43 results on '"Encheva V"'
Search Results
2. Protein expression diversity amongst serovars of Salmonella enterica
- Author
-
Encheva, V., Wait, R., Begum, S., Gharbia, S.E., and Shah, H.N.
- Subjects
Salmonella -- Comparative analysis ,Salmonella -- Varieties ,Salmonella -- Physiological aspects ,Biological diversity -- Research ,Proteomics -- Research ,Biological sciences - Abstract
Salmonella enterica is one of the most extensively studied bacterial species in terms of physiology, genetics, cell culture and development. As a very diverse group, the serovars of S. enterica display a spectrum of host specificities ranging from a broad host range to strictly host-adapted variants. This study utilized a classic proteomic approach combining 2D gel electrophoresis and mass spectrometry for the comparative analysis of the proteomes of serovars Typhimurium, Enteritidis, Choleraesuis, Pullorum and Dublin. The comparative analysis revealed species-specific protein factors with no significant change in expression amongst all isolates, as well as proteins with fluctuating expression levels between serovars and strains. Examples include an isoform of SodA specific for serovar Typhimurium, the third isoform of the lysine arginine ornithine (LAO)-binding amino acid transporter specific for serovar Pullorum, and the enzyme GabD found to be unique to serovar Choleraesuis. Overall the study demonstrated the importance of using multiple isolates when characterizing the expression patterns of bacteria in order to account for the intrinsic diversity of a bacterial population and revealed several factors with potential roles in host adaptation and pathogenicity of the serovars of S. enterica.
- Published
- 2007
3. Field evaluation of somaclonal variation in sunflower (Helianthus annuus L) and its application for crop improvement
- Author
-
Encheva, J., Köhler, H., Friedt, W., Tsvetkova, F., Ivanov, P., Encheva, V., and Shindrova, P.
- Published
- 2003
- Full Text
- View/download PDF
4. Sunflower commercial hybrid Yana, developed with mutant restorer line R12003
- Author
-
Encheva, J., primary, Shindrova, P., additional, Encheva, V., additional, and Penchev, E., additional
- Published
- 2012
- Full Text
- View/download PDF
5. Mutant sunflower line R12003, produced through in vitro mutagenesis
- Author
-
Encheva, J., primary, Shindrova, P., additional, Encheva, V., additional, and Valkova, D., additional
- Published
- 2012
- Full Text
- View/download PDF
6. New sunflower restorer lines developed by direct organogenesis method from interspecific cross Helianthus annuus L. (cv. Albena) × Helianthus salicifolius L.: Disease resistance, combining ability
- Author
-
Encheva, J., primary, Christov, M., additional, Shindrova, P., additional, Drumeva, M., additional, and Encheva, V., additional
- Published
- 2006
- Full Text
- View/download PDF
7. MUTANT SUNFLOWER LINE R 12003, PRODUCED THROUGH in vitro MUTAGENESIS.
- Author
-
Encheva, J., Shindrova, P., Encheva, V., and Valkova, D.
- Subjects
SUNFLOWER genetics ,MUTAGENESIS ,PLANT mutation ,PLANT embryology ,CHLOROPHYLL ,PLANT morphology ,BOTANICAL chemistry - Abstract
Immature sunflower ( Helianthus annuus L.) zygotic embryos of sunflower fertility restorer line R 2574 were treated with ultrasound before planting on the embryo culture medium. As a result some chlorophyll, leaf and inflorescence mutations were observed. New sunflower forms with inherited morphological, biochemical and phytopatological changes were obtained through selection and self-pollination. The line R 12003, subjected to investigation in this study, was characterized with significant changes concerning most of the studied characters. The contribution of the present research connected to the investigated mutation was the appearance of resistance to the parasite broomrape. The increased number of seeds per head in line R 12003, increased oil content in seed and 100% resistance to the parasite Orobanche are the desired combination in the breeding programme of sunflower. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
8. NEW SUNFLOWER RESTORER LINES DEVELOPED BY DIRECT ORGANOGENESIS METHOD FROM INTERSPECIFIC CROSS Helianthus annuus L. (cv. ALBENA) x Helianthus salicifolius L.-DISEASE RESISTANCE, COMBINING ABILITY.
- Author
-
Encheva, J., Christov, M., Shindrova, P., Drumeva, M., and Encheva, V.
- Subjects
MORPHOGENESIS ,SUNFLOWERS ,DISEASE resistance of plants ,PHOMOPSIS ,PHOMA ,ALTERNARIA ,PLANT hybridization ,PLANT breeding - Abstract
Copyright of Helia is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
- Published
- 2006
- Full Text
- View/download PDF
9. Antimeasles immunoglobulin G in sera of patients with otosclerosis is lower than that in healthy people.
- Author
-
Lolov, Stephan R., Encheva, Vessela I., Kyurkchiev, Stanimir D., Edrev, George E., Kehayov, Ivan R., Lolov, S R, Encheva, V I, Kyurkchiev, S D, Edrev, G E, and Kehayov, I R
- Published
- 2001
- Full Text
- View/download PDF
10. Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique
- Author
-
Appleton Hazel, Arnold Cath, Encheva Vesela, Karlsson Roger, Chooneea Darren, and Shah Haroun
- Subjects
Microbiology ,QR1-502 - Abstract
Abstract Background Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches. Results In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. Conclusion Using a multi-step digest approach the LPI™ technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI™ technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for Salmonella Typhimurium.
- Published
- 2010
- Full Text
- View/download PDF
11. Proteome analysis of serovars Typhimurium and Pullorum of Salmonella enterica subspecies I
- Author
-
Begum Shajna, Gharbia Saheer E, Wait Robin, Encheva Vesela, and Shah Haroun N
- Subjects
Microbiology ,QR1-502 - Abstract
Abstract Background Salmonella enterica subspecies I includes several closely related serovars which differ in host ranges and ability to cause disease. The basis for the diversity in host range and pathogenic potential of the serovars is not well understood, and it is not known how host-restricted variants appeared and what factors were lost or acquired during adaptations to a specific environment. Differences apparent from the genomic data do not necessarily correspond to functional proteins and more importantly differential regulation of otherwise identical gene content may play a role in the diverse phenotypes of the serovars of Salmonella. Results In this study a comparative analysis of the cytosolic proteins of serovars Typhimurium and Pullorum was performed using two-dimensional gel electrophoresis and the proteins of interest were identified using mass spectrometry. An annotated reference map was created for serovar Typhimurium containing 233 entries, which included many metabolic enzymes, ribosomal proteins, chaperones and many other proteins characteristic for the growing cell. The comparative analysis of the two serovars revealed a high degree of variation amongst isolates obtained from different sources and, in some cases, the variation was greater between isolates of the same serovar than between isolates with different sero-specificity. However, several serovar-specific proteins, including intermediates in sulphate utilisation and cysteine synthesis, were also found despite the fact that the genes encoding those proteins are present in the genomes of both serovars. Conclusion Current microbial proteomics are generally based on the use of a single reference or type strain of a species. This study has shown the importance of incorporating a large number of strains of a species, as the diversity of the proteome in the microbial population appears to be significantly greater than expected. The characterisation of a diverse selection of strains revealed parts of the proteome of S. enterica that alter their expression while others remain stable and allowed for the identification of serovar-specific factors that have so far remained undetected by other methods.
- Published
- 2005
- Full Text
- View/download PDF
12. Impact of fed-batch process intensification on the productivity and product quality of two CHO cell lines expressing unique novel molecular format proteins.
- Author
-
Wolnick NQ, Dickson MR, Webster TA, Connolly RP, Fernandes N, Encheva V, Crittenden H, Hodgkins J, Hadley BC, Palermo G, Hendrick SJ, Newell RA, Gray G, Siltanen C, Armstrong J, Downey BJ, and Mason C
- Subjects
- CHO Cells, Animals, Antibodies, Bispecific biosynthesis, Batch Cell Culture Techniques, Cricetinae, Recombinant Proteins biosynthesis, Cricetulus, Bioreactors
- Abstract
While monospecific antibodies have long been the foundational offering of protein therapeutics, recent advancements in antibody engineering have allowed for the development of far more complex antibody structures. Novel molecular format (NMF) proteins, such as bispecific antibodies (BsAbs), are structures capable of multispecific binding, allowing for expanded therapeutic functionality. As demand for NMF proteins continues to rise, biomanufacturers face the challenge of increasing bioreactor process productivity while simultaneously maintaining consistent product quality. This challenge is exacerbated when producing structurally complex proteins with asymmetric modalities, as seen in NMFs. In this study, the impact of a high inoculation density (HID) fed-batch process on the productivity and product quality attributes of two CHO cell lines expressing unique NMFs, a monospecific antibody with an Fc-fusion protein and a bispecific antibody, compared to low inoculation density (LID) platform fed-batch processes was evaluated. It was observed that an intensified platform fed-batch process increased product concentrations by 33 and 109% for the two uniquely structured complex proteins in a shorter culture duration while maintaining similar product quality attributes to traditional fed-batch processes., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
13. p97/VCP targets Toxoplasma gondii vacuoles for parasite restriction in interferon-stimulated human cells.
- Author
-
Clough B, Fisch D, Mize TH, Encheva V, Snijders A, and Frickel E-M
- Subjects
- Animals, Humans, Interferons metabolism, Vacuoles metabolism, Endothelial Cells, Interferon-gamma, Valosin Containing Protein metabolism, Toxoplasma metabolism, Parasites
- Abstract
Importance: Toxoplasma gondii (Tg) is a ubiquitous parasitic pathogen, infecting about one-third of the global population. Tg is controlled in immunocompetent people by mechanisms that are not fully understood. Tg infection drives the production of the inflammatory cytokine interferon gamma (IFNγ), which upregulates intracellular anti-pathogen defense pathways. In this study, we describe host proteins p97/VCP, UBXD1, and ANKRD13A that control Tg at the parasitophorous vacuole (PV) in IFNγ-stimulated endothelial cells. p97/VCP is an ATPase that interacts with a network of cofactors and is active in a wide range of ubiquitin-dependent cellular processes. We demonstrate that PV ubiquitination is a pre-requisite for recruitment of these host defense proteins, and their deposition directs Tg PVs to acidification in endothelial cells. We show that p97/VCP universally targets PVs in human cells and restricts Tg in different human cell types. Overall, these findings reveal new players of intracellular host defense of a vacuolated pathogen., Competing Interests: The authors declare no conflict of interest.
- Published
- 2023
- Full Text
- View/download PDF
14. PIM1 controls GBP1 activity to limit self-damage and to guard against pathogen infection.
- Author
-
Fisch D, Pfleiderer MM, Anastasakou E, Mackie GM, Wendt F, Liu X, Clough B, Lara-Reyna S, Encheva V, Snijders AP, Bando H, Yamamoto M, Beggs AD, Mercer J, Shenoy AR, Wollscheid B, Maslowski KM, Galej WP, and Frickel EM
- Subjects
- Humans, Virulence Factors metabolism, Macrophages immunology, 14-3-3 Proteins metabolism, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Immunity, Innate, Interferon-gamma metabolism, Proto-Oncogene Proteins c-pim-1 metabolism, Toxoplasma, Toxoplasmosis immunology, Host-Pathogen Interactions immunology
- Abstract
Disruption of cellular activities by pathogen virulence factors can trigger innate immune responses. Interferon-γ (IFN-γ)-inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense by attacking intracellular pathogens and by inducing programmed cell death. Working in human macrophages, we discovered that GBP1 expression in the absence of IFN-γ killed the cells and induced Golgi fragmentation. IFN-γ exposure improved macrophage survival through the activity of the kinase PIM1. PIM1 phosphorylated GBP1, leading to its sequestration by 14-3-3σ, which thereby prevented GBP1 membrane association. During Toxoplasma gondii infection, the virulence protein TgIST interfered with IFN-γ signaling and depleted PIM1, thereby increasing GBP1 activity. Although infected cells can restrain pathogens in a GBP1-dependent manner, this mechanism can protect uninfected bystander cells. Thus, PIM1 can provide a bait for pathogen virulence factors, guarding the integrity of IFN-γ signaling.
- Published
- 2023
- Full Text
- View/download PDF
15. Metabolic turnover and dynamics of modified ribonucleosides by 13 C labeling.
- Author
-
Gameiro PA, Encheva V, Dos Santos MS, MacRae JI, and Ule J
- Subjects
- Guanosine chemistry, Guanosine metabolism, Guanosine pharmacology, Isotope Labeling, Tandem Mass Spectrometry, Adenosine chemistry, Adenosine metabolism, Adenosine pharmacology, Carbon Isotopes chemistry, Carbon Isotopes pharmacology, Guanosine analogs & derivatives, RNA chemistry, RNA metabolism, RNA Processing, Post-Transcriptional
- Abstract
Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a
13 C labeling approach, called13 C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6 A) versus 7-methylguanosine (m7 G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m6 2 A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m6 2 A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus,13 C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
16. Systems level profiling of chemotherapy-induced stress resolution in cancer cells reveals druggable trade-offs.
- Author
-
Saavedra-García P, Roman-Trufero M, Al-Sadah HA, Blighe K, López-Jiménez E, Christoforou M, Penfold L, Capece D, Xiong X, Miao Y, Parzych K, Caputo VS, Siskos AP, Encheva V, Liu Z, Thiel D, Kaiser MF, Piazza P, Chaidos A, Karadimitris A, Franzoso G, Snijders AP, Keun HC, Oyarzún DA, Barahona M, and Auner HW
- Subjects
- Antineoplastic Agents pharmacology, Autophagy physiology, Cell Line, Tumor, Humans, Metabolome genetics, Mitochondria metabolism, Multiple Myeloma metabolism, Neoplasms metabolism, Neoplasms physiopathology, Proteasome Inhibitors pharmacology, Proteolysis, Proteome genetics, Systems Analysis, Transcriptome genetics, Neoplasms drug therapy, Stress, Physiological drug effects
- Abstract
Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization., Competing Interests: Competing interest statement: H.W.A. acknowledges research support by Amgen. Amgen did not have a role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript., (Copyright © 2021 the Author(s). Published by PNAS.)
- Published
- 2021
- Full Text
- View/download PDF
17. Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12.
- Author
-
Khan OM, Almagro J, Nelson JK, Horswell S, Encheva V, Keyan KS, Clurman BE, Snijders AP, and Behrens A
- Subjects
- Biocatalysis, Drug Resistance, Neoplasm, HCT116 Cells, HEK293 Cells, Humans, Lysine metabolism, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Protein Binding, Protein Processing, Post-Translational, Protein Stability, RNA, Small Interfering metabolism, Substrate Specificity, Ubiquitin-Conjugating Enzymes metabolism, Carrier Proteins metabolism, F-Box-WD Repeat-Containing Protein 7 metabolism, Proteasome Endopeptidase Complex metabolism, Proteolysis, Ubiquitin-Protein Ligases metabolism, Ubiquitination
- Abstract
The tumour suppressor FBW7 is a substrate adaptor for the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), that targets several oncoproteins for proteasomal degradation. FBW7 is widely mutated and FBW7 protein levels are commonly downregulated in cancer. Here, using an shRNA library screen, we identify the HECT-domain E3 ubiquitin ligase TRIP12 as a negative regulator of FBW7 stability. We find that SCF
FBW7 -mediated ubiquitylation of FBW7 occurs preferentially on K404 and K412, but is not sufficient for its proteasomal degradation, and in addition requires TRIP12-mediated branched K11-linked ubiquitylation. TRIP12 inactivation causes FBW7 protein accumulation and increased proteasomal degradation of the SCFFBW7 substrate Myeloid Leukemia 1 (MCL1), and sensitizes cancer cells to anti-tubulin chemotherapy. Concomitant FBW7 inactivation rescues the effects of TRIP12 deficiency, confirming FBW7 as an essential mediator of TRIP12 function. This work reveals an unexpected complexity of FBW7 ubiquitylation, and highlights branched ubiquitylation as an important signalling mechanism regulating protein stability.- Published
- 2021
- Full Text
- View/download PDF
18. Human GBP1 Differentially Targets Salmonella and Toxoplasma to License Recognition of Microbial Ligands and Caspase-Mediated Death.
- Author
-
Fisch D, Clough B, Domart MC, Encheva V, Bando H, Snijders AP, Collinson LM, Yamamoto M, Shenoy AR, and Frickel EM
- Subjects
- Cell Death immunology, HEK293 Cells, Humans, Inflammasomes immunology, Interferon-gamma pharmacology, Ligands, Salmonella Infections immunology, Salmonella Infections microbiology, THP-1 Cells, Toxoplasma genetics, Toxoplasmosis immunology, Toxoplasmosis microbiology, Vacuoles immunology, Caspases immunology, GTP-Binding Proteins immunology, Salmonella typhimurium immunology, Toxoplasma immunology
- Abstract
Interferon-inducible guanylate-binding proteins (GBPs) promote cell-intrinsic defense through host cell death. GBPs target pathogens and pathogen-containing vacuoles and promote membrane disruption for release of microbial molecules that activate inflammasomes. GBP1 mediates pyroptosis or atypical apoptosis of Salmonella Typhimurium (STm)- or Toxoplasma gondii (Tg)- infected human macrophages, respectively. The pathogen-proximal detection-mechanisms of GBP1 remain poorly understood, as humans lack functional immunity-related GTPases (IRGs) that assist murine Gbps. Here, we establish that GBP1 promotes the lysis of Tg-containing vacuoles and parasite plasma membranes, releasing Tg-DNA. In contrast, we show GBP1 targets cytosolic STm and recruits caspase-4 to the bacterial surface for its activation by lipopolysaccharide (LPS), but does not contribute to bacterial vacuole escape. Caspase-1 cleaves and inactivates GBP1, and a cleavage-deficient GBP1
D192E mutant increases caspase-4-driven pyroptosis due to the absence of feedback inhibition. Our studies elucidate microbe-specific roles of GBP1 in infection detection and its triggering of the assembly of divergent caspase signaling platforms., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
19. DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1.
- Author
-
Dirac-Svejstrup AB, Walker J, Faull P, Encheva V, Akimov V, Puglia M, Perkins D, Kümper S, Hunjan SS, Blagoev B, Snijders AP, Powell DJ, and Svejstrup JQ
- Subjects
- Aspartic Acid Proteases genetics, Binding Sites, CRISPR-Cas Systems, Cell Line, Gene Knockout Techniques, HEK293 Cells, Humans, Protein Biosynthesis, Proteolysis, Aspartic Acid Proteases metabolism, Nuclear Respiratory Factor 1 metabolism, Ubiquitin metabolism
- Abstract
The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
20. Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development.
- Author
-
Green JL, Wu Y, Encheva V, Lasonder E, Prommaban A, Kunzelmann S, Christodoulou E, Grainger M, Truongvan N, Bothe S, Sharma V, Song W, Pinzuti I, Uthaipibull C, Srichairatanakool S, Birault V, Langsley G, Schindelin H, Stieglitz B, Snijders AP, and Holder AA
- Subjects
- Humans, Plasmodium falciparum genetics, Protozoan Proteins genetics, Ubiquitin genetics, Merozoites metabolism, Plasmodium falciparum metabolism, Protozoan Proteins metabolism, Ubiquitin metabolism, Ubiquitination
- Abstract
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
- Full Text
- View/download PDF
21. Regulation of the RNAPII Pool Is Integral to the DNA Damage Response.
- Author
-
Tufegdžić Vidaković A, Mitter R, Kelly GP, Neumann M, Harreman M, Rodríguez-Martínez M, Herlihy A, Weems JC, Boeing S, Encheva V, Gaul L, Milligan L, Tollervey D, Conaway RC, Conaway JW, Snijders AP, Stewart A, and Svejstrup JQ
- Subjects
- DNA Repair, HEK293 Cells, Humans, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription, Genetic, Ubiquitination, Ultraviolet Rays, DNA Damage, RNA Polymerase II metabolism
- Abstract
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K
1268 ), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
22. Phenotypic proteomic profiling identifies a landscape of targets for circadian clock-modulating compounds.
- Author
-
Ray S, Lach R, Heesom KJ, Valekunja UK, Encheva V, Snijders AP, and Reddy AB
- Subjects
- Adenine analogs & derivatives, Adenine pharmacology, Anthracenes pharmacology, Cell Line, Tumor, Circadian Clocks drug effects, Circadian Rhythm genetics, Humans, Phenotype, Phosphorylation, Proteomics, Purines pharmacology, Roscovitine pharmacology, Signal Transduction drug effects, Signal Transduction genetics, Transcription Factors genetics, Circadian Clocks genetics, Circadian Rhythm drug effects, Drug Evaluation, Preclinical methods
- Abstract
Determining the exact targets and mechanisms of action of drug molecules that modulate circadian rhythms is critical to develop novel compounds to treat clock-related disorders. Here, we have used phenotypic proteomic profiling (PPP) to systematically determine molecular targets of four circadian period-lengthening compounds in human cells. We demonstrate that the compounds cause similar changes in phosphorylation and activity of several proteins and kinases involved in vital pathways, including MAPK, NGF, B-cell receptor, AMP-activated protein kinases (AMPKs), and mTOR signaling. Kinome profiling further indicated inhibition of CKId, ERK1/2, CDK2/7, TNIK, and MST4 kinases as a common mechanism of action for these clock-modulating compounds. Pharmacological or genetic inhibition of several convergent kinases lengthened circadian period, establishing them as novel circadian targets. Finally, thermal stability profiling revealed binding of the compounds to clock regulatory kinases, signaling molecules, and ubiquitination proteins. Thus, phenotypic proteomic profiling defines novel clock effectors that could directly inform precise therapeutic targeting of the circadian system in humans., (© 2019 Ray et al.)
- Published
- 2019
- Full Text
- View/download PDF
23. Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation.
- Author
-
Macpherson JA, Theisen A, Masino L, Fets L, Driscoll PC, Encheva V, Snijders AP, Martin SR, Kleinjung J, Barran PE, Fraternali F, and Anastasiou D
- Subjects
- Carrier Proteins chemistry, Carrier Proteins genetics, Cell Line, DNA Mutational Analysis, Enzyme Activators metabolism, Enzyme Inhibitors metabolism, Fructosediphosphates metabolism, Humans, Membrane Proteins chemistry, Membrane Proteins genetics, Molecular Dynamics Simulation, Phenylalanine metabolism, Protein Multimerization, Spectrum Analysis, Thyroid Hormones chemistry, Thyroid Hormones genetics, Thyroid Hormone-Binding Proteins, Allosteric Regulation, Carrier Proteins metabolism, Gene Expression Regulation, Enzymologic, Membrane Proteins metabolism, Thyroid Hormones metabolism
- Abstract
Several enzymes can simultaneously interact with multiple intracellular metabolites, however, how the allosteric effects of distinct ligands are integrated to coordinately control enzymatic activity remains poorly understood. We addressed this question using, as a model system, the glycolytic enzyme pyruvate kinase M2 (PKM2). We show that the PKM2 activator fructose 1,6-bisphosphate (FBP) alone promotes tetramerisation and increases PKM2 activity, but addition of the inhibitor L-phenylalanine (Phe) prevents maximal activation of FBP-bound PKM2 tetramers. We developed a method, AlloHubMat, that uses eigenvalue decomposition of mutual information derived from molecular dynamics trajectories to identify residues that mediate FBP-induced allostery. Experimental mutagenesis of these residues identified PKM2 variants in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings reveal residues involved in FBP-induced allostery that enable the integration of allosteric input from Phe and provide a paradigm for the coordinate regulation of enzymatic activity by simultaneous allosteric inputs., Competing Interests: JM, AT, LM, LF, PD, VE, AS, SM, JK, PB, FF, DA No competing interests declared, (© 2019, Macpherson et al.)
- Published
- 2019
- Full Text
- View/download PDF
24. Analysis of RNA polymerase II ubiquitylation and proteasomal degradation.
- Author
-
Tufegdzic Vidakovic A, Harreman M, Dirac-Svejstrup AB, Boeing S, Roy A, Encheva V, Neumann M, Wilson M, Snijders AP, and Svejstrup JQ
- Subjects
- Animals, DNA Damage, Humans, Mammals genetics, Mammals metabolism, Proteasome Endopeptidase Complex metabolism, Proteolysis, RNA Polymerase II antagonists & inhibitors, RNA Polymerase II genetics, Transcription, Genetic, Ultraviolet Rays, Yeasts enzymology, Yeasts genetics, Yeasts metabolism, RNA Polymerase II analysis, RNA Polymerase II metabolism, Ubiquitination
- Abstract
Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
25. LUBAC determines chemotherapy resistance in squamous cell lung cancer.
- Author
-
Ruiz EJ, Diefenbacher ME, Nelson JK, Sancho R, Pucci F, Chakraborty A, Moreno P, Annibaldi A, Liccardi G, Encheva V, Mitter R, Rosenfeldt M, Snijders AP, Meier P, Calzado MA, and Behrens A
- Subjects
- Adenocarcinoma of Lung drug therapy, Adenocarcinoma of Lung enzymology, Adenocarcinoma of Lung genetics, Adenocarcinoma of Lung pathology, Animals, Carcinoma, Squamous Cell drug therapy, Carcinoma, Squamous Cell genetics, Carcinoma, Squamous Cell pathology, Cisplatin pharmacology, Humans, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Lung Neoplasms pathology, Mice, Multienzyme Complexes genetics, Proto-Oncogene Proteins p21(ras) genetics, Proto-Oncogene Proteins p21(ras) metabolism, Carcinoma, Squamous Cell enzymology, Drug Resistance, Neoplasm, Lung Neoplasms enzymology, Multienzyme Complexes metabolism, Ubiquitination
- Abstract
Lung squamous cell carcinoma (LSCC) and adenocarcinoma (LADC) are the most common lung cancer subtypes. Molecular targeted treatments have improved LADC patient survival but are largely ineffective in LSCC. The tumor suppressor FBW7 is commonly mutated or down-regulated in human LSCC, and oncogenic KRasG12D activation combined with Fbxw7 inactivation in mice (KF model) caused both LSCC and LADC. Lineage-tracing experiments showed that CC10
+ , but not basal, cells are the cells of origin of LSCC in KF mice. KF LSCC tumors recapitulated human LSCC resistance to cisplatin-based chemotherapy, and we identified LUBAC-mediated NF-κB signaling as a determinant of chemotherapy resistance in human and mouse. Inhibition of NF-κB activation using TAK1 or LUBAC inhibitors resensitized LSCC tumors to cisplatin, suggesting a future avenue for LSCC patient treatment., (© 2019 Ruiz et al.)- Published
- 2019
- Full Text
- View/download PDF
26. SETDB1 Links the Meiotic DNA Damage Response to Sex Chromosome Silencing in Mice.
- Author
-
Hirota T, Blakeley P, Sangrithi MN, Mahadevaiah SK, Encheva V, Snijders AP, ElInati E, Ojarikre OA, de Rooij DG, Niakan KK, and Turner JMA
- Subjects
- Animals, Apoptosis, DNA Repair, Histone-Lysine N-Methyltransferase genetics, Histones metabolism, Male, Mice, Mice, Inbred C57BL, Tripartite Motif-Containing Protein 28 genetics, Tripartite Motif-Containing Protein 28 metabolism, Chromosome Pairing, DNA Damage, Gene Silencing, Histone Code, Histone-Lysine N-Methyltransferase metabolism
- Abstract
Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologs are transcriptionally inactivated by meiotic silencing, which serves a surveillance function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodeling and silencing fail, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1 and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis, and meiotic chromosome silencing., (Copyright © 2018 Francis Crick Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
27. Murine Gbp1 and Gbp2 are ubiquitinated independent of Toxoplasma gondii infection.
- Author
-
Encheva V, Foltz C, Snijders AP, and Frickel EM
- Subjects
- Animals, Embryo, Mammalian, Fibroblasts, Mice, Mice, Inbred C57BL, GTP-Binding Proteins metabolism, Toxoplasmosis, Ubiquitination
- Abstract
Objective: The intracellular parasite Toxoplasma gondii can invade any nucleated cell residing inside a parasitophorous vacuole (PV). Upon infection, the cytokine interferon gamma (IFNγ) is produced and elicits host defence mechanisms able to recognise the PV and destroy the parasite. Hereby, Guanylate binding proteins, ubiquitin and the E3 ubiquitin ligases Tripartite Motif Containing 21 (TRIM21) and TNF receptor associated factor 6 are targeted to the murine PV leading to its destruction. This study is the side product of research aiming to identify ubiquitinated substrates in a TRIM21-dependent fashion in murine cells infected with Toxoplasma., Results: We infected IFNγ-stimulated murine embryonic fibroblasts (MEFs) from either C57BL/6×129 wild-type (WT) mice or C57BL/6 TRIM21
-/- mice with Toxoplasma. Using mass spectrometry, we analysed proteins in both cell backgrounds presenting with the di-glycine remnant of ubiquitination. In addition, we compared peptide levels between WT and TRIM21-/- cells. In line with earlier reports, Gbp1 was expressed to higher levels in the C57BL/6×129 WT MEFs compared to the C57BL/6-only background TRIM21-/- MEFs. Protein expression differences in these different murine backgrounds thus precluded identification of TRIM21-dependent ubiquitinated substrates. Nevertheless, we identified and confirmed Gbp1 and Gbp2 as being ubiquitinated in a Toxoplasma-infection independent manner.- Published
- 2018
- Full Text
- View/download PDF
28. USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination.
- Author
-
Novellasdemunt L, Foglizzo V, Cuadrado L, Antas P, Kucharska A, Encheva V, Snijders AP, and Li VSW
- Subjects
- Amino Acid Sequence, Animals, Cell Differentiation drug effects, Cell Line, Tumor, Cell Proliferation drug effects, Colorectal Neoplasms pathology, HEK293 Cells, Humans, Mice, Organoids metabolism, Protein Binding drug effects, Protein Domains, Small Molecule Libraries pharmacology, Ubiquitin-Specific Peptidase 7 chemistry, Wnt Signaling Pathway drug effects, beta Catenin chemistry, beta-Transducin Repeat-Containing Proteins metabolism, Adenomatous Polyposis Coli Protein genetics, Colorectal Neoplasms genetics, Colorectal Neoplasms metabolism, Mutation genetics, Ubiquitin-Specific Peptidase 7 metabolism, Ubiquitination drug effects, beta Catenin metabolism
- Abstract
The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRCs), resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs., (Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
29. Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy.
- Author
-
Joachim J, Razi M, Judith D, Wirth M, Calamita E, Encheva V, Dynlacht BD, Snijders AP, O'Reilly N, Jefferies HBJ, and Tooze SA
- Subjects
- Apoptosis Regulatory Proteins, HEK293 Cells, Humans, Adaptor Proteins, Signal Transducing metabolism, Autophagy, Centrioles metabolism, Microtubule-Associated Proteins metabolism, Ubiquitination
- Abstract
Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
30. Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway.
- Author
-
Rapisarda V, Borghesan M, Miguela V, Encheva V, Snijders AP, Lujambio A, and O'Loghlen A
- Subjects
- Aged, 80 and over, Aging metabolism, Animals, Cell Line, Cells, Cultured, Child, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Extracellular Matrix metabolism, Fibroblasts metabolism, Humans, Integrin beta3 genetics, Isotope Labeling, Mice, Polycomb Repressive Complex 1 metabolism, Protein Subunits metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Tumor Suppressor Protein p53 metabolism, Up-Regulation genetics, Cellular Senescence genetics, Integrin beta3 metabolism, Signal Transduction, Transforming Growth Factor beta metabolism
- Abstract
Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or β3) is regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor β (TGF-β) pathway in a cell-autonomous and non-cell-autonomous manner. β3 levels are dynamically increased during oncogene-induced senescence (OIS) through CBX7 Polycomb regulation, and downregulation of β3 levels overrides OIS and therapy-induced senescence (TIS), independently of its ligand-binding activity. Moreover, cilengitide, an αvβ3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP) without affecting proliferation. Finally, we show an increase in β3 levels in a subset of tissues during aging. Altogether, our data show that integrin β3 subunit is a marker and regulator of senescence., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
31. A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair.
- Author
-
Ranes M, Boeing S, Wang Y, Wienholz F, Menoni H, Walker J, Encheva V, Chakravarty P, Mari PO, Stewart A, Giglia-Mari G, Snijders AP, Vermeulen W, and Svejstrup JQ
- Subjects
- Amino Acid Sequence, Cell Cycle, Cell Line, Cell Survival, Cluster Analysis, Gene Expression, Gene Expression Profiling, Genomic Instability, Humans, Mutation, Recombinant Fusion Proteins, Ubiquitination, Cockayne Syndrome genetics, Cockayne Syndrome metabolism, DNA Damage radiation effects, DNA Repair, Oxidative Stress, Transcription, Genetic
- Abstract
Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2016
- Full Text
- View/download PDF
32. Multiomic Analysis of the UV-Induced DNA Damage Response.
- Author
-
Boeing S, Williamson L, Encheva V, Gori I, Saunders RE, Instrell R, Aygün O, Rodriguez-Martinez M, Weems JC, Kelly GP, Conaway JW, Conaway RC, Stewart A, Howell M, Snijders AP, and Svejstrup JQ
- Subjects
- Chromatin metabolism, Databases, Factual, HEK293 Cells, Humans, Internet, Leupeptins pharmacology, Metabolic Networks and Pathways drug effects, Metabolic Networks and Pathways radiation effects, Nuclear Proteins metabolism, Phosphorylation radiation effects, Protein Serine-Threonine Kinases metabolism, Proteome drug effects, Proteome radiation effects, RNA Polymerase II metabolism, RNA, Small Interfering metabolism, Transcription, Genetic radiation effects, Ubiquitination radiation effects, User-Computer Interface, DNA Damage radiation effects, Proteomics, Ultraviolet Rays
- Abstract
In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
33. Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons.
- Author
-
Debaisieux S, Encheva V, Chakravarty P, Snijders AP, and Schiavo G
- Subjects
- Animals, Axonal Transport drug effects, Axonal Transport genetics, Axons drug effects, Axons pathology, Cell Differentiation drug effects, Cell Differentiation genetics, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Endocytosis genetics, Endosomes genetics, Endosomes metabolism, Endosomes pathology, Ferric Compounds administration & dosage, Ferric Compounds chemistry, Humans, Isotope Labeling, Metal Nanoparticles administration & dosage, Metal Nanoparticles chemistry, Metalloendopeptidases administration & dosage, Metalloendopeptidases chemistry, Mice, Motor Neurons drug effects, Motor Neurons pathology, Nerve Growth Factors genetics, Neurodegenerative Diseases metabolism, Neurodegenerative Diseases pathology, Signal Transduction, Tetanus Toxin administration & dosage, Tetanus Toxin chemistry, Axons metabolism, Motor Neurons metabolism, Nerve Growth Factors metabolism, Neurodegenerative Diseases genetics, Proteomics
- Abstract
Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
34. De novo DNA methylation drives 5hmC accumulation in mouse zygotes.
- Author
-
Amouroux R, Nashun B, Shirane K, Nakagawa S, Hill PW, D'Souza Z, Nakayama M, Matsuda M, Turp A, Ndjetehe E, Encheva V, Kudo NR, Koseki H, Sasaki H, and Hajkova P
- Subjects
- Animals, Biomarkers metabolism, Chromatography, Liquid, Cytosine metabolism, DNA (Cytosine-5-)-Methyltransferase 1, DNA (Cytosine-5-)-Methyltransferases genetics, DNA (Cytosine-5-)-Methyltransferases metabolism, DNA Methyltransferase 3A, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Dioxygenases, Embryo Culture Techniques, Fertilization in Vitro, Fluorescent Antibody Technique, Gene Expression Regulation, Developmental, Kinetics, Mass Spectrometry, Mice, Mice, Knockout, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism, 5-Methylcytosine metabolism, Cellular Reprogramming, Cytosine analogs & derivatives, DNA Methylation, Epigenesis, Genetic, Zygote metabolism
- Abstract
Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs 1-4). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation.
- Published
- 2016
- Full Text
- View/download PDF
35. The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression.
- Author
-
Silva N, Ferrandiz N, Barroso C, Tognetti S, Lightfoot J, Telecan O, Encheva V, Faull P, Hanni S, Furger A, Snijders AP, Speck C, and Martinez-Perez E
- Subjects
- Animals, Caenorhabditis elegans cytology, Chromosome Segregation physiology, Signal Transduction genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Cell Cycle Proteins metabolism, Chromosome Pairing genetics, Meiosis physiology, Signal Transduction physiology, Synaptonemal Complex metabolism
- Abstract
Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology, and poorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly. Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homolog pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology-independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of Mad2 in the spindle assembly checkpoint., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
36. Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia.
- Author
-
Thomas ER, Atanur SS, Norsworthy PJ, Encheva V, Snijders AP, Game L, Vandrovcova J, Siddiq A, Seed M, Soutar AK, and Aitman TJ
- Abstract
Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms.
- Published
- 2013
- Full Text
- View/download PDF
37. Naive pluripotency is associated with global DNA hypomethylation.
- Author
-
Leitch HG, McEwen KR, Turp A, Encheva V, Carroll T, Grabole N, Mansfield W, Nashun B, Knezovich JG, Smith A, Surani MA, and Hajkova P
- Subjects
- Animals, Benzamides pharmacology, Cell Differentiation, Cells, Cultured drug effects, DNA (Cytosine-5-)-Methyltransferases metabolism, DNA Methyltransferase 3A, DNA-Binding Proteins, Diphenylamine analogs & derivatives, Diphenylamine pharmacology, Enzyme Inhibitors pharmacology, Epigenesis, Genetic, Gene Expression Profiling, Genomic Imprinting, Germ Cells physiology, Germ Layers cytology, Glycogen Synthase Kinase 3 antagonists & inhibitors, Glycogen Synthase Kinase 3 metabolism, Glycogen Synthase Kinase 3 beta, MAP Kinase Kinase Kinases antagonists & inhibitors, MAP Kinase Kinase Kinases metabolism, Mice, Mice, Inbred C57BL, Pluripotent Stem Cells cytology, Pyridines pharmacology, Pyrimidines pharmacology, RNA-Binding Proteins, Transcription Factors genetics, Transcription Factors metabolism, Transcriptome, DNA Methyltransferase 3B, DNA Methylation, Embryonic Stem Cells physiology, Germ Cells cytology, Pluripotent Stem Cells physiology
- Abstract
Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Inhibition of MEK and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
- Published
- 2013
- Full Text
- View/download PDF
38. Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry.
- Author
-
Howard R, Encheva V, Thomson J, Bache K, Chan YT, Cowen S, Debenham P, Dixon A, Krause JU, Krishan E, Moore D, Moore V, Ojo M, Rodrigues S, Stokes P, Walker J, Zimmermann W, and Barallon R
- Subjects
- Humans, Polymerase Chain Reaction, Reproducibility of Results, Bone and Bones metabolism, DNA, Mitochondrial genetics, Sequence Analysis, DNA, Spectrometry, Mass, Electrospray Ionization methods, World War I
- Abstract
Mitochondrial DNA is commonly used in identity testing for the analysis of old or degraded samples or to give evidence of familial links. The Abbott T5000 mass spectrometry platform provides an alternative to the more commonly used Sanger sequencing for the analysis of human mitochondrial DNA. The robustness of the T5000 system has previously been demonstrated using DNA extracted from volunteer buccal swabs but the system has not been tested using more challenging sample types. For mass spectrometry to be considered as a valid alternative to Sanger sequencing it must also be demonstrated to be suitable for use with more limiting sample types such as old teeth, bone fragments, and hair shafts. In 2009 the Commonwealth War Graves Commission launched a project to identify the remains of 250 World War I soldiers discovered in a mass grave in Fromelles, France. This study characterises the performance of both Sanger sequencing and the T5000 platform for the analysis of the mitochondrial DNA extracted from 225 of these remains, both in terms of the ability to amplify and characterise DNA regions of interest and the relative information content and ease-of-use associated with each method., (Copyright © 2011 LGC Limited. Published by Elsevier Ireland Ltd.. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
39. Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique.
- Author
-
Chooneea D, Karlsson R, Encheva V, Arnold C, Appleton H, and Shah H
- Subjects
- Bacterial Outer Membrane Proteins metabolism, Carbonates chemistry, Cell Fractionation methods, Chromatography, Liquid, Immobilized Proteins metabolism, Lipoproteins chemistry, Lipoproteins metabolism, Mass Spectrometry, Microscopy, Electron, Proteome analysis, Proteomics methods, Salmonella typhi metabolism, Surface-Active Agents chemistry, Bacterial Outer Membrane Proteins chemistry, Immobilized Proteins chemistry, Salmonella typhi chemistry
- Abstract
Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches., Results: In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX., Conclusion: Using a multi-step digest approach the LPI technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for Salmonella Typhimurium.
- Published
- 2010
- Full Text
- View/download PDF
40. Proteomic analysis of the adaptive response of Salmonella enterica serovar Typhimurium to growth under anaerobic conditions.
- Author
-
Encheva V, Shah HN, and Gharbia SE
- Subjects
- Anaerobiosis, Bacterial Proteins biosynthesis, Carrier Proteins biosynthesis, Down-Regulation, Electrophoresis, Gel, Two-Dimensional, Fermentation, Gene Expression Regulation, Bacterial, Malate Dehydrogenase biosynthesis, Metabolic Networks and Pathways, Salmonella typhimurium growth & development, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Succinate Dehydrogenase biosynthesis, Up-Regulation, Adaptation, Physiological, Proteome analysis, Salmonella typhimurium metabolism
- Abstract
In order to survive in the host and initiate infection, Salmonella enterica needs to undergo a transition between aerobic and anaerobic growth by modulating its central metabolic pathways. In this study, a comparative analysis of the proteome of S. enterica serovar Typhimurium grown in the presence or absence of oxygen was performed. The most prominent changes in expression were measured in a semiquantitative manner using difference in-gel electrophoresis (DIGE) to reveal the main protein factors involved in the adaptive response to anaerobiosis. A total of 38 proteins were found to be induced anaerobically, while 42 were repressed. The proteins of interest were in-gel digested with trypsin and identified by MALDI TOF mass spectrometry using peptide mass fingerprinting. In the absence of oxygen, many fermentative enzymes catalysing reactions in the mixed-acid or arginine fermentations were overexpressed. In addition, the enzyme fumarate reductase, which is known to provide an alternative electron acceptor for the respiratory chains in the absence of oxygen, was shown to be induced. Increases in expression of several glycolytic and pentose phosphate pathway enzymes, as well as two malic enzymes, were detected, suggesting important roles for these in anaerobic metabolism. Substantial decreases in expression were observed for a large number of periplasmic transport proteins. The majority of these are involved in the uptake of amino acids and peptides, but permeases transporting iron, thiosulphate, glucose/galactose, glycerol 3-phosphate and dicarboxylic acids were also repressed. Decreases in expression were also observed for a superoxide dismutase, ATP synthase, inositol monophosphatase, and several chaperone and hypothetical proteins. The changes were monitored in two different isolates, and despite their very similar expression patterns, some variability in the adaptive response to anaerobiosis was also observed.
- Published
- 2009
- Full Text
- View/download PDF
41. High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells.
- Author
-
Rajakaruna L, Hallas G, Molenaar L, Dare D, Sutton H, Encheva V, Culak R, Innes I, Ball G, Sefton AM, Eydmann M, Kearns AM, and Shah HN
- Subjects
- Cell Culture Techniques, Databases, Genetic, Humans, RNA, Ribosomal, 16S genetics, Reproducibility of Results, Staphylococcal Infections diagnosis, Staphylococcus aureus chemistry, Staphylococcus aureus cytology, Staphylococcus aureus isolation & purification, Bacterial Proteins chemistry, Bacteriological Techniques, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Staphylococcal Infections microbiology, Staphylococcus aureus classification
- Abstract
Staphylococcus aureus remains an important human pathogen responsible for a high burden of disease in healthcare and community settings. The emergence of multidrug-resistant strains is of increasing concern world-wide. The identification of S. aureus is currently based upon phenotypic and genotypic methods. Here, an alternative approach involving mass spectral analysis of surface-associated proteins of intact bacterial cells by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was investigated using 95 isolates obtained directly from a clinical laboratory at The Royal London Hospital and 39 isolates from the Staphylococcal Reference Unit, Health Protection Agency, London. Results obtained indicate that clinical isolates share many common mass ions with-type/reference strains which allowed their correct identification when searched against a comprehensive database that has been in the process of development for several years. The existing database contains more than 5000 profiles of various bacterial pathogens, but comprises mainly type or reference strains. The MicrobeLynx software successfully identified all isolates to the correct genus and all but four to the correct species. These were misidentified in the first instance due to contamination or low mass ion intensity but once the cultures were purified and re-analysed they were confirmed as S. aureus by both MALDI-TOF-MS and 16S rRNA sequence analysis. The high percentage of correct identifications coupled with the high speed and the minimal sample preparation required, indicate that MALDI-TOF-MS has the potential to perform high throughput identification of clinical isolates of S. aureus despite the inherent diversity of this species. The method is, however, only reproducible if variable parameters such as sample preparation, media, growth condition, etc. are standardised.
- Published
- 2009
- Full Text
- View/download PDF
42. Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map.
- Author
-
Encheva V, Gharbia SE, Wait R, Begum S, and Shah HN
- Subjects
- Amino Acid Sequence, Antigens, Bacterial isolation & purification, Bacterial Proteins isolation & purification, Cholic Acids chemistry, Dithiothreitol chemistry, Electrophoresis, Gel, Two-Dimensional, Molecular Sequence Data, Sodium Dodecyl Sulfate chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Urea chemistry, Antigens, Bacterial metabolism, Bacterial Proteins metabolism, Proteome, Streptococcus pneumoniae chemistry
- Abstract
Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.
- Published
- 2006
- Full Text
- View/download PDF
43. Proteome analysis of serovars Typhimurium and Pullorum of Salmonella enterica subspecies I.
- Author
-
Encheva V, Wait R, Gharbia SE, Begum S, and Shah HN
- Subjects
- Serotyping, Bacterial Proteins metabolism, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Proteome analysis, Proteomics, Salmonella enterica classification, Salmonella enterica metabolism
- Abstract
Background: Salmonella enterica subspecies I includes several closely related serovars which differ in host ranges and ability to cause disease. The basis for the diversity in host range and pathogenic potential of the serovars is not well understood, and it is not known how host-restricted variants appeared and what factors were lost or acquired during adaptations to a specific environment. Differences apparent from the genomic data do not necessarily correspond to functional proteins and more importantly differential regulation of otherwise identical gene content may play a role in the diverse phenotypes of the serovars of Salmonella., Results: In this study a comparative analysis of the cytosolic proteins of serovars Typhimurium and Pullorum was performed using two-dimensional gel electrophoresis and the proteins of interest were identified using mass spectrometry. An annotated reference map was created for serovar Typhimurium containing 233 entries, which included many metabolic enzymes, ribosomal proteins, chaperones and many other proteins characteristic for the growing cell. The comparative analysis of the two serovars revealed a high degree of variation amongst isolates obtained from different sources and, in some cases, the variation was greater between isolates of the same serovar than between isolates with different sero-specificity. However, several serovar-specific proteins, including intermediates in sulphate utilisation and cysteine synthesis, were also found despite the fact that the genes encoding those proteins are present in the genomes of both serovars., Conclusion: Current microbial proteomics are generally based on the use of a single reference or type strain of a species. This study has shown the importance of incorporating a large number of strains of a species, as the diversity of the proteome in the microbial population appears to be significantly greater than expected. The characterisation of a diverse selection of strains revealed parts of the proteome of S. enterica that alter their expression while others remain stable and allowed for the identification of serovar-specific factors that have so far remained undetected by other methods.
- Published
- 2005
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.