The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Importin‐α proteins mediate the translocation of nuclear localization signal (NLS)‐containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN‐α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the autoimmune mutant snc1 (suppressor of npr1‐1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense‐regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope‐tagged MOS6 from pathogen‐challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR‐NBS‐type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type‐III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co‐immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN‐α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N‐terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN‐α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.     

The C. elegans TIA‐1/TIAR homolog TIAR‐1 is required to induce germ cell apoptosis

In Caenorhabditis elegans, physiological germ cell apoptosis eliminates more than half of the cells in the hermaphrodite gonad to support gamete quality and germline homeostasis by a still unidentified mechanism. External factors can also affect germ cell apoptosis. The BH3‐only protein EGL‐1 induces germ cell apoptosis when animals are exposed to pathogens or agents that produce DNA damage. DNA damage‐induced apoptosis also requires the nematode p53 homolog CEP‐1. Previously, we found that heat shock, oxidative, and osmotic stresses induce germ cell apoptosis through an EGL‐1 and CEP‐1 independent mechanism that requires the MAPKK pathway. However, we observed that starvation increases germ cell apoptosis by an unknown pathway. Searching for proteins that participate in stress‐induced apoptosis, we found the RNA‐binding protein TIAR‐1 (a homolog of the mammalian TIA‐1/TIAR family of proteins). Here, we show that TIAR‐1 in C. elegans is required to induce apoptosis in the germline under several conditions. We also show that TIAR‐1 acts downstream of CED‐9 (a BCL2 homolog) to induce apoptosis under stress conditions, and apparently does not seem to regulate ced‐4 or ced‐3 mRNAs accumulation directly. TIAR‐1 is expressed ubiquitously in the cytoplasm of the soma as well as the germline, where it sometimes associates with P granules. We show that animals lacking TIAR‐1 expression are temperature sensitive sterile due to oogenesis and spermatogenesis defects. Our work shows that TIAR‐1 is required for proper germline function and demonstrates that this protein is important to induce germ cell apoptosis under several conditions. genesis 51:690–707. © 2013 Wiley Periodicals, Inc.     

Caveolin‐1 down‐regulation is required for Wnt5a‐Frizzled 2 signalling in Ha‐RasV12‐induced cell transformation

Caveolin‐1 (Cav1) is down‐regulated during MK4 (MDCK cells harbouring inducible Ha‐Ras^V12 gene) transformation by Ha‐Ras^V12. Cav1 overexpression abrogates the Ha‐Ras^V12‐driven transformation of MK4 cells; however, the targeted down‐regulation of Cav1 is not sufficient to mimic this transformation. Cav1‐silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction‐related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I‐CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha‐Ras^V12‐inducing MK4 cells increased exosome‐like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I‐CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I‐CM (MK4+I‐EXs). Wnt5a, a downstream product of Ha‐Ras^V12, was markedly secreted by MK4+I‐CM and MK4+I‐EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha‐Ras^V12‐ and MK4+I‐CM‐induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down‐regulation, either by Ha‐Ras^V12 or targeted shRNA, increased frizzled‐2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I‐EXs in MDCK cells. These data suggest that Cav1‐dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha‐Ras^V12‐Wnt5a‐Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha‐Ras^V12‐driven cell transformation.     

Identification of differentially expressed proteins by treatment with PUGNAc in 3T3‐L1 adipocytes through analysis of ATP‐binding proteome

O‐GlcNAc (2‐acetamino‐2‐deoxy‐β‐D‐glucopyranose), an important modification for cellular processes, is catalyzed by O‐GlcNAc transferase and O‐GlcNAcase. O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene) amino‐N‐phenylcarbamate (PUGNAc) is a nonselective inhibitor of O‐GlcNAcase, which increases the level of protein O‐GlcNAcylation and is known to induce insulin‐resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3‐L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP‐bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP‐binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3‐L1 adipocytes following treatment with PUGNAc. For label‐free quantitation, a gel‐assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data‐independent (671 proteins identified) and data‐dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide‐binding proteins and we focused on some nucleotide‐binding proteins, ubiquitin‐activation enzyme 1 (E1), Hsp70, vasolin‐containing protein (Vcp), and Hsp90, involved in ubiquitin‐proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time‐dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3‐L1 cells.     

HMGB1 mediates hyperglycaemia‐induced cardiomyocyte apoptosis via ERK/Ets‐1 signalling pathway

Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up‐regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)‐induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG‐induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short‐hairpin RNA significantly decreased HG‐induced cell apoptosis by reducing caspase‐3 activation and ratio of Bcl2‐associated X protein to B‐cell lymphoma/leukemia‐2 (bax/bcl‐2). Furthermore, HG activated E26 transformation‐specific sequence‐1 (Ets‐1), and HMGB1 inhibition attenuated HG‐induced activation of Ets‐1 via extracellular signal‐regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets‐1 significantly decreased HG‐induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin‐treated diabetic mice. Inhibition of HMGB1 by short‐hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets‐1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia‐induced cardiomyocyte apoptosis by down‐regulating ERK‐dependent activation of Ets‐1.     

Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid,C8‐β‐D‐lactosylceramide

Plasma membrane (PM) microdomains, including caveolae and other cholesterol‐enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D‐erythro‐octanoyl‐lactosylceramide (C8‐D ‐e‐LacCer), stimulates endocytosis via caveolae and induces the appearance of micron‐size microdomains on the PM. To further understand the effects of C8‐D ‐e‐LacCer treatment on PM microdomains, we used a detergent‐free method to isolate microdomain‐enriched membranes from fibroblasts treated ±C8‐D ‐e‐LacCer, and performed 2‐DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain‐enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho‐associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D ‐e‐LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8‐D ‐e‐LacCer‐treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8‐L ‐threo‐lactosylceramide, a glycosphingolipid with non‐natural stereochemistry. This study identifies new microdomain‐associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.     

Crystal structure and nucleic acid‐binding activity of the CRISPR‐associated protein Csx1 of Pyrococcus furiosus

In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR‐associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C‐terminal domain of PF1127 has a unique β‐hairpin structure that protrudes out of an α‐helix and contains several positively charged residues. We demonstrate that PF1127 binds double‐stranded DNA and RNA and that this activity requires an intact β‐hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the β‐hairpin structure and exhibits no DNA‐binding properties under the same experimental conditions. Notably, the C‐terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N‐terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid‐binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Production and characterization of active recombinant interleukin‐12/eGFP fusion protein in stably‐transfected DF1 chicken cells

The adjuvant activity of chicken interleukin‐12 (chIL‐12) protein has been described as similar to that of mammalian IL‐12. Recombinant chIL‐12 can be produced using several methods, but chIL‐12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL‐12 which stably expressed a fusion protein, chIL‐12 and enhanced green fluorescent protein (eGFP) connected by a (G₄S)₃ linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10⁶ DF1/chIL‐12 cells were inoculated in a T‐175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL^?1 and 2,207 ± 3.28 ng mL^?1, respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN‐γ, which was measured using an enzyme‐linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL‐12 cells with DMSO or producing chIL‐12 in a fusion protein form does not have adverse effects on the bioactivity of chIL‐12. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:641–649, 2015     

Isolation and 2‐D‐DIGE proteomic analysis of intracellular and extracellular forms of Listeria monocytogenes

The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP‐1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2‐D‐DIGE approach followed by MALDI‐TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D ‐Ala‐D ‐Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde‐3‐phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti‐Listeria therapy.     

Jasmonoyl‐l‐isoleucine hydrolase 1 (JIH1) regulates jasmonoyl‐l‐isoleucine levels and attenuates plant defenses against herbivores

For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA‐Ile permanently inactivates JA‐Ile‐mediated signaling in plants, the alternative deactivation pathway of JA‐Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl‐l ‐isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA‐Ile and IAA‐Ala in vitro. When the herbivory‐inducible NaJIH1 gene was silenced by RNA interference, JA‐Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild‐type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild‐type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA‐Ile‐dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory‐elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA‐Ile‐hydroxylation and carboxylation of JA‐Ile, rapidly attenuates the JA‐Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.     

Temporal proteomic analysis of IGF‐1R signalling in MCF‐7 breast adenocarcinoma cells

Dysregulation of the insulin‐like growth factor 1 receptor signalling network is implicated in tumour growth and resistance to chemotherapy. We explored proteomic changes resulting from insulin‐like growth factor 1 stimulation of MCF‐7 adenocarcinoma cells as a function of time. Quantitative analysis using iTRAQ? reagents and 2‐D LC‐MS/MS analysis of three biological replicates resulted in the identification of 899 proteins (p≤0.05) with an estimated mean false‐positive rate of 2.6%. Quantitative protein expression was obtained from 681 proteins. Further analysis by supervised k‐means clustering identified five temporal clusters, which were submitted to the FuncAssociate server to assign overrepresented gene ontology terms. Proteins associated with vesicle transport were significantly overrepresented. We further analyzed our data set for proteins showing temporal significance using the software, extraction and analysis of differential gene expression, resulting in 20 significantly and temporally changing proteins (p≤0.1). These significant proteins play roles in, among others, altered glucose metabolism (lactate dehydrogenase A and pyruvate kinase M1/M2) and cellular stress (nascent polypeptide‐associated complex subunit α and heat shock (HSC70) proteins). We used multiple reaction monitoring to validate these interesting proteins and have revealed several differences in relative peptide expression corresponding to protein isoforms and variants.     

Early Origin of Genes Encoding Subunits of Biogenesis of Lysosome‐related Organelles Complex‐1, ‐2 and ‐3

Biogenesis of lysosome‐related organelles complex (BLOC)‐1, ‐2 and ‐3 are three multi‐subunit protein complexes that are deficient in various forms of Hermansky‐Pudlak syndrome, a human disease characterized by abnormal formation of lysosome‐related organelles. Contrasting views have arisen on the evolutionary origin of these protein complexes. One view is that the BLOCs represent a recent evolutionary ‘acquisition’ unique to metazoans. However, the yeast proteins Mon1, Ccz1 and She3 have been reported to display homology to the HPS1 and HPS4 subunits of BLOC‐3 and the BLOS2 subunit of BLOC‐1, respectively. In this work, we have systematically searched for orthologs of BLOC subunits in the annotated genomes of over 160 species of eukaryotes, including metazoans and fungi in the Opisthokonta group as well as highly divergent organisms. We have found orthologs of six of the eight BLOC‐1 subunits, two of the three BLOC‐2 subunits, and the two BLOC‐3 subunits, in some non‐opisthokonts such as Dictyostelium discoideum, suggesting an early evolutionary origin for these complexes. On the other hand, we have obtained no evidence in support of the notion that yeast She3 would be an ortholog of BLOS2, and found that yeast Mon1 and Ccz1, despite displaying restricted homology to portions of HPS1 and HPS4, are unlikely to represent the orthologs of these BLOC‐3 subunits. Potential orthologs of Mon1 and Ccz1 were found in humans and several other eukaryotes.     

Rm62, a DEAD‐box RNA helicase,complexes with DSP1 in Drosophila embryos

Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB‐like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD‐box RNA helicase, Rm62, as partner of DSP1 in a 250‐kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3‐ to 12‐h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes. genesis 48:244–253, 2010. © 2010 Wiley‐Liss, Inc.     

Germ cell survival in C. elegans and C. remanei is affected when the dead box rna helicases Vbh‐1 or Cre‐VBH‐1 are silenced

The Vasa family of proteins comprises several conserved DEAD box RNA helicases important for mRNA regulation whose exact function in the germline is still unknown. In Caenorhabditis elegans, there are six known members of the Vasa family, and all of them are associated with P granules. One of these proteins, VBH‐1, is important for oogenesis, spermatogenesis, embryo development, and the oocyte/sperm switch in this nematode. We decided to extend our previous work in C. elegans to sibling species Caenorhabditis remanei to understand what is the function of the VBH‐1 homolog in this gonochoristic species. We found that Cre‐VBH‐1 is present in the cytoplasm of germ cells and it remains associated with P granules throughout the life cycle of C. remanei. Several aspects between VBH‐1 and Cre‐VBH‐1 function are conserved like their role during oogenesis, spermatogenesis, and embryonic development. However, Cre‐vbh‐1 silencing in C. remanei had a stronger effect on spermatogenesis and spermatid activation than in C. elegans. An unexpected finding was that silencing of vbh‐1 in the C. elegans caused a decrease in germ cell apoptosis in the hermaphrodite gonad, while silencing of Cre‐vbh‐1 in C. remanei elicited germ cell apoptosis in the male gonad. These data suggest that VBH‐1 might play a role in germ cell survival in both species albeit it appears to have an opposite role in each one. genesis 1–18 2012. © 2012 Wiley Periodicals, Inc.