Publications 2015

Watson AS, Riffelmacher T, Stranks A, Williams O, De Boer J, Cain K, MacFarlane M, McGouran J, Kessler B, Khandwala S et al. 2015. Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia. Cell Death Discov, 1 (1), | Citations: 37 (Web of Science Lite) | Show Abstract | Read more

Decreased autophagy contributes to malignancies, however it is unclear how autophagy impacts on tumour growth. Acute myeloid leukemia (AML) is an ideal model to address this as (i) patient samples are easily accessible, (ii) the hematopoietic stem and progenitor population (HSPC) where transformation occurs is well characterized, and (iii) loss of the key autophagy gene Atg7 in hematopoietic stem and progenitor cells (HSPCs) leads to a lethal pre-leukemic phenotype in mice. Here we demonstrate that loss of Atg5 results in an identical HSPC phenotype as loss of Atg7, confirming a general role for autophagy in HSPC regulation. Compared to more committed/mature hematopoietic cells, healthy human and mouse HSCs displayed enhanced basal autophagic flux, limiting mitochondrial damage and reactive oxygen species in this long-lived population. Taken together, with our previous findings these data are compatible with autophagy limiting leukemic transformation. In line with this, autophagy gene losses are found within chromosomal regions that are commonly deleted in human AML. Moreover, human AML blasts showed reduced expression of autophagy genes, and displayed decreased autophagic flux with accumulation of unhealthy mitochondria indicating that deficient autophagy may be beneficial to human AML. Crucially, heterozygous loss of autophagy in an MLL-ENL model of AML led to increased proliferation in vitro, a glycolytic shift, and more aggressive leukemias in vivo. With autophagy gene losses also identified in multiple other malignancies, these findings point to low autophagy providing a general advantage for tumour growth.

Huang H, Fischer R, Secher N, Ravlo K, Soendergaard P, Norregaard R, Akhtar ZM, Kaisar M, Kessler B, Ploeg R, Jespersen B. 2015. USE OF PROTEOMICS TO IDENTIFY MOLECULAR PATHWAYS RELEVANT FOR REMOTE ISCHAEMIC CONDITIONING IN KIDNEY TRANSPLANTATION TRANSPLANT INTERNATIONAL, 28 pp. 64-64.

Kaisar M, Lo Faro L, Akhtar Z, Huang H, Leuvenink H, Kessler B, Ploeg R. 2015. IMPACT OF ORCHESTRATED DYSREGULATION OF DONOR METABOLIC PATHWAYS AFTER BRAIN DEATH WITH INCREASED CELL DEATH PROTEINS AND ROS SCAVENGER MOLECULES ON EARLY FUNCTION AFTER KIDNEY TRANSPLANTATION TRANSPLANT INTERNATIONAL, 28 pp. 620-620.

Kaisar M, Huang H, Akhtar Z, Lo Faro L, Leuvenink H, Kessler B, Ploeg R. 2015. USE OF PROTEOMIC SIGNATURES IN DONOR SERUM AND URINE TO DIFFERENTIATE BETWEEN IMMEDIATE FUNCTION AND DELAYED GRAFT FUNCTION AFTER KIDNEY TRANSPLANTATION TRANSPLANT INTERNATIONAL, 28 pp. 67-67.

Wijnhoven P, Konietzny R, Blackford AN, Travers J, Kessler BM, Nishi R, Jackson SP. 2015. USP4 Auto-Deubiquitylation Promotes Homologous Recombination. Mol Cell, 60 (3), pp. 362-373. | Citations: 28 (Web of Science Lite) | Show Abstract | Read more

Repair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.

Singleton DC, Rouhi P, Zois CE, Haider S, Li J-L, Kessler BM, Cao Y, Harris AL. 2015. Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis. Oncogene, 34 (36), pp. 4713-4722. | Citations: 12 (Scopus) | Show Abstract | Read more

Hypoxia is a common feature of locally advanced breast cancers that is associated with increased metastasis and poorer survival. Stabilisation of hypoxia-inducible factor-1α (HIF1α) in tumours causes transcriptional changes in numerous genes that function at distinct stages of the metastatic cascade. We demonstrate that expression of RIOK3 (RIght Open reading frame kinase 3) was increased during hypoxic exposure in an HIF1α-dependent manner. RIOK3 was localised to distinct cytoplasmic aggregates in normoxic cells and underwent redistribution to the leading edge of the cell in hypoxia with a corresponding change in the organisation of the actin cytoskeleton. Depletion of RIOK3 expression caused MDA-MB-231 to become elongated and this morphological change was due to a loss of protraction at the trailing edge of the cell. This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix. Proteomic analysis identified interactions of RIOK3 with actin and several actin-binding factors including tropomyosins (TPM3 and TPM4) and tropomodulin 3. Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments. Analysis of these filaments showed reduced association of TPM3, particularly during hypoxia, suggesting that RIOK3 regulates actin filament specialisation. RIOK3 depletion reduced the dissemination of MDA-MB-231 cells in both a zebrafish model of systemic metastasis and a mouse model of pulmonary metastasis. These findings demonstrate that RIOK3 is necessary for maintaining actin cytoskeletal organisation required for migration and invasion, biological processes that are necessary for hypoxia-driven metastasis.

Jallow S, Leligdowicz A, Kramer HB, Onyango C, Cotten M, Wright C, Whittle HC, McMichael A, Dong T, Kessler BM, Rowland-Jones SL. 2015. Correction. The presence of prolines in the flanking region of an immunodominant HIV-2 gag epitope influences the quality and quantity of the epitope generated. Eur J Immunol, 45 (9), pp. 2701. | Read more

Jallow S, Leligdowicz A, Kramer HB, Onyango C, Cotten M, Wright C, Whittle HC, McMichael A, Dong T, Kessler BM, Rowland-Jones SL. 2015. The presence of prolines in the flanking region of an immunodominant HIV-2 gag epitope influences the quality and quantity of the epitope generated (vol 45, pg 2232, 2015) EUROPEAN JOURNAL OF IMMUNOLOGY, 45 (9), pp. 2701-2701. | Read more

Zauri M, Berridge G, Thézénas M-L, Pugh KM, Goldin R, Kessler BM, Kriaucionis S. 2015. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer. Nature, 524 (7563), pp. 114-118. | Citations: 27 (Scopus) | Show Abstract | Read more

Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues.

Valli A, Harris AL, Kessler BM. 2015. Hypoxia metabolism in ageing. Aging (Albany NY), 7 (7), pp. 465-466. | Citations: 5 (Web of Science Lite) | Read more

Jallow S, Leligdowicz A, Kramer HB, Onyango C, Cotten M, Wright C, Whittle HC, McMichael A, Dong T, Kessler BM, Rowland-Jones SL. 2015. The presence of prolines in the flanking region of an immunodominant HIV-2 gag epitope influences the quality and quantity of the epitope generated. Eur J Immunol, 45 (8), pp. 2232-2242. | Citations: 4 (Web of Science Lite) | Show Abstract | Read more

Both the recognition of HIV-infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV-2 gag-specific T-cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165 DRFYKSLRA173 , within the highly conserved Major Homology Region of gag-p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag-p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag-specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165 DRFYKSLRA173 epitope. Our data demonstrate that the 165 DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral-load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV-2 encoding the "PPP-gag" sequence and both strong gag-specific CTL responses as well as lower viral load.

Plank M, Fischer R, Geoghegan V, Charles PD, Konietzny R, Acuto O, Pears C, Schofield CJ, Kessler BM. 2015. Expanding the yeast protein arginine methylome. Proteomics, 15 (18), pp. 3232-3243. | Citations: 13 (Scopus) | Show Abstract | Read more

Protein arginine methylation is a PTM involved in various cellular processes in eukaryotes. Recent discoveries led to a vast expansion of known sites in higher organisms, indicating that this modification is more widely spread across the proteome than previously assumed. An increased knowledge of sites in lower eukaryotes may facilitate the elucidation of its functions. In this study, we present the discovery of arginine mono-methylation sites in Saccharomyces cerevisiae by a combination of immunoaffinity enrichment and MS/MS. As detection of methylation is prone to yield false positives, we demonstrate the need for stringent measures to avoid elevated false discovery rates. To this end, we employed MethylSILAC in combination with a multistep data analysis strategy. We report 41 unambiguous methylation sites on 13 proteins. Our results indicate that, while substantially less abundant, arginine methylation follows similar patterns as in higher eukaryotes in terms of sequence context and functions of methylated proteins. The majority of sites occur on RNA-binding proteins participating in processes from transcription and splicing to translation and RNA degradation. Additionally, our data suggest a bias for localization of arginine methylation in unstructured regions of proteins, which frequently involves Arg-Gly-Gly motifs or Asn-rich contexts.

Dulneva A, Lee S, Oliver PL, Di Gleria K, Kessler BM, Davies KE, Becker EBE. 2015. The mutant Moonwalker TRPC3 channel links calcium signaling to lipid metabolism in the developing cerebellum. Hum Mol Genet, 24 (14), pp. 4114-4125. | Citations: 10 (Web of Science Lite) | Show Abstract | Read more

The Moonwalker (Mwk) mouse is a model of dominantly inherited cerebellar ataxia caused by a gain-of-function mutation in the transient receptor potential (TRP) channel TRPC3. Here, we report impairments in dendritic growth and synapse formation early on during Purkinje cell development in the Mwk cerebellum that are accompanied by alterations in calcium signaling. To elucidate the molecular effector pathways that regulate Purkinje cell dendritic arborization downstream of mutant TRPC3, we employed transcriptomic analysis of developing Purkinje cells isolated by laser-capture microdissection. We identified significant gene and protein expression changes in molecules involved in lipid metabolism. Consistently, lipid homeostasis in the Mwk cerebellum was found to be disturbed, and treatment of organotypic cerebellar slices with ceramide significantly improved dendritic outgrowth of Mwk Purkinje cells. These findings provide the first mechanistic insights into the TRPC3-dependent mechanisms, by which activated calcium signaling is coupled to lipid metabolism and the regulation of Purkinje cell development in the Mwk cerebellum.

Iglesias-Gato D, Chuan Y-C, Jiang N, Svensson C, Bao J, Shang Z, Paul I, Egevad L, Kessler BM, Wikström P et al. 2015. Erratum: OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo. Mol Cancer, 14 (1), pp. 88. | Citations: 2 (Web of Science Lite) | Read more

Fischer R, Kessler BM. 2015. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics, 15 (7), pp. 1224-1229. | Citations: 36 (Scopus) | Show Abstract | Read more

We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists.

Ternette N, Block PD, Sánchez-Bernabéu Á, Borthwick N, Pappalardo E, Abdul-Jawad S, Ondondo B, Charles PD, Dorrell L, Kessler BM, Hanke T. 2015. Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells. J Virol, 89 (11), pp. 5760-5771. | Citations: 13 (Scopus) | Show Abstract | Read more

UNLABELLED: Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv, which delivers HIV-1 conserved antigenic regions by using modified vaccinia virus Ankara (MVA). We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 h after MVA.HIVconsv infection. Four of the seven conserved peptides were monitored between 0 and 3.5 h of infection by using quantitative mass spectrometry (Q-MS), and their abundance in HLA class I associations reflected levels of the whole HIVconsv protein in the cell. While immunopeptides delivered by the incoming MVA vector proteins could be detected, all early HIVconsv-derived immunopeptides were likely synthesized de novo. MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance. IMPORTANCE: The vast changes in cellular antigen presentation after infection of cells with a vectored vaccine, as shown here for MVA.HIVconsv, highlight the complexity of factors that need to be considered for efficient antigen delivery and presentation. Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.

Markkanen E, Fischer R, Ledentcova M, Kessler BM, Dianov GL. 2015. Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability. Nucleic Acids Res, 43 (7), pp. 3667-3679. | Citations: 16 (Scopus) | Show Abstract | Read more

Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell 'hallmarks'. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells.

Welker F, Collins MJ, Thomas JA, Wadsley M, Brace S, Cappellini E, Turvey ST, Reguero M, Gelfo JN, Kramarz A et al. 2015. Ancient proteins resolve the evolutionary history of Darwin's South American ungulates. Nature, 522 (7554), pp. 81-84. | Citations: 91 (Scopus) | Show Abstract | Read more

No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin, who first collected their remains, they included perhaps the 'strangest animal[s] ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from 'condylarths', a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.

Bursomanno S, McGouran JF, Kessler BM, Hickson ID, Liu Y. 2015. Regulation of SUMO2 target proteins by the proteasome in human cells exposed to replication stress. J Proteome Res, 14 (4), pp. 1687-1699. | Citations: 6 (Web of Science Lite) | Show Abstract | Read more

In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair and replication, while the proteins stabilized upon SUMOylation were mainly involved in chromatin maintenance. In addition, we identified 43 SUMOylation sites in target proteins, of which 17 are located in the proximity of phosphorylated residues. Considering that DNA replication stress is a major source of genome instability, which is suggested to drive tumorigenesis and possibly aging, our data will facilitate future functional studies in the fields of DNA metabolism and cancer biology.

Valli A, Rodriguez M, Moutsianas L, Fischer R, Fedele V, Huang H-L, Van Stiphout R, Jones D, Mccarthy M, Vinaxia M et al. 2015. Hypoxia induces a lipogenic cancer cell phenotype via HIF1α-dependent and -independent pathways. Oncotarget, 6 (4), pp. 1920-1941. | Citations: 22 (Web of Science Lite) | Show Abstract | Read more

The biochemistry of cancer cells diverges significantly from normal cells as a result of a comprehensive reprogramming of metabolic pathways. A major factor influencing cancer metabolism is hypoxia, which is mediated by HIF1α and HIF2α. HIF1α represents one of the principal regulators of metabolism and energetic balance in cancer cells through its regulation of glycolysis, glycogen synthesis, Krebs cycle and the pentose phosphate shunt. However, less is known about the role of HIF1α in modulating lipid metabolism. Lipids serve cancer cells to provide molecules acting as oncogenic signals, energetic reserve, precursors for new membrane synthesis and to balance redox biological reactions. To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites. Untargeted metabolomics integrated with proteomics revealed that hypoxia induced many changes in lipids metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in a HIF1α-dependent fashion. Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner. Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.

Tomlinson PR, Zheng Y, Fischer R, Heidasch R, Gardiner C, Evetts S, Hu M, Wade-Martins R, Turner MR, Morris J et al. 2015. Identification of distinct circulating exosomes in Parkinson's disease. Ann Clin Transl Neurol, 2 (4), pp. 353-361. | Citations: 45 (Scopus) | Show Abstract | Read more

OBJECTIVE: Whether circulating microvesicles convey bioactive signals in neurodegenerative diseases remains currently unknown. In this study, we investigated the biochemical composition and biological function of exosomes isolated from sera of patients with Parkinson's disease (PD). METHODS: Proteomic analysis was performed on microvesicle preparations from grouped samples of patients with genetic and sporadic forms of PD, amyotrophic lateral sclerosis, and healthy subjects. Nanoparticle-tracking analysis was used to assess the number and size of exosomes between patient groups. To interrogate their biological effect, microvesicles were added to primary rat cortical neurons subjected to either nutrient deprivation or sodium arsenite. RESULTS: Among 1033 proteins identified, 23 exosome-associated proteins were differentially abundant in PD, including the regulator of exosome biogenesis syntenin 1. These protein changes were detected despite similar exosome numbers across groups suggesting that they may reflect exosome subpopulations with distinct functions. Accordingly, we showed in models of neuronal stress that Parkinson's-derived microvesicles have a protective effect. INTERPRETATION: Collectively, these data suggest for the first time that immunophenotyping of circulating exosome subpopulations in PD may lead to a better understanding of the systemic response to neurodegeneration and the development of novel therapeutics.

Iglesias-Gato D, Chuan Y-C, Jiang N, Svensson C, Bao J, Paul I, Egevad L, Kessler BM, Wikström P, Niu Y, Flores-Morales A. 2015. OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo. Mol Cancer, 14 (1), pp. 8. | Citations: 19 (Scopus) | Show Abstract | Read more

BACKGROUND: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored. METHODS: RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth. RESULTS: Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo. CONCLUSIONS: OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

Lugli EB, Correia RESM, Fischer R, Lundberg K, Bracke KR, Montgomery AB, Kessler BM, Brusselle GG, Venables PJ. 2015. Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis. Arthritis Res Ther, 17 (1), pp. 9. | Citations: 44 (Scopus) | Show Abstract | Read more

INTRODUCTION: Smoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4). METHODS: Lung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test. RESULTS: Within the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in α-enolase. CONCLUSIONS: We have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought.

Altun M, Walter TS, Kramer HB, Herr P, Iphöfer A, Boström J, David Y, Komsany A, Ternette N, Navon A et al. 2015. The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages. PLoS One, 10 (1), pp. e0115344. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

Antonopoulos AS, Margaritis M, Coutinho P, Shirodaria C, Psarros C, Herdman L, Sanna F, De Silva R, Petrou M, Sayeed R et al. 2015. Adiponectin as a link between type 2 diabetes and vascular NADPH oxidase activity in the human arterial wall: the regulatory role of perivascular adipose tissue. Diabetes, 64 (6), pp. 2207-2219. | Citations: 73 (Web of Science Lite) | Show Abstract | Read more

Oxidative stress plays a critical role in the vascular complications of type 2 diabetes. We examined the effect of type 2 diabetes on NADPH oxidase in human vessels and explored the mechanisms of this interaction. Segments of internal mammary arteries (IMAs) with their perivascular adipose tissue (PVAT) and thoracic adipose tissue were obtained from 386 patients undergoing coronary bypass surgery (127 with type 2 diabetes). Type 2 diabetes was strongly correlated with hypoadiponectinemia and increased vascular NADPH oxidase-derived superoxide anions (O2˙(-)). The genetic variability of the ADIPOQ gene and circulating adiponectin (but not interleukin-6) were independent predictors of NADPH oxidase-derived O2˙(-). However, adiponectin expression in PVAT was positively correlated with vascular NADPH oxidase-derived O2˙(-). Recombinant adiponectin directly inhibited NADPH oxidase in human arteries ex vivo by preventing the activation/membrane translocation of Rac1 and downregulating p22(phox) through a phosphoinositide 3-kinase/Akt-mediated mechanism. In ex vivo coincubation models of IMA/PVAT, the activation of arterial NADPH oxidase triggered a peroxisome proliferator-activated receptor-γ-mediated upregulation of the adiponectin gene in the neighboring PVAT via the release of vascular oxidation products. We demonstrate for the first time in humans that reduced adiponectin levels in individuals with type 2 diabetes stimulates vascular NADPH oxidase, while PVAT "senses" the increased NADPH oxidase activity in the underlying vessel and responds by upregulating adiponectin gene expression. This PVAT-vessel interaction is identified as a novel therapeutic target for the prevention of vascular complications of type 2 diabetes.

Maniam S, Coutts AS, Stratford MR, McGouran J, Kessler B, La Thangue NB. 2015. Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase. Cell Death Differ, 22 (1), pp. 156-163. | Citations: 7 (Web of Science Lite) | Show Abstract | Read more

Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration.

Total publications on this page: 26

Total citations for publications on this page: 521