Publications 2012

C. Trudgian D. 2012. ModLS: Post-Translational Modification Localization Scoring with Automatic Specificity Expansion Journal of Proteomics & Bioinformatics, 05 (12), pp. 285-291. | Citations: 5 (Scopus) | Show Abstract | Read more

Probability-based localization scoring of fragment mass-spectrum phosphorylation site identifications has become common practice to confirm search engine modification assignments, and indicate the degree of certainty with which they are defined. Localization of modifications other than phosphorylation is also required but is less commonly supported by current tools. These other modifications, such as hydroxylation, may have broad aminoacid specificity, and can be misassigned when the correct specificity is not considered in an MS database search. In addition, localization software is often specific to a particular MS/MS search engine, and cannot be used to localize modifications identified by multiple search engines. ModLS, a new tool within our freely-available Central Proteomics Facilities Pipeline (CPFP), applies a localization scoring method to arbitrary post-translational modifications (PTMs). As well as localising PTMs based on amino-acid specificities are included in the initial search, ModLS can automatically consider additional specificities from UniMod. This can help avoid 'correct modification, incorrect amino-acid' errors which can occur when data is searched using only a subset of PTM specificities. Localization scoring can be performed on the results from any search engine incorporated within the pipeline, or where the output of individual search engines is combined to give increased coverage. We demonstrate the performance of ModLS using a publicly available phosphorylated peptide dataset, showing that it outperforms the recently characterized Mascot Delta Score approach for CID and MSA data, and is comparable for HCD data. In addition, we show the utility of automatic specificity expansion using hydroxylated and methylated peptide data. ModLS is a user-friendly localization tool for arbitrary modifications. Its inclusion within CPFP allows PTM localization to be performed quickly and easily on large or small result sets, from multiple search engines. Specificity expansion, introduced in ModLS, allows misassignments of modifications due to incomplete consideration of specificities to be identified and Minimized. © 2012 Trudgian DC, et al.

Farcas AM, Blackledge NP, Sudbery I, Long HK, McGouran JF, Rose NR, Lee S, Sims D, Cerase A, Sheahan TW et al. 2012. KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. Elife, 1 (1), pp. e00205. | Citations: 201 (Scopus) | Show Abstract | Read more

CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs.DOI:http://dx.doi.org/10.7554/eLife.00205.001.

Ge W, Wolf A, Feng T, Ho C-H, Sekirnik R, Zayer A, Granatino N, Cockman ME, Loenarz C, Loik ND et al. 2012. Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans. Nat Chem Biol, 8 (12), pp. 960-962. | Citations: 80 (Scopus) | Show Abstract | Read more

The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

Frleta D, Ochoa CE, Kramer HB, Khan SA, Stacey AR, Borrow P, Kessler BM, Haynes BF, Bhardwaj N. 2012. HIV-1 infection-induced apoptotic microparticles inhibit human DCs via CD44. J Clin Invest, 122 (12), pp. 4685-4697. | Citations: 31 (Scopus) | Show Abstract | Read more

Acute HIV-1 infection results in dysregulated immunity, which contributes to poor control of viral infection. DCs are key regulators of both adaptive and innate immune responses needed for controlling HIV-1, and we surmised that factors elicited during acute HIV-1 infection might impede DC function. We derived immature DCs from healthy donor peripheral blood monocytes and treated them with plasma from uninfected control donors and donors with acute HIV-1 infections. We found that the plasma from patients with HIV specifically inhibited DC function. This suppression was mediated by elevated apoptotic microparticles derived from dying cells during acute HIV-1 infection. Apoptotic microparticles bound to and inhibited DCs through the hyaluronate receptor CD44. These data suggest that targeting this CD44-mediated inhibition by apoptotic microparticles could be a novel strategy to potentiate DC activation of HIV-specific immunity.

Kramer HB, Nicholson B, Kessler BM, Altun M. 2012. Detection of ubiquitin-proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development Biochimica et Biophysica Acta - Molecular Cell Research, 1823 (11), pp. 2029-2037. | Citations: 21 (Scopus) | Show Abstract | Read more

Background: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. © 2012 Elsevier B.V.

Parsons JL, Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Datta PK, Dianov GL. 2012. Phosphorylation of PNKP by ATM prevents its proteasomal degradation and enhances resistance to oxidative stress. Nucleic Acids Res, 40 (22), pp. 11404-11415. | Citations: 20 (Scopus) | Show Abstract | Read more

We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.

Leuchs S, Saalfrank A, Merkl C, Flisikowska T, Edlinger M, Durkovic M, Rezaei N, Kurome M, Zakhartchenko V, Kessler B et al. 2012. Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs. PLoS One, 7 (10), pp. e43323. | Citations: 34 (Web of Science Lite) | Show Abstract | Read more

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53(R167H) mutant allele, orthologous to oncogenic human mutant TP53(R175H) and mouse Trp53(R172H), that can be activated by Cre recombination. MSCs carrying the latent TP53(R167H) mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53(R167H) allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53(R167H) mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

Frleta D, Ochoa CE, Kramer HB, Khan SA, Stacey AR, Borrow P, Kessler BM, Haynes BF, Bhardwaj N. 2012. Apoptotic microparticles generated during acute HIV-1 infection inhibit human dendritic cells via CD44. Retrovirology, 9 (Suppl 2), pp. P183-P183. | Read more

Chauhan D, Tian Z, Nicholson B, Kumar KGS, Zhou B, Carrasco R, McDermott JL, Leach CA, Fulcinniti M, Kodrasov MP et al. 2012. A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell, 22 (3), pp. 345-358. | Citations: 226 (Scopus) | Show Abstract | Read more

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

Tannetta D, Mackeen M, Kessler B, Sargent I, Redman C. 2012. OS045. Multi-dimensional protein identification technology analysis of syncytiotrophoblast vesicles released from perfused preeclampsia placentas. Pregnancy Hypertens, 2 (3), pp. 201-202. | Citations: 4 (European Pubmed Central) | Show Abstract | Read more

INTRODUCTION: In pre-eclampsia, the consequences of poor placentation lead to the second stage of pre-eclampsia, which involves activation of a maternal systemic inflammatory response (MSIR). Endothelial and other inflammatory cellular dysfunction cause the diverse features which characterise the disorder. We have previously shown that syncytiotrophoblast microvesicles (STBM) are pro-inflammatory and circulate in increased amounts in pre-eclamptic women. We hypothesise that multiple placental "danger signals" are carried by STBM into the maternal circulation in increased amounts in PE with pro-inflammatory, anti-angiogenic and pro-coagulant activity, implicating STBM in the pathophysiology of PE. OBJECTIVES: To characterise the proteins carried by STBM from normal and PE placentas. For the first time multi-dimensional protein identification technology (MudPIT) was used to derive the proteome profiles of normal and PE placenta STBM. METHODS: STBM were prepared from placentas (normal term: n=9 and PE: n=5) by dual lobe perfusion, isolated by ultracentrifugation and stored at -80°C. Normal and PE derived placenta STBM pools were then subjected to MudPIT analysis. RESULTS: 538 proteins unique to PE STBM, 604 proteins unique to normal STBM and 1421 proteins common to both preparations were found. Preliminary analysis indicates the presence of alarmins (HSP70, and galectin 3), exosomal proteins (CD63,CD9,CD81), immunoregulatory molecules (CD26,CD200,CD47,Galectin 1), complement and complement regulatory molecules (C1q,C3,CD55,CD59 and vitronectin), amino acid transporters (CD98) and anti-angiogenic molecules (endoglin). Our analysis also reveals that proteins known to be elevated in blood before, or at, the time of pre-eclampsia are elevated or unique in STBM from PE placentas, including Fetuin A, Inter-alpha (globulin) inhibitor H4, Serum amyloid P component, Apolipoprotein H (or B2GP1) and Apolipoprotein AII. Thus, as predicted, a large number of circulating molecules are associated with STBM. The inter-relationships between proteins that are unique to either PE or normal pregnancy and the processes in which they are involved are being determined by Ingenuity Pathways Analysis software. In terms of biofunctions, preliminary analysis shows that proteins unique to PE STBM have a highly significant association (p<10(-11)) with 6 disease pathways including inflammatory, immunological, cardiovascular and reproductive system diseases and organ injury, whereas for proteins unique to normal STBM only protein synthesis was significant at the same level. CONCLUSION: STBM contain a heterogeneous population of vesicles that convey a large repertoire of placental proteins into the maternal circulation. The profound differences between PE and normal STBM indicate their pro-inflammatory potential.

Iphöfer A, Kummer A, Nimtz M, Ritter A, Arnold T, Frank R, van den Heuvel J, Kessler BM, Jänsch L, Franke R. 2012. Profiling ubiquitin linkage specificities of deubiquitinating enzymes with branched ubiquitin isopeptide probes. Chembiochem, 13 (10), pp. 1416-1420. | Citations: 24 (Web of Science Lite) | Read more

Huang H, Mackeen MM, Cook M, Oriero E, Locke E, Thézénas ML, Kessler BM, Nwakanma D, Casals-Pascual C. 2012. Proteomic identification of host and parasite biomarkers in saliva from patients with uncomplicated Plasmodium falciparum malaria. Malar J, 11 (1), pp. 178. | Citations: 10 (Scopus) | Show Abstract | Read more

BACKGROUND: Malaria cases attributed to Plasmodium falciparum account for approximately 600,000 deaths yearly, mainly in African children. The gold standard method to diagnose malaria requires the visualization of the parasite in blood. The role of non-invasive diagnostic methods to diagnose malaria remains unclear. METHODS: A protocol was optimized to deplete highly abundant proteins from saliva to improve the dynamic range of the proteins identified and assess their suitability as candidate biomarkers of malaria infection. A starch-based amylase depletion strategy was used in combination with four different lectins to deplete glycoproteins (Concanavalin A and Aleuria aurantia for N-linked glycoproteins; jacalin and peanut agglutinin for O-linked glycoproteins). A proteomic analysis of depleted saliva samples was performed in 17 children with fever and a positive-malaria slide and compared with that of 17 malaria-negative children with fever. RESULTS: The proteomic signature of malaria-positive patients revealed a strong up-regulation of erythrocyte-derived and inflammatory proteins. Three P. falciparum proteins, PFL0480w, PF08_0054 and PFI0875w, were identified in malaria patients and not in controls. Aleuria aurantia and jacalin showed the best results for parasite protein identification. CONCLUSIONS: This study shows that saliva is a suitable clinical specimen for biomarker discovery. Parasite proteins and several potential biomarkers were identified in patients with malaria but not in patients with other causes of fever. The diagnostic performance of these markers should be addressed prospectively.

Kramer HB, Nicholson B, Kessler BM, Altun M. 2012. Detection of ubiquitin-proteasome enzymatic activities in cells: application of activity-based probes to inhibitor development. Biochim Biophys Acta, 1823 (11), pp. 2029-2037. | Citations: 17 (Web of Science Lite) | Show Abstract | Read more

BACKGROUND: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. SCOPE OF REVIEW: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. MAJOR CONCLUSIONS: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. GENERAL SIGNIFICANCE: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Klymiuk N, Mundhenk L, Kraehe K, Wuensch A, Plog S, Emrich D, Langenmayer MC, Stehr M, Holzinger A, Kröner C et al. 2012. Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis. J Mol Med (Berl), 90 (5), pp. 597-608. | Citations: 43 (Web of Science Lite) | Show Abstract | Read more

Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.

Konietzny R, Fischer R, Ternette N, Wright CA, Turney BW, Chakera A, Hughes D, Kessler BM, Pugh CW. 2012. Detection of BK virus in urine from renal transplant subjects by mass spectrometry. Clin Proteomics, 9 (1), pp. 4. | Citations: 15 (Scopus) | Show Abstract | Read more

BACKGROUND: The diagnosis and management of BK virus (BKV) reactivation following renal transplantation continues to be a significant clinical problem. Following reactivation of latent virus, impaired cellular immunity enables sustained viral replication to occur in urothelial cells, which potentially leads to the development of BKV-associated nephropathy (BKVAN). Current guidelines recommend regular surveillance for BKV reactivation through the detection of infected urothelial cells in urine (decoy cells) or viral nucleic acid in urine or blood. However, these methods have variable sensitivity and cannot routinely distinguish between different viral subtypes. We therefore asked whether mass spectrometry might be able to overcome these limitations and provide an additional non-invasive technique for the surveillance of BKV and identification of recipients at increased risk of BKVAN. RESULTS: Here we describe a mass spectrometry (MS)-based method for the detection of BKV derived proteins directly isolated from clinical urine samples. Peptides detected by MS derived from Viral Protein 1 (VP1) allowed differentiation between subtypes I and IV. Using this approach, we observed an association between higher decoy cell numbers and the presence of the VP1 subtype Ib-2 in urine samples derived from a cohort of 20 renal transplant recipients, consistent with the hypothesis that certain viral subtypes may be associated with more severe BKVAN. CONCLUSIONS: This is the first study to identify BK virus proteins in clinical samples by MS and that this approach makes it possible to distinguish between different viral subtypes. Further studies are required to establish whether this information could lead to stratification of patients at risk of BKVAN, facilitate distinction between BKVAN and acute rejection (AR), and ultimately improve patient treatment and outcomes.

Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Parsons JL, Dianov GL. 2012. ATM-Dependent Downregulation of USP7/HAUSP by PPM1G Activates p53 Response to DNA Damage MOLECULAR CELL, 45 (6), pp. 801-813. | Citations: 77 (Scopus) | Show Abstract | Read more

The deubiquitylation enzyme USP7/HAUSP plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the DNA damage response. Despite this important role in controlling other proteins, USP7 itself has not been recognized as a target for regulation. Here, we report that USP7 regulation plays a central role in DNA damage signal transmission. We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2. Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53. After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53. Our findings provide a quantitative transmission mechanism of the DNA damage signal to coordinate a p53-dependent DNA damage response. © 2012 Elsevier Inc..

McGouran JF, Kramer HB, Mackeen MM, di Gleria K, Altun M, Kessler BM. 2012. Fluorescence-based active site probes for profiling deubiquitinating enzymes. Org Biomol Chem, 10 (17), pp. 3379-3383. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

Novel ubiquitin-based active site probes including a fluorescent tag have been developed and evaluated. A new, functionalizable electrophilic trap is utilized allowing for late stage diversification of the probe. Attachment of fluorescent dyes allowed direct detection of endogenous deubiquitinating enzyme (DUB) activities in cell extracts by in-gel fluorescence imaging.

Masson N, Singleton RS, Sekirnik R, Trudgian DC, Ambrose LJ, Miranda MX, Tian YM, Kessler BM, Schofield CJ, Ratcliffe PJ. 2012. The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity EMBO Reports, 13 (3), pp. 251-257. | Citations: 57 (Scopus) | Show Abstract | Read more

Hypoxic and oxidant stresses can coexist in biological systems, and oxidant stress has been proposed to activate hypoxia pathways through the inactivation of the oxygen-sensing hypoxia-inducible factor (HIF) prolyl and asparaginyl hydroxylases. Here, we show that despite reduced sensitivity to cellular hypoxia, the HIF asparaginyl hydroxylase-known as FIH, factor inhibiting HIF-is strikingly more sensitive to peroxide than the HIF prolyl hydroxylases. These contrasting sensitivities indicate that oxidant stress is unlikely to signal hypoxia directly to the HIF system, but that hypoxia and oxidant stress can interact functionally as distinct regulators of HIF transcriptional output. © 2012 European Molecular Biology Organization.

Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Parsons JL, Dianov GL. 2012. ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage. Mol Cell, 45 (6), pp. 801-813. | Citations: 68 (European Pubmed Central) | Show Abstract | Read more

The deubiquitylation enzyme USP7/HAUSP plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the DNA damage response. Despite this important role in controlling other proteins, USP7 itself has not been recognized as a target for regulation. Here, we report that USP7 regulation plays a central role in DNA damage signal transmission. We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2. Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53. After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53. Our findings provide a quantitative transmission mechanism of the DNA damage signal to coordinate a p53-dependent DNA damage response.

Cho E-C, Zheng S, Munro S, Liu G, Carr SM, Moehlenbrink J, Lu Y-C, Stimson L, Khan O, Konietzny R et al. 2012. Arginine methylation controls growth regulation by E2F-1. EMBO J, 31 (7), pp. 1785-1797. | Citations: 87 (Scopus) | Show Abstract | Read more

E2F transcription factors are implicated in diverse cellular functions. The founding member, E2F-1, is endowed with contradictory activities, being able to promote cell-cycle progression and induce apoptosis. However, the mechanisms that underlie the opposing outcomes of E2F-1 activation remain largely unknown. We show here that E2F-1 is directly methylated by PRMT5 (protein arginine methyltransferase 5), and that arginine methylation is responsible for regulating its biochemical and functional properties, which impacts on E2F-1-dependent growth control. Thus, depleting PRMT5 causes increased E2F-1 protein levels, which coincides with decreased growth rate and associated apoptosis. Arginine methylation influences E2F-1 protein stability, and the enhanced transcription of a variety of downstream target genes reflects increased E2F-1 DNA-binding activity. Importantly, E2F-1 is methylated in tumour cells, and a reduced level of methylation is evident under DNA damage conditions that allow E2F-1 stabilization and give rise to apoptosis. Significantly, in a subgroup of colorectal cancer, high levels of PRMT5 frequently coincide with low levels of E2F-1 and reflect a poor clinical outcome. Our results establish that arginine methylation regulates the biological activity of E2F-1 activity, and raise the possibility that arginine methylation contributes to tumourigenesis by influencing the E2F pathway.

Masson N, Singleton RS, Sekirnik R, Trudgian DC, Ambrose LJ, Miranda MX, Tian Y-M, Kessler BM, Schofield CJ, Ratcliffe PJ. 2012. The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity. EMBO Rep, 13 (3), pp. 251-257. | Citations: 57 (Web of Science Lite) | Show Abstract | Read more

Hypoxic and oxidant stresses can coexist in biological systems, and oxidant stress has been proposed to activate hypoxia pathways through the inactivation of the 'oxygen-sensing' hypoxia-inducible factor (HIF) prolyl and asparaginyl hydroxylases. Here, we show that despite reduced sensitivity to cellular hypoxia, the HIF asparaginyl hydroxylase--known as FIH, factor inhibiting HIF--is strikingly more sensitive to peroxide than the HIF prolyl hydroxylases. These contrasting sensitivities indicate that oxidant stress is unlikely to signal hypoxia directly to the HIF system, but that hypoxia and oxidant stress can interact functionally as distinct regulators of HIF transcriptional output.

Wright C, Sibani S, Trudgian D, Fischer R, Kessler B, LaBaer J, Bowness P. 2012. Detection of multiple autoantibodies in patients with ankylosing spondylitis using nucleic acid programmable protein arrays. Mol Cell Proteomics, 11 (2), pp. M9.00384. | Citations: 45 (Scopus) | Show Abstract | Read more

Ankylosing spondylitis (AS) is a common, inflammatory rheumatic disease that primarily affects the axial skeleton and is associated with sacroiliitis, uveitis, and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine whether plasma from patients with AS contained autoantibodies and, if so, characterize and quantify this response in comparison to patients with rheumatoid arthritis (RA) and healthy controls. Two high density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA, and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis to determine the patterns of signaling cascades or tissue origin. 44% of patients with ankylosing spondylitis demonstrated a broad autoantibody response, as compared with 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The autoantibody responses in the AS patients were targeted toward connective, skeletal, and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic acid programmable protein arrays constitute a powerful tool to study autoimmune diseases.

Cho E-C, Zheng S, Munro S, Liu G, Carr SM, Moehlenbrink J, Lu Y-C, Stimson L, Khan O, Konietzny R et al. 2012. Arginine methylation controls growth regulation by E2F-1 EMBO Journal, 31 (7), pp. 1785-1797.

Chen L, Fisher R, Kollnberger S, Kessler B, Bowness P. 2012. ENDOPLASMIC RETICULUM AMINOPEPTIDASE-1 (ERAP1) PLAYS A CRITICAL ROLE IN PEPTIDE BINDING AND ANTIGEN PRESENTATION BY HLA-B27 CLINICAL AND EXPERIMENTAL RHEUMATOLOGY, 30 (4), pp. 603-603.

Ge W, Wolf A, Feng T, Ho C-H, Sekirnik R, Zayer A, Granatino N, Cockman ME, Loenarz C, Loik ND et al. 2012. Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans Nature Chemical Biology,

Kemter E, Lieke T, Kessler B, Kurome M, Wuensch A, Summerfield A, Ayares D, Nagashima H, Baars W, Schwinzer R, Wolf E. 2012. Human TNF-related apoptosis-inducing ligand-expressing dendritic cells from transgenic pigs attenuate human xenogeneic T cell responses. Xenotransplantation, 19 (1), pp. 40-51. | Citations: 9 (European Pubmed Central) | Show Abstract | Read more

BACKGROUND: Efficient and precise techniques for the genetic modification of pigs facilitate the generation of tailored donor animals for xenotransplantation. Numerous transgenic pig lines exist with the focus on inhibition of the complement system and of humoral immune responses. In addition, immune cell-based responses need to be controlled to prevent pig-to-primate xenograft rejection. Expression of human (hu) TNF-related apoptosis-inducing ligand (TRAIL) on porcine cells has the potential to ameliorate human T cell responses. METHODS: We generated transgenic pigs expressing human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (huTRAIL) under the control of either the mouse H2K(b) promoter or a CMV enhancer/chicken β-actin (CAG) promoter, the latter one (CAG-huTRAIL) on a GGTA1 knockout/huCD46 transgenic background. The biological activity of huTRAIL was demonstrated by its apoptosis-inducing effect on Jurkat lymphoma cells. To clarify whether huTRAIL affects also primary immune cells and whether its effects depend on the presence of co-stimulatory molecules, we exposed human peripheral blood mononuclear cells (PBMC) or isolated T cells to huTRAIL-expressing porcine fibroblasts or dendritic cells in vitro. RESULTS: H2Kb-huTRAIL transgenic pigs express huTRAIL mainly in the spleen and secondary lymphoid tissues. The CAG-huTRAIL construct facilitated huTRAIL expression in multiple organs, the level being at least one order of magnitude higher than in H2Kb-huTRAIL transgenic pigs. Incubation with huTRAIL-expressing H2Kb-huTRAIL transgenic porcine dendritic cells decreased human T cell proliferation significantly without any signs of apoptosis. In spite of the high transgene expression level, CAG-huTRAIL transgenic fibroblasts did not affect proliferation of human PBMC, independent of their activation state. CONCLUSIONS: These results suggest huTRAIL expression on porcine dendritic cells as a possible strategy to attenuate T cell responses against pig-to-primate xenografts.

Mavridou DAI, Stevens JM, Mönkemeyer L, Daltrop O, di Gleria K, Kessler BM, Ferguson SJ, Allen JWA. 2012. A pivotal heme-transfer reaction intermediate in cytochrome c biogenesis. J Biol Chem, 287 (4), pp. 2342-2352. | Citations: 16 (Web of Science Lite) | Show Abstract | Read more

c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway.

Fischer R, Trudgian DC, Wright C, Thomas G, Bradbury LA, Brown MA, Bowness P, Kessler BM. 2012. Discovery of candidate serum proteomic and metabolomic biomarkers in ankylosing spondylitis. Mol Cell Proteomics, 11 (2), pp. M111.013904. | Citations: 52 (Scopus) | Show Abstract | Read more

Ankylosing Spondylitis (AS) is a common inflammatory rheumatic disease with a predilection for the axial skeleton, affecting 0.2% of the population. Current diagnostic criteria rely on a composite of clinical and radiological changes, with a mean time to diagnosis of 5 to 10 years. In this study we employed nano liquid-chromatography mass spectrometry analysis to detect and quantify proteins and small compounds including endogenous peptides and metabolites in serum from 18 AS patients and nine healthy individuals. We identified a total of 316 proteins in serum, of which 22 showed significant up- or down-regulation (p < 0.05) in AS patients. Receiver operating characteristic analysis of combined levels of serum amyloid P component and inter-α-trypsin inhibitor heavy chain 1 revealed high diagnostic value for Ankylosing Spondylitis (area under the curve = 0.98). We also depleted individual sera of proteins to analyze endogenous peptides and metabolic compounds. We detected more than 7000 molecular features in patients and healthy individuals. Quantitative MS analysis revealed compound profiles that correlate with the clinical assessment of disease activity. One molecular feature identified as a Vitamin D3 metabolite-(23S,25R)-25-hydroxyvitamin D3 26,23-peroxylactone-was down-regulated in AS. The ratio of this vitamin D metabolite versus vitamin D binding protein serum levels was also altered in AS as compared with controls. These changes may contribute to pathological skeletal changes in AS. Our study is the first example of an integration of proteomic and metabolomic techniques to find new biomarker candidates for the diagnosis of Ankylosing Spondylitis.

Meisenberg C, Tait PS, Dianova II, Wright K, Edelmann MJ, Ternette N, Tasaki T, Kessler BM, Parsons JL, Kwon YT, Dianov GL. 2012. Ubiquitin ligase UBR3 regulates cellular levels of the essential DNA repair protein APE1 and is required for genome stability. Nucleic Acids Res, 40 (2), pp. 701-711. | Citations: 32 (Scopus) | Show Abstract | Read more

APE1 (Ref-1) is an essential human protein involved in DNA damage repair and regulation of transcription. Although the cellular functions and biochemical properties of APE1 are well characterized, the mechanism involved in regulation of the cellular levels of this important DNA repair/transcriptional regulation enzyme, remains poorly understood. Using an in vitro ubiquitylation assay, we have now purified the human E3 ubiquitin ligase UBR3 as a major activity that polyubiquitylates APE1 at multiple lysine residues clustered on the N-terminal tail. We further show that a knockout of the Ubr3 gene in mouse embryonic fibroblasts leads to an up-regulation of the cellular levels of APE1 protein and subsequent genomic instability. These data propose an important role for UBR3 in the control of the steady state levels of APE1 and consequently error free DNA repair.

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