protein 90 (Hsp90), in cytokinesis regulation by stabilizing regulator of chromosome condensation 2 (RCC2) at the midbody in mammalian cells. NudCL2 localizes at the midbody, and its downregulation results in cytokinesis failure, multinucleation, and midbody disorganization. Using iTRAQ-based quantitative proteomic analysis, we find that RCC2 levels are decreased in NudCL2 knockout (KO) cells. Moreover, Hsp90 forms a complex with NudCL2 to stabilize RCC2, which is essential for cytokinesis. RCC2 depletion mirrors phenotypes observed in NudCL2-downregulated cells. Importantly, ectopic expression of RCC2 rescues the cytokinesis defects induced by NudCL2 deletion, but not vice versa. Together, our data reveal the significance of the NudCL2/Hsp90/RCC2 pathway in cytokinesis at the midbody.

Inhibitory activity and antioomycete mechanism of citral against Phytophthora capsici

The natural terpenoid citral has antifungal activity against multiple fungi, but its bioactivity against oomycetes is unclear. Therefore, this study investigated the antioomycete activity and mechanism of citral against Phytophthora capsici, a highly destructive invasive oomycete. Results showed that citral not only had a great inhibition on the mycelial growth of P. capsici (EC50 = 94.15 mg/L), but also had a significant inhibition on multiple spores, such as sporangia formation, zoospore discharge and zoospore germination. Citral at 4000 mg/L exhibited favorable protective (73.33%) and curative efficacy (55.11%) against pepper Phytophthora blight. Citral significantly damaged the hyphal morphology, disrupted the cell membrane integrity, increased the permeability of cell membrane, and increased the glycerol content in P. capsici. A total of 250 upregulated and 288 downregulated proteins were identified in iTRAQ-based quantitative proteomic analysis. Downregulated proteins were mostly enriched in pathways of ABC transporters, cyanoamino acid metabolism and starch and sucrose metabolism, suggesting an inhibition of citral on transmembrane transporter (e.g., ABC transporters) and pathogenicity (e.g., β-glucosidases) proteins. Upregulated proteins were enriched in biosynthesis of unsaturated fatty acids, pyruvate metabolism and glycolysis/gluconeogenesis, suggesting an activation of citral on energy generation proteins, including acyl-CoA oxidase, D-lactate dehydrogenase, pyruvate kinase, acetyl-CoA synthetase and phosphoenolpyruvate carboxykinase. Biochemical and iTRAQ analysis suggested that cell membrane may be the target of citral in P. capsici.

Comparative physiological and proteomic analysis reveals different responding mechanisms of phosphate deficiency between two clones of Pinus elliottii × P. caribaea

Enhancing efficiency of phosphorus uptake and utilization in forest trees through genetic improvement is crucial for ecological conservation and climate change mitigation. Pinus species exhibit significant genetic variation in phosphorus efficiency (PE). However, potential regulation mechanisms of hybrid pine (Pinus elliottii × P. caribaea) under phosphate deficiency are still unclear. Two distinct hybrid pine genotypes, clone 38 and clone 31, were screened out from 28 hybrid pine genotypes based on morphological and physiological changes. Clone 38 showed superior heights and diameters under –Pi condition than +Pi condition, while Clone 31 showed a slight advantage under +Pi condition than –Pi condition. Root-shoot fresh weight (FW) and dry weight (DW) ratios in clone 38 under Pi (inorganic phosphate) deficiency increased more than that in clone 31. Clone 38 and clone 31 could be classified as the genotypes with high and low Pi efficient, respectively. In order to explore the mechanisms that hybrid pine employed to response to phosphate starvation, iTRAQ-based quantitative proteomics analysis of shoots of two clones (clone 31 and clone 38) was conducted. There were 94 and 106 proteins showed differential accumulation patterns under Pi deficient condition in clone 38 and clone 31, respectively. Pi deficit in shoots of high Pi efficient clone 38 resulted in the induction of proteins associated with amylopectin, sulfolipid, and sucrose metabolism. Inversely, proteins associated with photosynthesis were decreased in shoots of low Pi efficient clone 31. The proteins that are accumulated differently amongst genotypes with varying Pi efficiency provide a potential and foundation for developing hybrid pine genotypes with higher phosphate efficiency.

MiR-29a efficiently suppresses the generation of reactive oxygen species and α-synuclein in a cellular model of Parkinson's disease by potentially targeting GSK-3β

MicroRNA-29a (miR-29a) has been suggested to serve a potential protective function against Parkinson's disease (PD); however, the exact molecular mechanisms remain elusive. This study explored the protective role of miR-29a in a cellular model of PD using SH-SY5Y cell lines through iTRAQ-based quantitative proteomic and biochemistry analysis. The findings showed that using a miR-29a mimic in SH-SY5Y cells treated with 1-methyl-4-phenylpyridinium (MPP+) significantly decreased cell death and increased mitochondrial membrane potential. It also reduced mitochondrial reactive oxygen species (ROS) and the production of α-synuclein. Subsequent heatmap analysis using iTRAQ-based quantitative proteomics revealed remarkably contrasting protein expression profiles for 882 genes when comparing the groups treated with miR-29a mimic plus MPP + against the control group treated solely with MPP+. The KEGG pathway analysis of these 882 genes indicated the substantial role of miR-29a in the PD pathway (P = 1.58x10−5) and highlighted its function in mitochondrial genes. Furthermore, treatment with a miR-29a mimic in SH-SY5Y cells reduced the levels of GSK-3β, phosphorylated GSK-3β, and cleaved caspase-7 following exposure to MPP+. The miR-29a mimic also upregulated the expressions of α-synuclein clearance proteins FYCO1 and Rab7 in this cellular PD model, thereby inhibiting the production of α-synuclein. Luciferase activity analysis confirmed the specific binding of miR-29a to the 3′ untranslated region (3′UTR) of GSK-3β, leading to its repression. Our findings demonstrated miR-29a′s neuroprotective role in mitochondrial function and highlighted its potential to inhibit ROS and α-synuclein production, offering possible therapeutic avenues for PD treatment.

ITRAQ-based quantitative proteomic analysis of defense responses of two tea cultivars to <i>Empoasca onukii</i> Matsuda feeding

iTRAQ-based quantitative proteomic analysis of defense responses of two tea cultivars to <i>Empoasca onukii</i> Matsuda feeding

ITRAQ-based quantitative proteomic analysis of the antibacterial mechanism of silver nanoparticles against multidrug-resistant Streptococcus suis.

The increase in antibiotic resistance of bacteria has become a major concern in clinical treatment. Silver nanoparticles (AgNPs) have significant antibacterial effects against Streptococcus suis. Therefore, this study aimed to investigate the antibacterial activity and mechanism of action of AgNPs against multidrug-resistant S. suis. The effect of AgNPs on the morphology of multidrug-resistant S. suis was observed using scanning electron microscopy (SEM). Differentially expressed proteins were analyzed by iTRAQ quantitative proteomics, and the production of reactive oxygen species (ROS) was assayed by H2DCF-DA staining. SEM showed that AgNPs disrupted the normal morphology of multidrug-resistant S. suis and the integrity of the biofilm structure. Quantitative proteomic analysis revealed that a large number of cell wall synthesis-related proteins, such as penicillin-binding protein and some cell cycle proteins, such as the cell division protein FtsZ and chromosomal replication initiator protein DnaA, were downregulated after treatment with 25 μg/mL AgNPs. Significant changes were also observed in the expression of the antioxidant enzymes glutathione reductase, alkyl hydroperoxides-like protein, α/β superfamily hydrolases/acyltransferases, and glutathione disulfide reductases. ROS production in S. suis positively correlated with AgNP concentration. The potential antibacterial mechanism of AgNPs may involve disrupting the normal morphology of bacteria by inhibiting the synthesis of cell wall peptidoglycans and inhibiting the growth of bacteria by inhibiting the cell division protein FtsZ and Chromosomal replication initiator protein DnaA. High oxidative stress may be a significant cause of bacterial death. The potential mechanism by which AgNPs inhibit S. suis biofilm formation may involve affecting bacterial adhesion and interfering with the quorum sensing system.

An iTRAQ-based quantitative proteomic analysis reveals the role of mitochondrial complex I subunits in S-type cytoplasmic male sterility of maize

Maize S-type cytoplasmic male sterility (CMS-S) arises from the activity of the mitochondrial gene orf355. This sterility can be counteracted by the nuclear fertility-restoring allele, Rf3. CMS-S occurs when bicellular pollen collapses following microspore mitosis. Despite this knowledge, the precise protein alterations in CMS-S mitochondria preceding pollen collapse are not well-understood. Our study compared the mitochondrial proteomic profiles between CMS-S and maintainer lines. We found that oxidative phosphorylation (OXPHOS) and glycolysis were significantly associated with CMS-S. A detailed analysis of the differentially abundant proteins (DAPs) associated with the OXPHOS pathway revealed that complex I (CI) subunits play a vital role in the degenerative process of maize CMS-S. Among these, the CI-B8 subunit exhibited abundant presence in CMS-S and displayed earlier transcriptional changes compared to other DAP genes. A yeast two-hybrid assay revealed a physical interaction between another DAP, the CI-13kDa subunit, and orf355 in yeast. These findings highlight the pivotal role played by complex I in the sterility mechanism of CMS-S in maize microspores.

Tissue proteome analysis for profiling proteins associated with lymph node metastasis in gallbladder cancer

Lymph node (LN) metastasis is the earliest sign of metastatic spread and an established predictor of poor outcome in gallbladder cancer (GBC). Patients with LN positive GBC have a significantly worse survival (median survival- 7 months) than patients with LN negative disease (median survival- ~ 23 months) in spite of standard treatment which includes extended surgery followed by chemotherapy, radiotherapy and targeted therapy. This study aims at understanding the underlying molecular processes associated with LN metastasis in GBC. Here, we used iTRAQ-based quantitative proteomic analysis using tissue cohort comprising of primary tumor of LN negative GBC (n = 3), LN positive GBC (n = 4) and non-tumor controls (Gallstone disease, n = 4), to identify proteins associated with LN metastasis. A total of 58 differentially expressed proteins (DEPs) were found to be specifically associated with LN positive GBC based on the criteria of p value ≤ 0.05, fold change ≥ 2 and unique peptides ≥ 2. These include the cytoskeleton and associated proteins such as keratin, type II cytoskeletal 7 (KRT7), keratin type I cytoskeletal 19 (KRT19), vimentin (VIM), sorcin (SRI) and nuclear proteins such as nucleophosmin Isoform 1 (NPM1), heterogeneous nuclear ribonucleoproteins A2/B1 isoform X1 (HNRNPA2B1). Some of them are reported to be involved in promoting cell invasion and metastasis. Bioinformatic analysis of the deregulated proteins in LN positive GBC using STRING database identified ‘neutrophil degranulation’ and ‘HIF1 activation’ to be among the top deregulated pathways. Western blot and IHC analysis showed a significant overexpression of KRT7 and SRI in LN positive GBC in comparison to LN negative GBC. KRT7, SRI and other proteins may be further explored for their diagnostics and therapeutic applications in LN positive GBC.

ITRAQ-Based Quantitative Proteomic Analysis of Arthrobacter simplex in Response to Cortisone Acetate and Its Mutants with Improved Δ1-Dehydrogenation Efficiency.

Arthrobacter simplex is extensively used for cortisone acetate (CA) biotransformation in industry, but the Δ1-dehydrogenation molecular fundamental remains unclear. Herein, the comparative proteome revealed several proteins with the potential role in this reaction, which were mainly involved in lipid or amino acid transport and metabolism, energy production and conversion, steroid degradation, and transporter. The influences of six proteins were further confirmed, where pps, MceGA, yrbE4AA, yrbE4BA, and hyp2 showed positive impacts, while hyp1 exhibited a negative effect. Additionally, KsdD5 behaved as the best catalytic enzyme. By the combined manipulation in multiple genes under the control of a stronger promoter, an optimal strain with better catalytic enzyme activity, substrate transportation, and cell stress tolerance was created. After biotechnology optimization, the production peak and productivity were, respectively, boosted by 4.1- and 4.0-fold relative to the initial level. Our work broadens the understanding of the Δ1-dehydrogenation mechanism, also providing effective strategies for excellent steroid-transforming strains.

Proteins in pregnant swine serum promote the African swine fever virus replication: an iTRAQ-based quantitative proteomic analysis

African swine fever (ASF) is a severe infectious disease caused by the African swine fever virus (ASFV), seriously endangering the global pig industry. ASFV possesses a large genome, strong mutation ability, and complex immune escape mechanisms. Since the first case of ASF was reported in China in August 2018, it has had a significant impact on social economy and food safety. In the present study, pregnant swine serum (PSS) was found to promote viral replication; differentially expressed proteins (DEPs) in PSS were screened and identified using the isobaric tags for relative and absolute quantitation technology and compared with those in non-pregnant swine serum (NPSS). The DEPs were analyzed using Gene Ontology functional annotation, Kyoto Protocol Encyclopedia of Genes and Genome pathway enrichment, and protein–protein interaction networks. In addition, the DEPs were validated via western blot and RT-qPCR experiments. And the 342 of DEPs were identified in bone marrow-derived macrophages cultured with PSS compared with the NPSS. The 256 were upregulated and 86 of DEPs were downregulated. The primary biological functions of these DEPs involved signaling pathways that regulate cellular immune responses, growth cycles, and metabolism-related pathways. An overexpression experiment showed that the PCNA could promote ASFV replication whereas MASP1 and BST2 could inhibit it. These results further indicated that some protein molecules in PSS were involved in the regulation of ASFV replication. In the present study, the role of PSS in ASFV replication was analyzed using proteomics, and the study will be provided a basis for future detailed research on the pathogenic mechanism and host interactions of ASFV as well as new insights for the development of small-molecule compounds to inhibit ASFV.

Quantitative tissue proteome profile reveals neutrophil degranulation and remodeling of extracellular matrix proteins in early stage gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy of the gastrointestinal tract with a poor prognosis. It is important to understand the molecular processes associated with the pathogenesis of early stage GBC and identify proteins useful for diagnostic and therapeutic strategies. Here, we have carried out an iTRAQ-based quantitative proteomic analysis of tumor tissues from early stage GBC cases (stage I, n=7 and stage II, n=5) and non-tumor controls (n=6) from gallstone disease (GSD). We identified 357 differentially expressed proteins (DEPs) based on ≥ 2 unique peptides and ≥ 2 fold change with p value < 0.05. Pathway analysis using the STRING database showed, 'neutrophil degranulation' to be the major upregulated pathway that includes proteins such as MPO, PRTN3, S100A8, MMP9, DEFA1, AZU, and 'ECM organization' to be the major downregulated pathway that includes proteins such as COL14A1, COL1A2, COL6A1, COL6A2, COL6A3, BGN, DCN. Western blot and/or IHC analysis confirmed the elevated expression of MPO, PRTN3 and S100A8 in early stage of the disease. Based on the above results, we hypothesize that there is an increased neutrophil infiltration in tumor tissue and neutrophil degranulation leading to degradation of extracellular matrix (ECM) proteins promoting cancer cell invasion in the early stage GBC. Some of the proteins (MPO, MMP9, DEFA1) associated with 'neutrophil degranulation' showed the presence of 'signal sequence' suggesting their potential as circulatory markers for early detection of GBC. Overall, the study presents a protein dataset associated with early stage GBC.

Extracellular Vesicle Membrane Protein Profiling and Targeted Mass Spectrometry Unveil CD59 and Tetraspanin 9 as Novel Plasma Biomarkers for Detection of Colorectal Cancer.

Extracellular vesicles (EVs) are valuable sources for the discovery of useful cancer biomarkers. This study explores the potential usefulness of tumor cell-derived EV membrane proteins as plasma biomarkers for early detection of colorectal cancer (CRC). EVs were isolated from the culture supernatants of four CRC cell lines by ultracentrifugation, and their protein profiles were analyzed by LC-MS/MS. Bioinformatics analysis of identified proteins revealed 518 EV membrane proteins in common among at least three CRC cell lines. We next used accurate inclusion mass screening (AIMS) in parallel with iTRAQ-based quantitative proteomic analysis to highlight candidate proteins and validated their presence in pooled plasma-generated EVs from 30 healthy controls and 30 CRC patients. From these, we chose 14 potential EV-derived targets for further quantification by targeted MS assay in a separate individual cohort comprising of 73 CRC and 80 healthy subjects. Quantitative analyses revealed significant increases in ADAM10, CD59 and TSPAN9 levels (2.19- to 5.26-fold, p < 0.0001) in plasma EVs from CRC patients, with AUC values of 0.83, 0.95 and 0.87, respectively. Higher EV CD59 levels were significantly correlated with distant metastasis (p = 0.0475), and higher EV TSPAN9 levels were significantly associated with lymph node metastasis (p = 0.0011), distant metastasis at diagnosis (p = 0.0104) and higher TNM stage (p = 0.0065). A two-marker panel consisting of CD59 and TSPAN9 outperformed the conventional marker CEA in discriminating CRC and stage I/II CRC patients from healthy controls, with AUC values of 0.98 and 0.99, respectively. Our results identify EV membrane proteins in common among CRC cell lines and altered plasma EV protein profiles in CRC patients and suggest plasma EV CD59 and TSPAN9 as a novel biomarker panel for detecting early-stage CRC.

ITRAQ-based quantitative proteome analysis in liver of large yellow croaker Larimichthys crocea under high temperature stress

In order to explore the changes in protein expression of large yellow croaker Larimichthys crocea under high temperature stress, isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to perform proteome analysis on the liver between thermal-tolerant and thermal-sensitive groups of large yellow croaker. A total of 23,269 peptides and 3,388 proteins were identified, and 442 differentially expressed proteins (DEPs) were screened, among which 171 proteins were up-regulated and 271 proteins were down-regulated with fold change ≥ 1.2 or < 0.83 and P < 0.05 as the standard. Twelve randomly selected proteins were verified by PRM and four proteins were verified by Western blot, and the results were consistent with the results of iTRAQ quantitative proteome analysis. Through bioinformatics analysis, DEPs mainly enriched in GO terms such as translation, electron transport chain, ribosome, NADH dehydrogenase activity, and KEGG pathways such as protein processing in endoplasmic reticulum, ribosomes, oxidative phosphorylation, steroid biosynthesis, and biosynthesis of unsaturated fatty acid. In summary, high temperature stress had significant effect on protein synthesis, energy metabolism and lipid metabolism in liver of large yellow croaker, the protein synthesis had decreased, energy metabolism changed from oxidative phosphorylation to glycolysis, and fatty acid biosynthesis increased and fatty acid metabolism decreased in thermal-tolerant group of large yellow croaker. The results will help to study molecular regulatory mechanisms of thermal adaptation in large yellow croaker.

ITRAQ-based quantitative proteomics analysis of forest musk deer with pneumonia.

Pneumonia can seriously threaten the life of forest musk deer (FMD, an endangered species). To gain a comprehensive understanding of pneumonia pathogenesis in FMD, iTRAQ-based proteomics analysis was performed in diseased (Pne group) lung tissues of FMD that died of pneumonia and normal lung tissues (Ctrl group) of FMD that died from fighting against each other. Results showed that 355 proteins were differentially expressed (fold change ≥ 1.2 and adjusted P-value < 0.05) in Pne vs. Ctrl. GO/KEGG annotation and enrichment analyses showed that dysregulated proteins might play vital roles in bacterial infection and immunity. Given the close association between bacterial infection and pneumonia, 32 dysregulated proteins related to Staphylococcus aureus infection, bacterial invasion of epithelial cells, and pathogenic Escherichia coli infection were screened out. Among these 32 proteins, 13 proteins were mapped to the bovine genome. Given the close phylogenetic relationships of FMD and bovine, the protein-protein interaction networks of the above-mentioned 13 proteins were constructed by the String database. Based on the node degree analysis, 5 potential key proteins related to pneumonia-related bacterial infection in FMD were filtered out. Moreover, 85 dysregulated proteins related to the immune system process were identified given the tight connection between immune dysregulation and pneumonia pathogenesis. Additionally, 12 proteins that might function as crucial players in pneumonia-related immune response in FMD were screened out using the same experimental strategies described above. In conclusion, some vital proteins, biological processes, and pathways in pneumonia development were identified in FMD.

MbtD and celA1 association with ethambutol resistance in Mycobacterium tuberculosis: A multiomics analysis

Ethambutol (EMB) is a first-line antituberculosis drug currently being used clinically to treat tuberculosis. Mutations in the embCAB operon are responsible for EMB resistance. However, the discrepancies between genotypic and phenotypic EMB resistance have attracted much attention. We induced EMB resistance in Mycobacterium tuberculosis in vitro and used an integrated genome–methylome–transcriptome–proteome approach to study the microevolutionary mechanism of EMB resistance. We identified 509 aberrantly methylated genes (313 hypermethylated genes and 196 hypomethylated genes). Moreover, some hypermethylated and hypomethylated genes were identified using RNA-seq profiling. Correlation analysis revealed that the differential methylation of genes was negatively correlated with transcription levels in EMB-resistant strains. Additionally, two hypermethylated candidate genes (mbtD and celA1) were screened by iTRAQ-based quantitative proteomics analysis, verified by qPCR, and corresponded with DNA methylation differences. This is the first report that identifies EMB resistance-related genes in laboratory-induced mono-EMB-resistant M. tuberculosis using multi-omics profiling. Understanding the epigenetic features associated with EMB resistance may provide new insights into the underlying molecular mechanisms.

ITRAQ-based quantitative proteomic analysis of the liver regeneration termination phase after partial hepatectomy in mice

Liver regeneration (LR) is an important biological process after liver injury. As the “brake” in the process of LR, the termination phase of LR not only suppresses the continuous increase in liver volume but also effectively promotes the recovery of liver function. However, the mechanisms underlying the termination phase of LR are still not clear. In our study, we used isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis to determine the protein expression profiles of livers in the termination phase of mouse LR after partial hepatectomy (PH). We found that the expression of 197 proteins increased gradually during LR; in addition, 187 proteins were upregulated and 264 proteins were downregulated specifically in the termination phase of LR. The GO analysis of the proteins revealed the upregulation of “cell–cell adhesion” and “translation” and the downregulation of the “oxidation–reduction process”. The KEGG pathway analysis showed that “biosynthesis of antibiotics” and “ribosomes” were significantly upregulated, while “metabolic pathways” were significantly downregulated. These analyses indicated that the termination phase of LR mainly focuses on restoring cellular structure and function. Differentially expressed proteins such as SNX5 were also screened out from biological processes. SignificanceThe key regulatory factors in the termination phase of LR were studied by iTRAQ-based proteomics to lay a foundation for further study of the molecular mechanism and biomarkers of the termination phase of LR. This study will guide the clinical perioperative management of patients after hepatectomy.

Comparative iTRAQ-based quantitative proteomic analysis of spotted seal (Phoca largha) pups inhabiting different environments

Captive breeding is among the most effective approaches for ensuring the conservation of numerous endangered species. However, only a few studies have examined the effects of captive maintenance of animals at the molecular level. In this study, we adopted the isobaric tags for relative and absolute quantification (iTRAQ) approach to compare protein expression in the blood of endangered spotted seal pups (Phoca largha) inhabiting different environments. We identified 519 proteins from 2,628 peptides, the expression of 158 of which differed significantly between short-term captive and wild seals. In addition, 140 proteins were identified as being differentially expressed between short-term and long-term captive seals, whereas 235 proteins were differentially expressed between the wild and long-term captive groups (p < 0.05). In wild seal pups, proteins associated with the maintenance of cardiomyocyte cell stability and integrity, protection of the heart, and prevention of anemia were upregulated, reflecting the high stress and energetic costs associated with movement and foraging. In addition, significant differences were detected among the three groups with respect to immune-related proteins. The results of this study advance our current understanding of protein expression in spotted seals and provide data that will contribute to the future conservation of this species.

ITRAQ-based quantitative proteomic analysis reveals that VPS35 promotes the expression of MCM2-7 genes in HeLa cells

Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that regulates endosomal trafficking in eukaryotic cells. Recent studies have shown that VPS35 promotes tumor cell proliferation and affects the nuclear accumulation of its interacting partner. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry were used to measure the changes in nuclear protein abundance in VPS35-depleted HeLa cells. A total of 47 differentially expressed proteins were identified, including 27 downregulated and 20 upregulated proteins. Gene ontology (GO) analysis showed that the downregulated proteins included several minichromosome maintenance (MCM) proteins described as cell proliferation markers, and these proteins were present in the MCM2-7 complex, which is essential for DNA replication. Moreover, we validated that loss of VPS35 reduced the mRNA and protein expression of MCM2-7 genes. Notably, re-expression of VPS35 in VPS35 knockout HeLa cells rescued the expression of these genes. Functionally, we showed that VPS35 contributes to cell proliferation and maintenance of genomic stability of HeLa cells. Therefore, these findings reveal that VPS35 is involved in the regulation of MCM2-7 gene expression and establish a link between VPS35 and cell proliferation.

ITRAQ-based quantitative proteomic analysis provides insight into the drought-stress response in maize seedlings

Drought is a major abiotic stress that harms plant cell physiology and limits the growth and productivity of crops. Maize (Zea mays L.), one of the most drought-susceptible crops, is a major food source for humans and an important resource for industrial bioenergy production; therefore, understanding the mechanisms of the drought response is essential for maize improvement. Using isotopic tagging relative quantitation (iTRAQ)-based protein labeling technology, we detected the proteomic changes in maize leaves under drought stress. Among the 3063 proteins that were identified, the abundance of 214 and 148 proteins increased and decreased, respectively, after three days of drought treatment. These differentially abundant proteins (DAPs) were mainly involved in cell redox homeostasis, cell wall organization, photosynthesis, abscisic acid biosynthesis, and stress-response processes. Furthermore, some of the DAP abundances still differed from the control six days after the drought treatment, most of which were molecular chaperones, heat shock proteins, metabolism-related enzymes, hydrolases, and transmembrane signal receptors. The expression level of some DAPs returned to normal when the water supply was restored, but for others it did not. A significant correlation between the protein and transcript levels was observed following an RT-qPCR analysis. Finally, our research provides insights into the overall mechanism of drought-stress tolerance, and important information for breeding of drought-tolerant maize.

ITRAQ proteomic analysis of the inhibitory effect of 1,6-O,O-diacetylbritannilactone on the plant pathogenic oomycete Phytophthora capsici

Phytophthora capsici is a highly destructive oomycete of vegetables; its management is challenging due to its broad host range, rapid dispersion, resilient spores and severe fungicide resistance. Identifying an effective alternative fungicide is important for the control of P. capsici. 1,6-O,O-diacetylbritannilactone (ABLOO), one of the secondary metabolites of Inula Britannica, showed a favorable inhibitory activity against P. capsici at different developmental stages, with a sensitivity order as follows: sporangia formation (30.45 mg/L) > zoospore discharge (77.69 mg/L) > mycelial growth (93.18 mg/L) > cystospore germination (591.48 mg/L). To investigate the mode of action of ABLOO in P. capsici, iTRAQ-based quantitative proteomic analysis was performed by comparing the expression levels of proteins in the control and ABLOO-treated (400 mg/L, inhibition rate of 80%) mycelial groups. A total of 65 downregulated and 75 upregulated proteins were identified in the proteomic analysis. Functional enrichment analyses showed that proteins with transmembrane transport activity were significantly inhibited, while proteins involved in energy production were significantly increased, including proteins involved in ubiquinone and other terpenoid-quinone biosynthesis, oxidative phosphorylation, and glycolysis/gluconeogenesis. The morphological results indicated that ABLOO treatment could decrease the thickness of the cell walls of P. capsici mycelia. Correspondingly, biochemical results showed that ABLOO treatment reduced the β-1,3-glucan contents (the key component of the cell wall of P. capsici) and increased the cell membrane permeability of P. capsici. ABLOO may exhibit antioomycete activity by destroying the cell membrane of P. capsici. This study provides new evidence regarding the inhibitory mechanisms of ABLOO against P. capsici.