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AIM The present study aimed to investigate the salivary proteome profiles of pregnant women with gingivitis (PG) or without gingivitis (HP) and non-pregnant healthy controls (HC) by employing iTRAQ-based proteomics. MATERIALS AND METHODS Saliva samples were collected from 30 Chinese women comprising 10 subjects in each of the three groups (PG, HP, and HC). The samples were subjected to iTRAQ-based proteomics analysis, and ELISA was performed to validate the results. The subsequent observations were validated in a cohort of 48 subjects. RESULTS Pathways associated with neutrophil-mediated immune response and antioxidant defence mechanism were significantly higher in PG than HC. The abundance of salivary cystatins (S, SA, and SN) and antimicrobials were significantly decreased in PG and HP, while cystatin C and D were additionally decreased in PG. Cystatin C was mapped to all the major catabolic pathways and was the most re-wired protein in pregnancy gingivitis. Further validation demonstrated cystatin C to be significantly lower in PG than HC. CONCLUSIONS While the decrease in levels of salivary cystatins and antimicrobial proteins may predispose healthy pregnant women to pregnancy gingivitis, it may cause persistence of inflammation in pregnant women with gingivitis.

Both intraflagellar transport (IFT) and lipidated protein intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility, and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies have revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei, the causative agent of African sleeping sickness. TbArl13 catalyzes two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 has been identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119. We showed that TbUnc119 binds to a flagellar arginine kinase TbAK3 in a myristoylation-dependent manner and is responsible for its targeting to and enrichment in the flagellum. Interestingly, only TbArl3A, but not TbArl3C interacted with TbUnc119 in a GTP-dependent manner, suggesting functional specialization of Arl3-GTPases in T. brucei. These results establish the function of TbUnc119 as a myristoylated cargo carrier and support the presence of a conserved LIFT pathway in T. brucei.

Treatment failures with artemisinin combination therapies (ACTs) threaten global efforts to eradicate malaria. They highlight the importance of identifying drug targets and new inhibitors and of studying how existing antimalarial classes work.Herein we report the successful development of an heterologous expression-based compound screening tool. Validated drug target P. falciparum calcium ATPase6 (PfATP6) and a mammalian ortholog (SERCA1a) were functionally expressed in yeast providing a robust, sensitive, and specific screening tool. Whole-cell and in vitro assays consistently demonstrated inhibition and labelling of PfATP6 by artemisinins. Mutations in PfATP6 resulted in fitness costs that were ameliorated in the presence of artemisinin derivatives when studied in the yeast model.As previously hypothesised, PfATP6 is a target of artemisinins. Mammalian SERCA1a can be mutated to become more susceptible to artemisinins. The inexpensive, low technology yeast screening platform has identified unrelated classes of druggable PfATP6 inhibitors. Resistance to artemisinins may depend on mechanisms that can concomitantly address multi-targeting by artemisinins and fitness costs of mutations that reduce artemisinin susceptibility.

Gram-negative pathogens like Enteropathogenic Escherichia coli (EPEC) utilize the type three secretion system (T3SS) to translocate various effector proteins that are needed to "hijack" the host system for pathogenic survival. Specialized T3SS chaperones inside bacterial cells stabilize these effector proteins and facilitate their translocation. CesT is a unique multi-cargo chaperone that interacts with and translocates ~10 different effector proteins. Here, we report the specific interaction between CesT and its key effector, NleH2, and explore the potential role of NleH2 as a kinase for CesT phosphorylation. First, we identified the chaperone-binding domain (CBD; 19-97aa) of NleH2, and mapped the specific interaction sites for both CesT and NleH2. The N- and C-terminal residues of the CBD interact with the dimeric interface of CesT. Further, we compared the CesT binding to NleH2, to that of another key effector Tir and with the global carbon regulator CsrA. Notably, the effectors have the binding regions at the β-sheet core and dimer interface of CesT, whereas the CsrA regulator interacts predominantly through the C-terminal region, which is found ~17 A away from the effectors-binding sites. Next, we showed that NleH2 remains an active kinase even as a complex with CesT and is responsible for its autophosphorylation as well as phosphorylation of CesT at Tyr153. Collectively, our findings enhance the understanding of the role of multi-cargo chaperone CesT in orchestrating effector translocation through T3SS.

Singapore grouper iridovirus (SGIV) is a lethal grouper virus containing 162 predicted ORFs. Previous proteomic studies led to identification of 73 SGIV structural proteins. Here, SDS-assisted tube-gel digestion and DOC-assisted in-solution digestion coupled with LC-ESI-MS/MS were applied to further profile the SGIV structural proteome. We identified a total of 90 SGIV structural proteins including 24 newly reported proteins. Additionally, several PTMs were identified, including 26 N-terminal acetylated proteins, three phosphorylated proteins, and one myristoylated protein. Importantly, 47 of the proteins that were identified are predicted to contain conserved domains. Our work greatly expands the repertoire of the SGIV structural proteome and provides more insight into the biology of SGIV.

Background:The molecular mechanisms or valuable biomarkers for early diagnosis of colorectal cancer (CRC) are not fully elucidated yet.Objective:To understand the proteomic changes at the global level in the carcinogenesis of CRC, differentially expressed proteins between normal intestinal epithelial cells CCD841 and colorectal cancer cells HCT116 were identified.Method:The isobaric tags for relative and absolute quantitation (iTRAQ) coupled with 2D LC-MS/MS proteomic approach were performed for screening the altered proteins between cells CCD841 and HCT116.Results:A total of 1947 proteins were identified after filtering and using a 1% false discovery rate. Based on a final cutoff (> 3.16 and < 0.32), 229 proteins were found to be significantly altered, among which 95 (41%) were up-regulated while 134 (59%) were down-regulated. Gene Ontology analysis revealed that the differentially expressed proteins were mainly cell part proteins involved in cellular process and binding in terms of subcellular distribution, biological process, and molecular function. KEGG analysis indicated that the differentially expressed proteins were significantly involved in the process of focal adhesion, pathogenic Escherichia coli infection, leukocyte transendothelial migration, bacterial invasion of epithelial cells, regulation of actin cytoskeleton, DNA replication and so on.Conclusion:Collectively, our data identified differentially expressed proteins in colon cancer carcinogenesis, which could provide the clues on unraveling the molecular mechanism of CRC.

Tobacco mosaic virus (TMV) is a positive single-stranded RNA virus. Its 5′ end ORF codes for the replicase proteins, namely 126 kDa and 183 kDa, respectively. These proteins interact with many host proteins to form a virus replication complex (VRC). This study aims to dissect the proteome profile of TMV-infected Nicotiana tabacum in host cellular and molecular pathways. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyse the differential global proteomic profile of TMV infected and mock infected plants. Out of 1897 total proteins, we identified 407 differentially abundant proteins and grouped them into three functional categories, namely metabolism, cellular processes and signalling processing. Our results showed that photosynthesis, carbon metabolism, plant defence, protein synthesis, and protein processing in the endoplasmic reticulum were significantly altered. Carbon metabolism and photosynthesis were present in very low abundance, whereas accumulation of reactive oxygen species and misfolded proteins lead to the accumulation of thioredoxin H-type 1. In conclusion, we identified several key host proteins that are involved in TMV infection/replication in N. tabacum plants. Significance of the study TMV is one of the most widely studied plant virus. It is used as a tool to study host-virus interaction. There are several host proteins reported that facilitate VRC formation and replication of TMV. However, there is limited knowledge in the expression regulation of these host proteins upon TMV infection. This study is the first report that investigates the response of host protein expression involved in TMV infection through a quantitative proteomics technique iTRAQ, combined with LC-MS/MS analysis. We used TMV-infected Nicotiana tabacum plants to investigate the effects of TMV infection on host proteins. Our results revealed differential abundance of proteins involving various pathways in protein translation, protein processing, photosynthesis and plant defence. There was a high abundance of thioredoxin H-type 1, a protein that counters oxidative stress and accelerated regulation of fatty acid synthesis to provide additional lipid molecules for VRC formation. There was a significant reduction in abundance of psaA and psbB proteins in the photosynthetic pathways. Our results identified key candidate host proteins involved in TMV-infected N. tabacum for functional studies in future.

Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13-24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.

Pregnancy gingivitis peaks during mid-pregnancy and resolves transiently towards the postpartum period. However, the role of maternal immune response in orchestrating gingival inflammation has not yet been fully understood. Hence, in this study, we examined the salivary protein profile during the three trimesters of pregnancy, in context to pregnancy gingivitis, employing iTRAQ-based quantitative proteomics. Unstimulated saliva was collected from 10 subjects in each trimester of pregnancy and postpartum period. Samples were analysed using iTRAQ analysis and ELISA and SEM was performed to validate results. Neutrophil mediated immune response was overrepresented in all three trimesters of pregnancy, despite the decrease in phagocytic responses during the second and third trimesters. ELISA showed a significantly higher Neutrophil Extracellular Traps (NETs) formation in the third trimester of pregnancy coinciding with the resolution of pregnancy gingivitis. The NETs-associated proteins (neutrophil elastase and myeloperoxidase) showed a positive correlation with estrogen hormones, which was also highest during the third trimester. Sex hormone-driven NETs formation could be the mainstay of defence that contributes to the remission of pregnancy gingivitis. This study has provided a new insight into the role of immune-modulation in pregnancy gingivitis, which will aid development of new therapeutics for managing pregnancy gingivitis in future.

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13–24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets., Liew et al. describe an efficient monoclonal antibody (mAb) production method and as a proof-of-concept, develop mAbs against multiple epitopes on hANKRD1 in a single hybridoma production cycle. With rapid screening and validation techniques, this pipeline shows promise of general applicability for antibody panel generation.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5—its highly homologous counterpart—is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F′ loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent Vmax increased and Km decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and β of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

Trypanosoma brucei contains a large array of single-copied organelles and structures. Through extensive interorganelle connections, these structures replicate and divide following a strict temporal and spatial order. A microtubule quartet (MtQ) originates from the basal bodies and extends toward the anterior end of the cell, stringing several cytoskeletal structures together along its path. In this study, we examined the interaction network of TbSpef1, the only protein specifically located to the MtQ. We identified an interaction between TbSpef1 and a basal body protein TbSAF1, which is required for MtQ anchorage to the basal bodies. This study thus provides the first molecular description of MtQ association with the basal bodies, since the discovery of this association ∼30 years ago. The results also reveal a general mechanism of the evolutionarily conserved Spef1/CLAMP, which achieves specific cellular functions via their conserved microtubule functions and their diverse molecular interaction networks., Sperm flagellar protein 1 (Spef1, also known as CLAMP) is a microtubule-associated protein involved in various microtubule-related functions from ciliary motility to polarized cell movement and planar cell polarity. In Trypanosoma brucei, the causative agent of trypanosomiasis, a single Spef1 ortholog (TbSpef1) is associated with a microtubule quartet (MtQ), which is in close association with several single-copied organelles and is required for their coordinated biogenesis during the cell cycle. Here, we investigated the interaction network of TbSpef1 using BioID, a proximity-dependent protein-protein interaction screening method. Characterization of selected candidates provided a molecular description of TbSpef1-MtQ interactions with nearby cytoskeletal structures. Of particular interest, we identified a new basal body protein TbSAF1, which is required for TbSpef1-MtQ anchorage to the basal bodies. The results demonstrate that MtQ-basal body anchorage is critical for the spatial organization of cytoskeletal organelles, as well as the morphology of the membrane-bound flagellar pocket where endocytosis takes place in this parasite.

The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell.

Both intraflagellar transport (IFT) and lipidated intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies revealed a single Arl13b ortholog in the evolutionarily divergentTrypanosoma brucei. TbArl13 catalyses two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 was identified inT. bruceigenome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119. We showed that TbUnc119 binds to a flagellar arginine kinase TbAK3 in a myristoylation-dependent manner and is responsible for its targeting and enrichment in the flagellum. Interestingly, only TbArl3A, but not TbArl3C interacts with TbUnc119 in a GTP-dependant manner, suggesting functional specialization of Arl3-GTPases inT. brucei. This study establishes the function of TbUnc119 as a myristoylated cargo carrier and supports the presence of a conserved LIFT pathway inT. brucei.

Pyrazinamide is a sterilizing first-line tuberculosis drug. Genetic, metabolomic and biophysical analyses previously demonstrated that pyrazinoic acid, the bioactive form of the prodrug pyrazinamide (PZA), interrupts biosynthesis of coenzyme A in Mycobacterium tuberculosis by binding to aspartate decarboxylase PanD. While most drugs act by inhibiting protein function upon target binding, we find here that pyrazinoic acid is only a weak enzyme inhibitor. We show that binding of pyrazinoic acid to PanD triggers degradation of the protein by the caseinolytic protease ClpC1-ClpP. Thus, the old tuberculosis drug pyrazinamide exerts antibacterial activity by acting as a target degrader, a mechanism of action that has recently emerged as a successful strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA., It has been shown that the bioactive component of pyrazinamide, pyrazinoic acid (POA), blocks coenzyme A biosynthesis in M. tuberculosis by binding to the aspartate decarboxylase PanD. Here the authors show that pyrazinamide triggers degradation of PanD by stimulating its degradation by the caseinolytic protease Clp.

TFEB (transcription factor EB) is a master regulator of lysosomal biogenesis, function and autophagy. The transcriptional activity of TFEB is mainly controlled by its phosphorylation status mediated by the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) complex 1 (MTORC1). At present, little is known whether other forms of posttranslational modifications (PTMs) such as acetylation also affects is transcriptional activity. In this study, we first observed that a well-established histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) activated lysosomal function in human cancer cells, a process independent of the MTORC1 pathway. Second, SAHA treatment activated TFEB transcriptional activity, as evidenced by increased TFEB luciferase activity and expression of its target genes. Third and more importantly, we observed the enhanced TFEB acetylation in SAHA-treated cells, with identification of 4 acetylation sites. Mutation of these 4 sites markedly diminished TFEB transcriptional activity and lysosomal function induced by SAHA. Finally, we found that TFEB acetylation was functionally implicated in SAHA-mediated autophagy and cell death in cancer cells. Taken together, our results demonstrate that TFEB acetylation is a novel form of PTMs in TFEB that plays an important role in determining its transcriptional activity, lysosomal function and autophagy in cancer cells. Abbreviations: ACAT1: acetyl-coenzyme A acetyltransferase 1; AHA: L-azidohomoalanine; AO: acidic orange; ATG: autophagy related; CLEAR: Coordinated Lysosomal Expression and Regulation; CQ: chloroquine; CTSB: cathepsin B; HATs: histone acetyltransferases; HDACIs: HDACs inhibitors; HDACs: histone deacetylases; IP: immunoprecipitation; MEFs: mouse embryonic fibroblasts; MS: mass spectrometry; MTOR: mechanistic target of rapamycin (serine/threonine kinase); MTORC1: mechanistic target of rapamycin (serine/threonine kinase) complex 1; PTMs: posttranslational modifications; SAHA: suberoylanilidehydroxamic acid; TFEB: transcription factor EB

With the development of intensive aquaculture, economic losses increasingly result from fish mortality due to pathogen infection. In recent years, a growing number of researchers have used transcriptomic and proteomic analyses to study fish immune responses to exogenous pathogen infection. Integrating transcriptomic and proteomic analyses provides a better understanding of the fish immune system including gene expression, regulation, and the intricate biological processes underlying immune responses against infection. This review focuses on the recent advances in the fields of transcriptomics and proteomics, which have contributed to our understanding of fish immunity to exogenous pathogens.

Plant viruses trigger numerous responses in their insect vectors. Using iTRAQ-based quantitative proteomics analysis, early responses of the insect vector, the small brown planthopper (Laodelphax striatellus Fallén, SBPH), after acquiring Rice black-streaked dwarf virus (RBSDV) at 3 days and 5 days post first access to diseased plants (padp) were revealed. A total of 582 differentially abundant proteins (DAPs) in SBPH with a fold change1.500 or0.667 (p-value 0.05) were identified. The proteomic analysis in SBPH at 3 days padp revealed 106 highly abundant proteins and 193 of low abundance, while 5 days padp revealed 214 highly abundant proteins and 182 of low abundance. Among them, 51 highly abundant proteins and 42 of low abundance were shown consistently at both 3 days and 5 days padp. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapping and Gene Ontology (GO) term classification suggested impairment of mitochondria in SBPH after RBSDV acquisition, and the 77 out of 582 differentially abundant SBPH proteins analyzed by the STRING program revealed the interaction network of the mitochondrial DAPs, showing an overall down-regulation of mitochondrial proteins including the electron transport chain proteins and mitochondrial ribosome proteins. The high abundance of Parkin at 5 days padp suggests that activation of mitophagy induced degradation of mitochondria occurred. Further verification of autophagy/mitophagy-related genes by reverse-transcription quantitative RT-PCR (RT-qPCR) in SBPH after RBSDV acquisition showed up-regulation of the autophagy receptors Optineurin (OPTN), Sequestosome-1 (SQSTM1, also known as p62) and Tax1-binding protein 1 (TAX1BP1) which targets ubiquitinated damaged mitochondria during mitophagy. The phosphorylation of the three autophagy receptors may be up-regulated through an increase of transcription level TRAF-associated NFκB activator (TANK)-binding kinase 1 (TBK1). As a result, an overall reduction in the abundance of mitochondrial proteins was observed and the selective autophagic degradation was up-regulated through increased transcription level of OPTN, p62/SQSTM1, TAX1BP1 and TBK1. Therefore, acquisition of RBSDV associated with up-regulated autophagy and selective mitochondrial degradation in SBPH suggest prevention of mitochondrial-mediated apoptosis and extension of the vector life span. BIOLOGICAL SIGNIFICANCE: RBSDV causes severe yield loss in rice plants. RBSDV is transmitted efficiently only through SBPH. It is important to understand how RBSDV infects SBPH in a persistent, circulative and propagative manner. However, there has been no study on the interaction between RBSDV and SBPH at the early acquisition stage using a proteomics approach. In this study, we combined iTRAQ technique and LC-MS/MS to analyze the vector proteomics at both the initial and latent infection stages after RBSDV acquisition and verified the results by RT-qPCR. Our results revealed that significantly low DAPs were involved in various pathways, including biosynthesis of secondary metabolites, ribosomes, carbon metabolism, biosynthesis of amino acids and TCA cycle. Further clustering of the DAPs revealed significant changes in SBPH mitochondria, including decreased proteins in mitochondrial ribosomes and electron transport chain complex I, II and V. On the other hand, there was a high abundance of Parkin, suggesting the occurrence of mitochondria damage and subsequent Parkin-mediated mitophagy for clearance of impaired mitochondria. Moreover, the decreased level of PMPCB in terms of gene expression and protein abundance suggested decreased PINK1 turnover, promoting Parkin/PINK1-mediated mitophagy. Further analysis on autophagy/mitophagy-related gene transcription level indicated up-regulation of OPTN, p62/SQSTM1, TAX1BP1 and TBK1, promoting selective autophagy in SBPH after RBSDV acquisition. These findings provided new insights into the effects of RBSDV on SBPH after early acquisition by selective degradation of mitochondria, especially on reprogramming of energy metabolism and decreased mitochondria biogenesis, to prevent apoptosis and prolong the life span of SBPH post virus acquisition.

Macroautophagy/autophagy is an evolutionarily well-conserved cellular degradative process with important biological functions that is closely implicated in health and disease. In recent years, quantitative mass spectrometry-based proteomics and chemical proteomics have emerged as important tools for the study of autophagy, through large-scale unbiased analysis of the proteome or through highly specific and accurate analysis of individual proteins of interest. At present, a variety of approaches have been successfully applied, including (i) expression and interaction proteomics for the study of protein post-translational modifications, (ii) investigating spatio-temporal dynamics of protein synthesis and degradation, and (iii) direct determination of protein activity and profiling molecular targets in the autophagic process. In this review, we attempted to provide an overview of principles and techniques relevant to the application of quantitative and chemical proteomics methods to autophagy, and outline the current landscape as well as future outlook of these methods in autophagy research.

Salinity affects growth and development of plants, but mangroves exhibit exceptional salt tolerance. With direct exposure to salinity, mangrove roots possess specific adaptations to tolerate salt stress. Therefore, studying the early effects of salt on mangrove roots can help us better understand the tolerance mechanisms. Using two-month-old greenhouse-grown seedlings of the mangrove tree Avicennia officinalis subjected to NaCl treatment, we profiled gene expression changes in the roots by RNA-sequencing. Of the 6547 genes that were differentially regulated in response to salt treatment, 1404 and 5213 genes were significantly up- and down-regulated, respectively. By comparative genomics, 93 key salt tolerance-related genes were identified of which 47 were up-regulated. Upon placing all the differentially expressed genes (DEG) in known signaling pathways, it was evident that most of the DEGs involved in ethylene and auxin signaling were up-regulated while those involved in ABA signaling were down-regulated. These results imply that ABA-independent signaling pathways also play a major role in salt tolerance of A. officinalis. Further, ethylene response factors (ERFs) were abundantly expressed upon salt treatment and the Arabidopsis mutant aterf115, a homolog of AoERF114 is characterized. Overall, our results would help in understanding the possible molecular mechanism underlying salt tolerance in plants.

Intracellular calcium (Ca2+) mobilization after engagement of the BCR has been proposed to play an important role in B cell development and function. BCR activation causes an initial Ca2+ release from the endoplasmic reticulum that is mediated by inositol 1,4,5-trisphosphate receptor (IP3R) and then triggers store-operated Ca2+ entry once endoplasmic reticulum Ca2+ store is depleted. Store-operated Ca2+ entry has been shown to regulate B cell function but is dispensable for B cell development. By contrast, the function of IP3R-mediated Ca2+ release in B cells remains to be determined. In this study, we generated a B cell–specific IP3R triple-knockout (IP3R-TKO) mouse model and revealed that loss of IP3Rs increased transitional B cell numbers and reduced recirculating mature B cell numbers in bone marrow. In the peripheral tissues, the numbers of conventional B2 B cells and B1 B cells were both significantly decreased in IP3R-TKO mice. Ablation of IP3Rs also dramatically reduced BCR-mediated B cell proliferation and survival. Furthermore, T cell–dependent and T cell–independent Ab responses were altered in IP3R-TKO mice. In addition, deletion of IP3Rs reduced IL-10–producing regulatory B cell numbers and led to defects in NFAT activation, which together resulted in decreased IL-10 secretion. Taken together, our study demonstrated for the first time, to our knowledge, that IP3R-mediated Ca2+ release plays an essential role in regulating B cell development, proliferation, Ab production, and B cell regulatory function in vivo.

Artemisinin and its derivatives (collectively termed as artemisinins) are among the most important and effective antimalarial drugs, with proven safety and efficacy in clinical use. Beyond their antimalarial effects, artemisinins have also been shown to possess selective anticancer properties, demonstrating cytotoxic effects against a wide range of cancer types both in vitro and in vivo. These effects appear to be mediated by artemisinin-induced changes in multiple signaling pathways, interfering simultaneously with multiple hallmarks of cancer. Great strides have been taken to characterize these pathways and to reveal their anticancer mechanisms of action of artemisinin. Moreover, encouraging data have also been obtained from a limited number of clinical trials to support their anticancer property. However, there are several key gaps in knowledge that continue to serve as significant barriers to the repurposing of artemisinins as effective anticancer agents. This review focuses on important and emerging aspects of this field, highlighting breakthroughs in unresolved questions as well as novel techniques and approaches that have been taken in recent studies. We discuss the mechanism of artemisinin activation in cancer, novel and significant findings with regards to artemisinin target proteins and pathways, new understandings in artemisinin-induced cell death mechanisms, as well as the practical issues of repurposing artemisinin. We believe these will be important topics in realizing the potential of artemisinin and its derivatives as safe and potent anticancer agents.

Development of sustainable biobutanol production platforms from lignocellulosic materials is impeded by inefficient five carbon sugar uptake by solventogenic bacteria. The recently isolated Clostridium sp. strain BOH3 is particularly advantaged in this regard as it serves as a model organism which can simultaneously utilize both glucose and xylose for high butanol (>15 g/L) production. Strain BOH3 was therefore investigated for its metabolic mechanisms for efficient five carbon sugar uptake using a quantitative proteomics based approach. The proteomics data show that proteins within the CAC1341-1349 operon play a pivotal role for efficient xylose uptake within the cells to produce butanol. Furthermore, up-regulation of key enzymes within the riboflavin synthesis pathway explained that xylose could induce higher riboflavin production capability of the bacteria (e.g., ∼80 mg/L from glucose vs ∼120 mg/L from xylose). Overall results from the present experimental approach indicated that xylose-fed BOH3 cultures are subjected to high levels of redox stress which coupled with the solvent stress - trigger a sporulation response within the cells earlier than the glucose-fed cultures. The study lays the platform for metabolic engineering strategies in designing organisms for efficient butanol and other value-added chemicals such as riboflavin production. This article is protected by copyright. All rights reserved

Background: Zebrafish is a well-recognised organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. Results: In this study, we developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the LC-MS-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately two-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3-4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and KEGG analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. Conclusion: This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

Candida albicans is a major fungal pathogen, accounting for approximately 15% of healthcare infections with associated mortality as high as 40% in the case of systemic candidiasis. Antifungal agents for C. albicans infections are limited, and rising resistance is an inevitable problem. Therefore, understanding the mechanism behind antifungal responses is among the top research focuses in combating Candida infections. Herein, the recently developed C. albicans haploid model is employed to examine the association between mitochondrial fission, regulated by Dnm1, and the pathogen's response to antifungals. Proteomic analysis of dnm1Δ and its wild-type haploid parent, GZY803, reveal changes in proteins associated with mitochondrial structures and functions, cell wall, and plasma membrane. Antifungal susceptibility testing revealed that dnm1Δ is more susceptible to SM21, a novel antifungal, than GZY803. Analyses of reactive oxygen species release, antioxidant response, lipid peroxidation, and membrane damages uncover an association between dnm1Δ and the susceptibility to SM21. Dynasore-induced mitochondrial inhibition in SC5314 diploids corroborate the findings. Interestingly, Dynasore-primed SC5314 cultures exhibit increased susceptibility to all antifungals tested. These data suggest an important contribution of mitochondrial fission in antifungal susceptibility of C. albicans. Hence, mitochondrial fission can be a potential target for combined therapy in anti-C. albicans treatment.

Candida albicans, a major opportunistic fungal pathogen, is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood caries (tooth decay), a prevalent pediatric oral disease. However, the impact of this cross-kingdom relationship on C. albicans remains largely uncharacterized. Here, we employed a novel quantitative proteomics approach in conjunction with transcriptomic profiling to unravel molecular pathways of C. albicans when cocultured with S. mutans in mixed biofilms., Candida albicans, a major opportunistic fungal pathogen, is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood caries (tooth decay), a prevalent pediatric oral disease. However, the impact of this cross-kingdom relationship on C. albicans remains largely uncharacterized. Here, we employed a novel quantitative proteomics approach in conjunction with transcriptomic profiling to unravel molecular pathways of C. albicans when cocultured with S. mutans in mixed biofilms. RNA sequencing and iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomics revealed that C. albicans genes and proteins associated with carbohydrate metabolism were significantly enhanced, including sugar transport, aerobic respiration, pyruvate breakdown, and the glyoxylate cycle. Other C. albicans genes and proteins directly and indirectly related to cell morphogenesis and cell wall components such as mannan and glucan were also upregulated, indicating enhanced fungal activity in mixed-species biofilm. Further analyses revealed that S. mutans-derived exoenzyme glucosyltransferase B (GtfB), which binds to the fungal cell surface to promote coadhesion, can break down sucrose into glucose and fructose that can be readily metabolized by C. albicans, enhancing growth and acid production. Altogether, we identified key pathways used by C. albicans in the mixed biofilm, indicating an active fungal role in the sugar metabolism and environmental acidification (key virulence traits associated with caries onset) when interacting with S. mutans, and a new cross-feeding mechanism mediated by GtfB that enhances C. albicans carbohydrate utilization. In addition, we demonstrate that comprehensive transcriptomics and quantitative proteomics can be powerful tools to study microbial contributions which remain underexplored in cross-kingdom biofilms.

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that is a major threat to grouper aquaculture. The pathogenesis of SGIV is not well understood so far. Previous studies have revealed that ICP18, an immediate early protein encoded by SGIV ORF086R gene, promotes viral replication by regulating cell proliferation and virus assembly. In the present study, the potential functions of ICP18 were further explored by probing into its interactors using a proximity-dependent BioID method. Since our in-house grouper embryonic cells (a natural host cell of SGIV) could not be efficiently transfected with the plasmid DNA, and the grouper genome data for mass spectrometry-based protein identification is not currently available, we chosen a non-permissive cell (HEK293 T) as a substitute for this study. A total of 112 cellular proteins that potentially bind to ICP18 were identified by mass spectrometry analysis. Homology analysis showed that among these identified proteins, 110 candidate ICP18-interactors had homologous proteins in zebrafish (a host of SGIV), and shared high sequence identity. Further analysis revealed that the identified ICP18-interacting proteins modulate various cellular processes such as cell cycle and cell adhesion. In addition, the interaction between ICP18 and its candidate interactor, i.e., cyclin-dependent kinase1 (CDK1), was confirmed using Co-immunoprecipitation (Co-IP) and Pull-down assays. Collectively, our present data provides additional insight into the biological functions of ICP18 during viral infection, which could help in further unraveling the pathogenesis of SGIV.

Many protein extraction methods have been developed for plant proteome analysis but information is limited on the optimal protein extraction method from algae species. This study evaluated four protein extraction methods, i.e. direct lysis buffer method, TCA-acetone method, phenol method, and phenol/TCA-acetone method, using green algae Chlorella vulgaris for proteome analysis. The data presented showed that phenol/TCA-acetone method was superior to the other three tested methods with regards to shotgun proteomics. Proteins identified using shotgun proteomics were validated using sequential window acquisition of all theoretical fragment-ion spectra (SWATH) technique. Additionally, SWATH provides protein quantitation information from different methods and protein abundance using different protein extraction methods was evaluated. These results highlight the importance of green algae protein extraction method for subsequent MS analysis and identification.

Cross-protection is a phenomenon in which infection with a mild virus strain protects host plants against subsequent infection with a closely related severe virus strain. This study showed that a mild strain mutant virus, Tobacco mosaic virus (TMV)-43A could cross protect Nicotiana benthamiana plants against wild-type TMV. Furthermore, we investigated the host responses at the proteome level to identify important host proteins involved in cross-protection. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyze the proteome profiles of TMV, TMV-43A and cross-protected plants at different time-points. Our results showed that TMV-43A can cross-protect N. benthamiana plants from TMV. In cross-protected plants, photosynthetic activities were augmented, as supported by the increased accumulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) enzymes, which are crucial for chlorophyll biosynthesis. The increased abundance of ROS scavenging enzymes like thioredoxins and L-ascorbate peroxidase would prevent oxidative damage in cross-protected plants. Interestingly, the abundance of defence-related proteins (14-3-3 and NbSGT1) decreased, along with a reduction in virus accumulation during cross-protection. In conclusion, we have identified several important host proteins that are crucial in cross-protection to counter TMV infection in N. benthamiana plants. BIOLOGICAL SIGNIFICANCE: TMV is the most studied model for host-virus interaction in plants. It can infect wide varieties of plant species, causing significant economic losses. Cross protection is one of the methods to combat virus infection. A few cross-protection mechanisms have been proposed, including replicase/coat protein-mediated resistance, RNA silencing, and exclusion/spatial separation between virus strains. However, knowledge on host responses at the proteome level during cross protection is limited. To address this knowledge gap, we have leveraged on a global proteomics analysis approach to study cross protection. We discovered that TMV-43A (protector) protects N. benthamiana plants from TMV (challenger) infection through multiple host pathways: secondary metabolite biosynthesis, photosynthesis, defence, carbon metabolism, protein translation and processing and amino acid biosynthesis. In the secondary metabolite biosynthesis pathway, enzymes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) play crucial roles in chlorophyll biosynthesis during cross protection. In addition, accumulation of ROS scavenging enzymes was also found in cross-protected plants, providing rescues from excessive oxidative damage. Reduced abundance of plant defence proteins is correlated to reduced virus accumulation in host plants. These findings have increased our knowledge in host responses during cross-protection.

Background: Human leptospirosis, or commonly known as “rat urine disease” is a zoonotic disease that is caused by the bacteria called Leptospira sp. The incidence rate of leptospirosis has been under-reported due to its unspecific clinical symptoms and the limitations of current laboratory diagnostic methods. Leptospirosis can be effectively treated with antibiotics in the early stage, and it is a curable disease but the accuracy to diagnose the infection is rarely achieved. Methods: The present pilot study investigated plasma protein profiles of leptospirosis patients and compared them against two control groups which consisted of dengue patients and healthy individuals. The plasma protein digests were analyzed using shotgun approach by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein abundances were estimated from the exponentially modified protein abundance index (emPAI) values. Plasma proteins in leptospirosis patients with at least two-fold differential expression compared to dengue and healthy control groups (p

Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography&ndash, tandem mass spectrometry (LC&ndash, MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3&ndash, 4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

The small GTPase Arl13b is one of the most conserved and ancient ciliary proteins. In human and animals, Arl13b is primarily associated with the ciliary membrane, where it acts as a Guanine-nucleotide Exchange Factor (GEF) for Arl3 and is implicated in a variety of ciliary and cellular functions. We have identified and characterized TbArl13, the sole Arl13b homologue in the evolutionarily divergent, protozoan parasite Trypanosoma brucei. TbArl13 has conserved flagellar functions and exhibits catalytic activity towards two different TbArl3 homologues. However, TbArl13 is distinctly associated with the axoneme through a dimerization/docking (D/D) domain. Replacing the D/D domain with a flagellar membrane protein created a viable alternative to the wild-type TbArl13 in our RNA interference (RNAi)-based rescue assay. Therefore, flagellar enrichment is crucial for TbArl13 but mechanisms to achieve this could be flexible. Our findings thus extended the understanding of Arl13b and Arl13b-Arl3 pathway in a divergent flagellate of medical importance.

Traditional toxicological screens based on the zebrafish model use observable phenotypic endpoints during their development to determine the toxicity of teratogens. Yet toxicity does not always translate to obvious phenotypic changes and the criteria used to score the toxicity of a teratogen are frequently subjected to human perception. The advancement in omics-based technologies has allowed us to quantitatively and objectively determine the toxicity of a teratogen based on biomolecular changes. The field of proteomics has been gaining popularity as a valuable tool in toxicology. Hence, in this chapter, we described a protocol for both label-free and label-based proteomic methods to analyse proteomic changes in both embryos and adult livers of zebrafish exposed to the teratogen TCDD (tetrachlorodibenzo-p-dioxin) as an example.

Preparation of proteins from salt-gland-rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high-quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland-rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland-rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol-based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D-LC-MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol-based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high-throughput proteomic analyses involving LC-MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.

The artemisinin compounds, which are well-known for their potent therapeutic antimalarial activity, possess in vivo and in vitro antitumor effects. Although the anticancer effect of artemisinin compounds has been extensively reported, the precise mechanisms underlying its cytotoxicity remain under intensive study. In the present study, a high-throughput quantitative proteomics approach was applied to identify differentially expressed proteins of HCT116 colorectal cancer cell line with artesunate (ART) treatment. Through Ingenuity Pathway Analysis, we discovered that the top-ranked ART-regulated biological pathways are abrogation of fatty acid biosynthetic pathway and mitochondrial dysfunction. Subsequent assays showed that ART inhibits HCT116 cell proliferation through suppressing the fatty acid biosynthetic pathway and activating the mitochondrial apoptosis pathway. In addition, ART also regulates several proteins that are involved in NF-κB pathway, and our subsequent assays showed that ART suppresses the NF-κB pathway. These proteomic findings will contribute to improving our understanding of the underlying molecular mechanisms of ART for its therapeutic cytotoxic effect towards cancer cells.

Background: Andrographolide (ADR), the main active component of Andrographis paniculata, displays anticancer activity in various cancer cell lines, among which leukemia cell lines exhibit the highest sensitivity to ADR. In particular, ADR was also reported to have reduced drug resistance in multidrug resistant cell lines. However, the mechanism of action (MOA) of ADR’s anticancer and anti-drug-resistance activities remain elusive. Methods: In this study, we used the MV4-11 cell line, a FLT3 positive acute myeloid leukemia (AML) cell line that displays multidrug resistance, as our experimental system. We first evaluated the effect of ADR on MV4-11 cell proliferation. Then, a quantitative proteomics approach was applied to identify differentially expressed proteins in ADR-treated MV4-11 cells. Finally, cellular processes and signal pathways affected by ADR in MV4-11 cell were predicted with proteomic analysis and validated with in vitro assays. Results: ADR inhibits MV4-11 cell proliferation in a dose- and time-dependent manner. With a proteomic approach, we discovered that ADR inhibited fatty acid synthesis, cellular iron uptake and FLT3 signaling pathway in MV4-11 cells. Conclusions: ADR inhibits MV4-11 cell proliferation through inhibition of fatty acid synthesis, iron uptake and protein synthesis. Furthermore, ADR reduces drug resistance by blocking FLT3 signaling.

The antimalarial artemisinin (ART) possesses anticancer activity, but its underlying mechanism remains largely unclear. Using a chemical proteomics approach with artemisinin-based activity probes, we identified over 300 specific ART targets. This reveals an anticancer mechanism whereby ART promiscuously targets multiple critical biological pathways and leads to cancer cell death. The specific cytotoxicity of ART against colorectal cancer (CRC) cells rather than normal colon epithelial cells is due to the elevated capacity of heme synthesis in the cancer cells. Guided by this mechanism, the specific cytotoxicity of ART toward CRC cells can be dramatically enhanced with the addition of aminolevulinic acid (ALA), a clinically used heme synthesis precursor, to increase heme levels. Importantly, this novel ART/ALA combination therapy proves to be more effective than an ART monotherapy in a mouse xenograft CRC model. Thus, ART can be repurposed and potentiated by exploitation of its mechanism of action and the metabolic features of the CRC cells., Chemical proteomics reveals a role of heme in the activation and anticancer activity of artemisinin. Based on this mechanism, we propose a novel combination treatment to enhance its efficacy.

Centromeric identity and chromosome segregation are determined by the precise centromeric targeting of CENP-A, the centromere-specific histone H3 variant. The significance of the amino-terminal domain (NTD) of CENP-A in this process remains unclear. Here, we assessed the functional significance of each residue within the NTD of CENP-A from Schizosaccharomyces pombe (SpCENP-A) and identified a proline-rich ‘GRANT’ (Genomic stability-Regulating site within CENP-A N-Terminus) motif that is important for CENP-A function. Through sequential mutagenesis, we show that GRANT proline residues are essential for coordinating SpCENP-A centromeric targeting. GRANT proline-15 (P15), in particular, undergoes cis–trans isomerization to regulate chromosome segregation fidelity, which appears to be carried out by two FK506-binding protein (FKBP) family prolyl cis–trans isomerases. Using proteomics analysis, we further identified the SpCENP-A-localizing chaperone Sim3 as a SpCENP-A NTD interacting protein that is dependent on GRANT proline residues. Ectopic expression of sim3+ complemented the chromosome segregation defect arising from the loss of these proline residues. Overall, cis–trans proline isomerization is a post-translational modification of the SpCENP-A NTD that confers precise propagation of centromeric integrity in fission yeast, presumably via targeting SpCENP-A to the centromere.

Epigallocatechin-3-gallate (EGCG), a bioactive compound in green tea, is the most abundant and biologically active catechin, and it exerts multiple effects in humans through mechanisms that remain to be clarified. The present study used bioinformatics to identify possible mechanisms by which EGCG reduces risk of breast cancer. Possible human protein targets of EGCG were identified in the PubChem database, possible human gene targets were identified in the NCBI database, and then both sets of targets were analyzed using Ingenuity Pathway Analysis to predict molecular networks affected by EGCG in breast cancer. The results suggest that signaling proteins affected by EGCG in breast cancer, which include JUN, FADD, NFKB1, Bcl-2, GNAO1, and MMP14, are involved primarily in cell death and survival; DNA replication, recombination and repair; and the cell cycle. The main networks affected by EGCG are predicted to involve the cell cycle; cellular assembly and organization; DNA replication, recombination and repair; and cell death and survival. These results identify several specific proteins and pathways that may be affected by EGCG in breast cancer, and they illustrate the power of integrative bioinformatics and chemical fragment analysis for focusing mechanistic studies.

Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l -azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a “click” reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy.

Biological membranes with cubic symmetry are a hallmark of virus-infected or diseased cells. The mechanisms of formation and specific cellular functions of cubic membranes, however, are unclear. The best-documented cubic membrane formation occurs in the free-living giant amoeba Chaos carolinense. In that system, mitochondrial inner membranes undergo a reversible structural change from tubular to cubic membrane organization upon starvation of the organism. As a prerequisite to further analyze the structural and functional features of cubic membranes, we adapted protocols for the isolation of mitochondria from starved amoeba and have identified buffer conditions that preserve cubic membrane morphology in vitro. The requirement for high concentration of ion-chelating agents in the isolation media supports the importance of a balanced ion milieu in establishing and maintaining cubic membranes in vivo.

Drug target identification is a critical step toward understanding the mechanism of action of a drug, which can help one improve the drug's current therapeutic regime and expand the drug's therapeutic potential. However, current in vitro affinity-chromatography-based and in vivo activity-based protein profiling approaches generally face difficulties in discriminating specific drug targets from nonspecific ones. Here we describe a novel approach combining isobaric tags for relative and absolute quantitation with clickable activity-based protein profiling to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. Our findings, validated through cell migration and invasion assays, showed that Andro has a potential novel application as a tumor metastasis inhibitor. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analog synthesis, protein engineering, and mass-spectrometry-based approaches and determined the drug-binding sites of two protein targets, NF-κB and actin.

Saltexclusionattherootsandsaltsecretionintheleaveswere examined in a mangrove, Avicennia officinalis. The non- secretor mangrove Bruguiera cylindrica was used for com- parative study of hydrophobic barrier formation in the roots. Bypass flow was reduced when seedlings were previously treated with high salt concentration. A biseriate exodermis was detected in the salt-treated roots,along with an enhanced deposition of hydrophobic barriers in the endodermis.These barriers reduced Na + loading into the xylem,accounting for a 90-95% salt exclusion in A. officinalis. Prominent barriers werefoundintherootsofB. cylindricaevenintheabsenceof salt treatment. A cytochrome P450 gene that may regulate suberin biosynthesis was up-regulated within hours of salt treatment in A. officinalis roots and leaves, corresponding with increased suberin deposition. X-ray microanalysis showed preferential deposition of Na + and Cl − in the root cortex compared with the stele, suggesting that the endodermisistheprimarysiteofsaltexclusion.Enhancedsalt secretion and increased suberin deposition surrounding the saltglandswereseenintheleaveswithsalttreatment.Overall, these data show that the deposition of apoplastic barriers increases resistance to bypass flow leading to efficient salt exclusion at the roots in mangroves.

Tobacco mosaic virus (TMV) is a positive, single-stranded RNA virus. It encodes two replicases (126 kDa and 183 kDa), a movement protein and a coat protein. These proteins interact with host proteins for successful infection. Some host proteins such as eEF1α, Tm-1, TOM1, 14–3-3 proteins directly interact with Tobamovirus replication proteins. There are host proteins in the virus replication complex which do not interact with viral replicases directly, such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. We have used Proximity-dependent biotin identification (BioID) technique to screen for transient or weak protein interactions of host proteins and viral replicase in vivo. We transiently expressed BirA* tagged TMV 126 kDa replicase in TMV infected Nicotiana benthamiana plants. Among 18 host proteins, we identified NbSGT1 as a potential target for further characterization. Silencing of NbSGT1 in N. benthamiana plants increased its susceptibility to TMV infection, and overexpression of NbSGT1 increased resistance to TMV infection. There were weak interactions between NbSGT1 and TMV replicases but no interaction between them was found in Y2H assay. It suggests that the interaction might be transient or indirect. Therefore, the BioID technique is a valuable method to identify weak or transient/indirect interaction(s) between pathogen proteins and host proteins in plants. Biological significance TMV is a well characterized positive-strand RNA virus model for study of virus-plant host interactions. It infects >350 plant species and is one of the significant pathogens of crop loss globally. Many host proteins are involved in TMV replication complex formation. To date there are few techniques available for identifying interacting host proteins to viral proteins. There is limited knowledge on transient or non-interacting host proteins during virus infection/replication. In this study, we used agroinfiltration-mediated in planta BioID technique to identify transiently or non-interacting host proteins to viral proteins in TMV-infected N. benthamiana plants. This technique allowed us to identify potential candidate proteins in the vicinity of TMV 126 kDa replicase. We have selected NbSGT1 and its overexpression suppresses TMV replication and increase plant resistance. NbSGT1 is believed to interact transiently or indirectly with TMV replicases in the presence of Hsp90/Hsp70. BioID is a novel and powerful technique to identify transiently or indirectly interacting proteins in the study of pathogen-host interactions.

Keeping continuity with our previous study that revealed direct correlations between CRC metastasis and enhanced CacyBP protein levels, here we attempt to improve our understanding of the mechanisms involved within this enigmatic process. Overexpression of CacyBP (CacyBP-OE) in primary CRC cell and its knock down (CacyBP-KD) in the metastatic CRC cells revealed (through phenotypic studies) the positive impact of the protein on metastasis. Additionally, two individual 4-plex iTRAQ based comparative proteomics experiments were carried out on the CacyBP-OE and CacyBP-KD cells, each with two biological replicates. Mining of proteomics data identified total 279 (63.80% up-regulated and 36.20% down-regulated) proteins to be significantly altered in expression level for the OE set and in the KD set, this number was 328 (48.78% up-regulated and 51.22% down-regulated). Functional implications of these significantly regulated proteins were related to metastatic phenotypes such as cell migration, invasion, adhesion and proliferation. Gene ontology analysis identified integrin signaling as the topmost network regulated within CacyBP-OE. Further detection of caveolar mediated endocytosis in the top hit list correlated this phenomenon with the dissociation of integrins from the focal adhesion complex which are known to provide the traction force for cell movement when transported back to the leading edge. This finding was further supported by the data obtained from CacyBP-KD data set showing down-regulation of proteins necessary for integrin endocytosis. Furthermore, intracellular calcium levels (known to influence integrin mediated cell migration) were found to be lowered in CacyBP-KD cells indicating decreased cell motility and vice versa for the CacyBP-OE cells. Actin nucleation by ARP-WASP complex, known to promote cell migration, was also identified as one of the top regulated pathways in CacyBP-OE cells. In short, this study presents CacyBP as a promising candidate biomarker for CRC metastasis and also sheds light on the underlying molecular mechanism by which CacyBP promotes CRC metastasis.

We have evaluated the effect of heart extracellular matrix (ECM) on the cardiomyocyte differentiation of mouse embryonic stem cells (ES cells) using de-cellularized heart tissue. Several lines of evidence indicate that ECM plays significant roles in cell proliferation, cell death and differentiation, but role of ECM possessing a 3D structure in differentiation has not been studied in detail. We found that there are substantial differences in the quantitative protein profiles of ECM in SDS-treated heart tissue compared to that of liver tissue, as assessed by iTRAQ™ quantitative proteomics analysis. When mouse ES cells were cultured on thin (60 μm) sections of de-cellularized tissue, the expression of cardiac myosin heavy chain (cMHC) and cardiac troponin I (cTnI) was high in ES cells cultured on heart ECM compared with those cultured on liver ECM. In addition, the protein expression of cMHC and cTnI was detected in cells on heart ECM after 2 weeks, which was not detectable in cells on liver ECM. These results indicate that heart ECM plays a critical role in the cardiomyocyte differentiation of ES cells. We propose that tissue-specific ECM induced cell lineage specification through mechano-transduction mediated by the structure, elasticity and components of ECM.

The specialized salt glands on the epidermis of halophytic plants secrete excess salts from tissues by a mechanism that is poorly understood. We examined the salt glands as putative salt and water bi-regulatory units that can respond swiftly to altering environmental cues. The tropical mangrove tree species (Avicennia officinalis) is able to grow under fluctuating salinities (0.7-50.0 dS m(-1)) at intertidal zones, and its salt glands offer an excellent platform to investigate their dynamic responses under rapidly changing salinities. Utilizing a novel epidermal peel system, secretion profiles of hundreds of individual salt glands examined revealed that these glands could secrete when exposed to varying salinities. Notably, rhythmic fluctuations observed in secretion rates were reversibly inhibited by water channel (aquaporin) blocker, and two aquaporin genes (PIP and TIP) preferentially expressed in the salt gland cells were rapidly induced in response to increasing salt concentration. We propose that aquaporins are involved and contribute to the re-absorption of water during salt removal in Avicennia officinalis salt glands. This constitutes an adaptive feature that contributes to salt balance of trees growing in saline environments where freshwater availability is limited.