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author:("wattie, Ruddy")
1.  Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005 
PLoS ONE  2014;9(6):e99076.
The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.
doi:10.1371/journal.pone.0099076
PMCID: PMC4051694  PMID: 24914774
2.  Proteomic Study of HPV-Positive Head and Neck Cancers: Preliminary Results 
BioMed Research International  2014;2014:430906.
Human papillomavirus (HPV) was recently recognized as a new risk factor for head and neck squamous cell carcinoma. For oropharyngeal cancers, an HPV+ status is associated with better prognosis in a subgroup of nonsmokers and nondrinkers. However, HPV infection is also involved in the biology of head and neck carcinoma (HNC) in patients with a history of tobacco use and/or alcohol consumption. Thus, the involvement of HPV infection in HN carcinogenesis remains unclear, and further studies are needed to identify and analyze HPV-specific pathways that are involved in this process. Using a quantitative proteomics-based approach, we compared the protein expression profiles of two HPV+ HNC cell lines and one HPV− HNC cell line. We identified 155 proteins that are differentially expressed (P < 0.01) in these three lines. Among the identified proteins, prostate stem cell antigen (PSCA) was upregulated and eukaryotic elongation factor 1 alpha (EEF1α) was downregulated in the HPV+ cell lines. Immunofluorescence and western blotting analyses confirmed these results. Moreover, PSCA and EEF1α were differentially expressed in two clinical series of 50 HPV+ and 50 HPV− oral cavity carcinomas. Thus, our study reveals for the first time that PSCA and EEF1α are associated with the HPV-status, suggesting that these proteins could be involved in HPV-associated carcinogenesis.
doi:10.1155/2014/430906
PMCID: PMC3955617  PMID: 24719866
3.  How Does Pollen Chemistry Impact Development and Feeding Behaviour of Polylectic Bees? 
PLoS ONE  2014;9(1):e86209.
Larvae and imagos of bees rely exclusively on floral rewards as a food source but host-plant range can vary greatly among bee species. While oligolectic species forage on pollen from a single family of host plants, polylectic bees, such as bumblebees, collect pollen from many families of plants. These polylectic species contend with interspecific variability in essential nutrients of their host-plants but we have only a limited understanding of the way in which chemicals and chemical combinations influence bee development and feeding behaviour. In this paper, we investigated five different pollen diets (Calluna vulgaris, Cistus sp., Cytisus scoparius, Salix caprea and Sorbus aucuparia) to determine how their chemical content affected bumblebee colony development and pollen/syrup collection. Three compounds were used to characterise pollen content: polypeptides, amino acids and sterols. Several parameters were used to determine the impact of diet on micro-colonies: (i) Number and weight of larvae (total and mean weight of larvae), (ii) weight of pollen collected, (iii) pollen efficacy (total weight of larvae divided by weight of the pollen collected) and (iv) syrup collection. Our results show that pollen collection is similar regardless of chemical variation in pollen diet while syrup collection is variable. Micro-colonies fed on S. aucuparia and C. scoparius pollen produced larger larvae (i.e. better mates and winter survivors) and fed less on nectar compared to the other diets. Pollen from both of these species contains 24-methylenecholesterol and high concentrations of polypeptides/total amino acids. This pollen nutritional “theme” seems therefore to promote worker reproduction in B. terrestris micro-colonies and could be linked to high fitness for queenright colonies. As workers are able to selectively forage on pollen of high chemical quality, plants may be evolutionarily selected for their pollen content, which might attract and increase the degree of fidelity of generalist pollinators, such as bumblebees.
doi:10.1371/journal.pone.0086209
PMCID: PMC3897652  PMID: 24465963
4.  Proteomic Characterization of Murid Herpesvirus 4 Extracellular Virions 
PLoS ONE  2013;8(12):e83842.
Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi’s sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.
doi:10.1371/journal.pone.0083842
PMCID: PMC3875534  PMID: 24386290
5.  Expression of calgranulin A/B heterodimer after acute inhalation of endotoxin: proteomic approach and validation 
Background
The acute inhalation of endotoxin mimicks several aspects of the inflammation related to chronic obstructive pulmonary disease (COPD). The aim of the current study was to identify and to validate biomarkers of endotoxin-induced airways’ inflammation.
Methods
The cellular count in the induced-sputum, was measured before and after an inhalation of 20 mcg endotoxin, in 8 healthy volunteers. A proteomic analysis was applied to identify the more relevant proteins expression, before measurement by ELISA. The amplitude and the repeatability of the markers were evaluated among another population of 12 healthy subjects.
Results
There was a significant rise of viable cells (p <0.01), macrophages (p <0.05), and neutrophils (p <0.02) 24 hours after endotoxin inhalation, and of neutrophils (p <0.02) and lymphocytes (p <0.05) at 6 hours. Among the highest amplitude responses, the two dimensional electrophoretic separation shown proteolytic activity and overexpression of protein spots. By MALDI-TOF mass spectrometry, the last were identified as calgranulin A and B. The expression of the bioactive A/B heterodimeric complex was confirmed by ELISA both in the sputum (p <0.01) and at the blood level (p <0.01). The intra-subject repeatability of the sputum calgranulin A/B was highly significant (p <0.0001).
Conclusion
In healthy subjects, the inhalation of endotoxin induced expression of sputum calgranulin A/B that could be a biomarker of the endotoxin response/exposure.
doi:10.1186/1471-2466-13-65
PMCID: PMC4225611  PMID: 24237763
Endotoxin; Inflammation; Sputum; Proteomic; Calgranulin; Neutrophils
6.  Shotgun Redox Proteomics: Identification and Quantitation of Carbonylated Proteins in the UVB-Resistant Marine Bacterium, Photobacterium angustum S14 
PLoS ONE  2013;8(7):e68112.
UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems.
doi:10.1371/journal.pone.0068112
PMCID: PMC3706606  PMID: 23874515
7.  Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions 
Journal of Virology  2012;86(21):11567-11580.
Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host's antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi's sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.
doi:10.1128/JVI.00456-12
PMCID: PMC3486312  PMID: 22896609
8.  The IL-10 homologue encoded by cyprinid herpesvirus 3 is essential neither for viral replication in vitro nor for virulence in vivo 
Veterinary Research  2013;44(1):53.
Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae, is the causative agent of a lethal disease in common and koi carp. CyHV-3 ORF134 encodes an interleukin-10 (IL-10) homologue. The present study was devoted to this ORF. Transcriptomic analyses revealed that ORF134 is expressed as a spliced gene belonging to the early-late class. Proteomic analyses of CyHV-3 infected cell supernatant demonstrated that the ORF134 expression product is one of the most abundant proteins of the CyHV-3 secretome. To investigate the role of ORF134 in viral replication in vitro and in virulence in vivo, a deleted strain and a derived revertant strain were produced using BAC cloning technologies. The recombinant ORF134 deleted strain replicated in vitro comparably to the parental and the revertant strains. Infection of fish by immersion in water containing the virus induced comparable CyHV-3 disease for the three virus genotypes tested (wild type, deleted and revertant). Quantification of viral DNA by real time TaqMan PCR (in the gills and the kidney) and analysis of carp cytokine expression (in the spleen) by RT-qPCR at different times post-infection did not revealed any significant difference between the groups of fish infected with the three virus genotypes. Similarly, histological examination of the gills and the kidney of infected fish revealed no significant differences between fish infected with ORF134 deleted virus versus fish infected with the control parental or revertant strains. All together, the results of the present study demonstrate that the IL-10 homologue encoded by CyHV-3 is essential neither for viral replication in vitro nor for virulence in common carp.
doi:10.1186/1297-9716-44-53
PMCID: PMC3750702  PMID: 23865540
9.  FSHD Myotubes with Different Phenotypes Exhibit Distinct Proteomes 
PLoS ONE  2012;7(12):e51865.
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder linked to a contraction of the D4Z4 repeat array in the 4q35 subtelomeric region. This deletion induces epigenetic modifications that affect the expression of several genes located in the vicinity. In each D4Z4 element, we identified the double homeobox 4 (DUX4) gene. DUX4 expresses a transcription factor that plays a major role in the development of FSHD through the initiation of a large gene dysregulation cascade that causes myogenic differentiation defects, atrophy and reduced response to oxidative stress. Because miRNAs variably affect mRNA expression, proteomic approaches are required to define the dysregulated pathways in FSHD. In this study, we optimized a differential isotope protein labeling (ICPL) method combined with shotgun proteomic analysis using a gel-free system (2DLC-MS/MS) to study FSHD myotubes. Primary CD56+ FSHD myoblasts were found to fuse into myotubes presenting various proportions of an atrophic or a disorganized phenotype. To better understand the FSHD myogenic defect, our improved proteomic procedure was used to compare predominantly atrophic or disorganized myotubes to the same matching healthy control. FSHD atrophic myotubes presented decreased structural and contractile muscle components. This phenotype suggests the occurrence of atrophy-associated proteolysis that likely results from the DUX4-mediated gene dysregulation cascade. The skeletal muscle myosin isoforms were decreased while non-muscle myosin complexes were more abundant. In FSHD disorganized myotubes, myosin isoforms were not reduced, and increased proteins were mostly involved in microtubule network organization and myofibrillar remodeling. A common feature of both FSHD myotube phenotypes was the disturbance of several caveolar proteins, such as PTRF and MURC. Taken together, our data suggest changes in trafficking and in the membrane microdomains of FSHD myotubes. Finally, the adjustment of a nuclear fractionation compatible with mass spectrometry allowed us to highlight alterations of proteins involved in mRNA processing and stability.
doi:10.1371/journal.pone.0051865
PMCID: PMC3525578  PMID: 23272181
10.  Purification and Characterization of the Acetone Carboxylase of Cupriavidus metallidurans Strain CH34 
Applied and Environmental Microbiology  2012;78(12):4516-4518.
Acetone carboxylase (Acx) is a key enzyme involved in the biodegradation of acetone by bacteria. Except for the Helicobacteraceae family, genome analyses revealed that bacteria that possess an Acx, such as Cupriavidus metallidurans strain CH34, are associated with soil. The Acx of CH34 forms the heterohexameric complex α2β2γ2 and can carboxylate only acetone and 2-butanone in an ATP-dependent reaction to acetoacetate and 3-keto-2-methylbutyrate, respectively.
doi:10.1128/AEM.07974-11
PMCID: PMC3370517  PMID: 22492439
11.  Differential proteomics and physiology of Pseudomonas putida KT2440 under filament-inducing conditions 
BMC Microbiology  2012;12:282.
Background
Pseudomonas putida exerts a filamentous phenotype in response to environmental stress conditions that are encountered during its natural life cycle. This study assessed whether P. putida filamentation could confer survival advantages. Filamentation of P. putida was induced through culturing at low shaking speed and was compared to culturing in high shaking speed conditions, after which whole proteomic analysis and stress exposure assays were performed.
Results
P. putida grown in filament-inducing conditions showed increased resistance to heat and saline stressors compared to non-filamented cultures. Proteomic analysis showed a significant metabolic change and a pronounced induction of the heat shock protein IbpA and recombinase RecA in filament-inducing conditions. Our data further indicated that the associated heat shock resistance, but not filamentation, was dependent of RecA.
Conclusions
This study provides insights into the altered metabolism of P. putida in filament-inducing conditions, and indicates that the formation of filaments could potentially be utilized by P. putida as a survival strategy in its hostile, recurrently changing habitat.
doi:10.1186/1471-2180-12-282
PMCID: PMC3538555  PMID: 23186381
Pseudomonas putida KT2440; Filamentation; Elongation; SOS response; RecA; Shaking speed; Stress resistance
12.  Proteome Analysis of the UVB-Resistant Marine Bacterium Photobacterium angustum S14 
PLoS ONE  2012;7(8):e42299.
The proteome of the marine bacterium Photobacterium angustum S14 was exposed to UVB and analyzed by the implementation of both the post-digest ICPL labeling method and 2D-DIGE technique using exponentially growing cells. A total of 40 and 23 proteins were quantified in all replicates using either the ICPL or 2D-DIGE methods, respectively. By combining both datasets from 8 biological replicates (4 biological replicates for each proteomics technique), 55 proteins were found to respond significantly to UVB radiation in P. angustum. A total of 8 UVB biomarkers of P. angustum were quantified in all replicates using both methods. Among them, the protein found to present the highest increase in abundance (almost a 3-fold change) was RecA, which is known to play a crucial role in the so-called recombinational repair process. We also observed a high number of antioxidants, transport proteins, metabolism-related proteins, transcription/translation regulators, chaperonins and proteases. We also discuss and compare the UVB response and global protein expression profiles obtained for two different marine bacteria with trophic lifestyles: the copiotroph P. angustum and oligotroph Sphingopyxis alaskensis.
doi:10.1371/journal.pone.0042299
PMCID: PMC3411663  PMID: 22870314
13.  Chemical Basis of Prey Recognition in Thamnophiine Snakes: The Unexpected New Roles of Parvalbumins 
PLoS ONE  2012;7(6):e39560.
Detecting and locating prey are key to predatory success within trophic chains. Predators use various signals through specialized visual, olfactory, auditory or tactile sensory systems to pinpoint their prey. Snakes chemically sense their prey through a highly developed auxiliary olfactory sense organ, the vomeronasal organ (VNO). In natricine snakes that are able to feed on land and water, the VNO plays a critical role in predatory behavior by detecting cues, known as vomodors, which are produced by their potential prey. However, the chemical nature of these cues remains unclear. Recently, we demonstrated that specific proteins–parvalbumins–present in the cutaneous mucus of the common frog (Rana temporaria) may be natural chemoattractive proteins for these snakes. Here, we show that parvalbumins and parvalbumin-like proteins, which are mainly intracellular, are physiologically present in the epidermal mucous cells and mucus of several frog and fish genera from both fresh and salt water. These proteins are located in many tissues and function as Ca2+ buffers. In addition, we clarified the intrinsic role of parvalbumins present in the cutaneous mucus of amphibians and fishes. We demonstrate that these Ca2+-binding proteins participate in innate bacterial defense mechanisms by means of calcium chelation. We show that these parvalbumins are chemoattractive for three different thamnophiine snakes, suggesting that these chemicals play a key role in their prey-recognition mechanism. Therefore, we suggest that recognition of parvalbumin-like proteins or other calcium-binding proteins by the VNO could be a generalized prey-recognition process in snakes. Detecting innate prey defense mechanism compounds may have driven the evolution of this predator-prey interaction.
doi:10.1371/journal.pone.0039560
PMCID: PMC3384659  PMID: 22761824
14.  A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16▿† 
Applied and Environmental Microbiology  2011;77(24):8754-8764.
The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the phenylurea herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested phenylurea herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a phenylurea herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.
doi:10.1128/AEM.06162-11
PMCID: PMC3233098  PMID: 22003008
15.  Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells 
Veterinary Research  2011;42(1):92.
Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.
doi:10.1186/1297-9716-42-92
PMCID: PMC3166907  PMID: 21816061
16.  Identification and localization of the structural proteins of anguillid herpesvirus 1 
Veterinary Research  2011;42(1):105.
Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.
doi:10.1186/1297-9716-42-105
PMCID: PMC3203048  PMID: 21975111
17.  Overexpression, purification, crystallization and crystallographic analysis of CopK of Cupriavidus metallidurans  
Overexpression, purification and crystallization of C. metallidurans CopK allowed the collection of a complete data set to 2.2 Å resolution.
CopK of Cupriavidus metallidurans is a 93-amino-acid protein whose mature form (73 amino acids) has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li2SO4, 20%(w/v) glycerol, 13%(w/v) PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 57.53, b = 128.65, c = 49.77 Å, and diffract to 2.2 Å resolution using synchrotron radiation.
doi:10.1107/S174430910502316X
PMCID: PMC1978110  PMID: 16511169
CopK; copper; Ralstonia; Cupriavidus metallidurans
18.  Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor–induced Nur77 expression and apoptosis 
The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH2 terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.
doi:10.1084/jem.20042034
PMCID: PMC2212830  PMID: 15738054
19.  Key Role of Ser562/661 in Snf1-Dependent Regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis 
Molecular and Cellular Biology  2004;24(10):4083-4091.
Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes. We demonstrate that KlSnf1p and KlCat8p from K. lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p. Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation. Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site. Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed. A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect. Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1. The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S. cerevisiae. The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p. Unlike that of S. cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K. lactis.
doi:10.1128/MCB.24.10.4083-4091.2004
PMCID: PMC400452  PMID: 15121831
20.  The yggH Gene of Escherichia coli Encodes a tRNA (m7G46) Methyltransferase 
Journal of Bacteriology  2003;185(10):3238-3243.
We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-l-methionine-dependent formation of N7-methylguanosine at position 46 (m7G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m7G46) methyltransferase activity.
doi:10.1128/JB.185.10.3238-3243.2003
PMCID: PMC154064  PMID: 12730187
21.  Proteomics as the Tool to Search for Lung Disease Markers in Bronchoalveolar Lavage 
Disease Markers  2002;17(4):271-284.
Most lung disorders are known to be associated to considerable modifications of surfactant composition. Numerous of these abnormalities have been exploited in the past to diagnose lung diseases, allowing proper treatment and follow-up. Diagnosis was then based on phospholipid content, surface tension and cytological features of the epithelial lining fluid (ELF), sampled by bronchoalveolar lavage (BAL) during fiberoscopic bronchoscopy. Today, it appears that the protein content of ELF displays a remarkably high complexity, not only due to the wide variety of the proteins it contains but also because of the great diversity of their cellular origins. The significance of the use of proteome analysis of BAL fluid for the search for new lung disease marker proteins and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.
doi:10.1155/2001/607263
PMCID: PMC3850871  PMID: 11790894
22.  Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells 
Nucleic Acids Research  2000;28(4):868-874.
The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [125I]ODN and by photolabelling of living cells with a [125I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by the two methods. Its labelling was saturable and competed for by unlabelled ODN of various sequences and irrespective of the presence of a phosphodiester or phosphorothioate backbone. This protein remained sedimentable after carbonate extraction, indicating strong membrane association. About half of the total cell amount resisted extensive surface proteolysis, suggesting a dual localisation at the plasma membrane and cytoplasmic vesicles. The protein was purified using a biotinylated ODN-benzophenone conjugate by photocrosslinking followed by streptavidin affinity purification. A sequence obtained by Edman degradation showed no homology with known proteins. Using anti-peptide antisera, labelling by western blotting revealed at 66 kDa a band with comparable properties as found by ligand blotting. Thus, a new membrane protein acting as an ODN receptor has been demonstrated.
PMCID: PMC102580  PMID: 10648777
23.  Phosphorylation of the Human T-Cell Leukemia Virus Type 1 Transactivator Tax on Adjacent Serine Residues Is Critical for Tax Activation 
Journal of Virology  1999;73(1):738-745.
The Tax transactivator protein of human T-cell leukemia virus type 1 (HTLV-1) plays a central role in the activation of viral gene expression. In addition, Tax is capable of activating the expression of specific cellular genes and is involved in the transformation of T-lymphocytes resulting in the development of adult T-cell leukemia. Tax is a phosphoprotein that colocalizes in nuclear bodies with RNA polymerase II, splicing complexes, and specific transcription factors including members of the ATF/CREB and NF-κB families. In this study, we identified adjacent serine residues at positions 300 and 301 in the carboxy terminus of Tax as the major sites for phosphorylation. Phosphorylation of at least one of these serine residues is required for Tax localization in nuclear bodies and for Tax-mediated activation of gene expression via both the ATF/CREB and NF-κB pathways. Introduction of amino acid substitutions which are phosphoserine mimetics at positions 300 and 301 restored the ability of a phosphorylation-defective Tax mutant to form nuclear bodies and to activate gene expression. These studies define sites for regulatory phosphorylation events in Tax which are critical for its ability to activate gene transcription.
PMCID: PMC103881  PMID: 9847380

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