Publications 2018

Lee R, Charles PD, Lapolla P, Cassimjee I, Huang H, Kessler B, Fischer R, Handa A, Oxford Abdominal Aortic Aneurysm Study, the Oxford Regional Vascular Services. 2018. Integrated Physiological and Biochemical Assessments for the Prediction of Growth of Abdominal Aortic Aneurysms in Humans. Ann Surg, | Read more

Nishi R, Wijnhoven PWG, Kimura Y, Matsui M, Konietzny R, Wu Q, Nakamura K, Blundell TL, Kessler BM. 2018. The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage. Sci Rep, 8 (1), pp. 17891. | Show Abstract | Read more

Non-homologous end-joining (NHEJ), which can promote genomic instability when dysfunctional, is a major DNA double-strand break (DSB) repair pathway. Although ubiquitylation of the core NHEJ factor, Ku (Ku70-Ku80), which senses broken DNA ends, is important for its removal from sites of damage upon completion of NHEJ, the mechanism regulating Ku ubiquitylation remains elusive. We provide evidence showing that the ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) interacts with and directly deubiquitylates one of the Ku heterodimer subunits, Ku80. Additionally, depleting UCHL3 resulted in reduced Ku80 foci formation, Ku80 binding to chromatin after DSB induction, moderately sensitized cells to ionizing radiation and decreased NHEJ efficiencies. Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity. Collectively, these data suggest that UCHL3 facilitates cellular viability after DSB induction by antagonizing Ku80 ubiquitylation to enhance Ku80 retention at sites of damage.

Yuzhalin AE, Gordon-Weeks AN, Tognoli ML, Jones K, Markelc B, Konietzny R, Fischer R, Muth A, O'Neill E, Thompson PR et al. 2018. Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix. Nat Commun, 9 (1), pp. 4783. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.

Buono M, Thézénas M-L, Ceroni A, Fischer R, Nerlov C. 2018. Bi-directional signaling by membrane-bound KitL induces proliferation and coordinates thymic endothelial cell and thymocyte expansion. Nat Commun, 9 (1), pp. 4685. | Show Abstract | Read more

The ligand for the c-Kit receptor, KitL, exists as a membrane-associated (mKitL) and a soluble form (sKitL). KitL functions outside c-Kit activation have not been identified. We show that co-culture of c-Kit- and mKitL-expressing NIH3T3 cells results in signaling through mKitL: c-Kit-bound mKitL recruits calcium-modulating cyclophilin ligand (CAML) to selectively activate Akt, leading to CREB phosphorylation, mTOR pathway activation, and increased cell proliferation. Activation of mKitL in thymic vascular endothelial cells (VECs) induces mKitL- and Akt-dependent proliferation, and genetic ablation of mKitL in thymic VECs blocks their c-Kit responsiveness and proliferation during neonatal thymic expansion. Therefore, mKitL-c-Kit form a bi-directional signaling complex that acts in the developing thymus to coordinate thymic VEC and early thymic progenitor (ETP) expansion by simultaneously promoting ETP survival and VEC proliferation. This mechanism may be relevant to both normal tissues and malignant tumors that depend on KitL-c-Kit signaling for their proliferation.

Thompson AG, Gray E, Mager I, Fischer R, Thézénas M-L, Charles PD, Talbot K, El Andaloussi S, Kessler BM, Wood M, Turner MR. 2018. UFLC-Derived CSF Extracellular Vesicle Origin and Proteome. Proteomics, 18 (24), pp. e1800257. | Show Abstract | Read more

Cerebrospinal fluid (CSF) extracellular vesicles (EVs) show promise as a source of neurological disease biomarkers, although their precise origin is poorly understood. Current extraction techniques produce disappointing yield and purity. This study describes the application of ultrafiltration LC (UFLC) to CSF-EVs, compared with ultracentrifugation (UC), and explores CSF-EV origin. EVs are extracted from human CSF by UC and UFLC and characterized using nanoparticle tracking analysis, electron microscopy, and immunoblotting. EV and CSF proteomes are analyzed by LC-MS/MS. UFLC-isolated particles have size, morphology, and marker expression characteristic of EVs. UFLC provides greater EV yield (UFLC 7.90 × 108  ± SD 1.31 × 108 EVs mL-1 CSF, UC 1.06 × 108  ± 0.57 × 108 p < 0.001). UFLC enhances purity, proteomic depth (UFLC 622 ± 49, UC 298 ± 50, p = 0.001), and consistency of quantification (CV 17% vs 23%). EVs contain more intracellular proteins (Odds ratio [OR] 2.63 p < 0.001) and fewer plasma proteins than CSF (OR 0.60, p < 0.001). CSF and EV-enriched proteomes show overrepresentation of brain-specific proteins (EV OR 3.18, p < 0.001; CSF OR 3.37, p < 0.001). Overrepresentation of cerebral white matter (OR 1.99, p = 0.015) and choroid plexus proteins (OR 1.87, p<0.001) is observed in EVs. UFLC improves yield and purity of CSF-EVs. The EV-enriched proteome better reflects the intracellular and white matter proteome than whole CSF.

Howells A, McGouran JF, Kessler BM, Luhn K. 2018. Dengue virus stimulation of dendritic cells induces phosphorylation- and proteolysis-dependent signalling Malaysian Journal of Microbiology, 14 (Specialissue6), pp. 474-482. | Show Abstract | Read more

© 2018 Universiti Sains Malaysia. Aims: Dengue virus (DENV) infection is commonly observed in countries with tropical climates and remains a significant health hazard. No real cure has been established for the infection to date. Methodology and results: To better understand the very early molecular events during the initial infection process, we exposed primary dendritic cells with Dengue virus and analysed proteins with increased phosphorylation signatures in the first 10 min using phospho-protein enrichment and tandem mass spectrometry analysis. Upon initial viral interaction, strong phosphorylation was observed for Endoplasmin, BiP, BID, Dok-2, GEF-H1 and Calpain-2. Reduced phosphorylation was noted for Importin-5, ERp72 and Rho-GDI. Knockdown of Calpain-2, a protease activated by calcium flux, reduced DENV infection rates of primary dendritic cells as measured by focus-forming units (FFUs). Conclusion, significance and impact of study: We conclude that Calpain-2, BID, Importin 5 and ATP/GTPases are all active along the apoptosis pathway axis, indicating that dendritic cells commit to early signalling steps of cell death upon initial viral contact.

Jailani NM, Abdu Sani AA, Fischer R, Huang H, Aizat WM, Nathan S, Kramer HB, Kessler BM, Mackeen MM. 2018. Comparison of gel-aided sample preparation (GASP) and two in-solution digestion workflows for proteomic analysis using Saccharomyces cerevisiae lysate Malaysian Journal of Microbiology, 14 (Specialissue6), pp. 579-584. | Show Abstract | Read more

© 2018 Universiti Sains Malaysia. Using Saccharomyces cerevisiae lysate, two in-solution trypsin digestions (chloroform-methanol-water precipitation and RapiGest) were compared to the recently reported gel-aided sample preparation (GASP) workflow. Our proteomic results showed that GASP afforded the highest number of overall protein identifications and peptide spectrum matches without systematic bias towards peptide or protein size.

D’Ascenzio M, Pugh K, Konietzny R, Berridge G, Tallant C, Hashem S, Monteiro O, Thomas J, Schirle M, Knapp S et al. 2018. An Activity-based Probe Targeting Non-catalytic, Highly Conserved Amino Acid Residues Within Bromodomains | Show Abstract | Read more

<b>Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from physiological recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule based approach to covalently modify recombinant and endogenous bromodomain-containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag, via copper-catalyzed alkyne azide coupling, to an alkyne handle on the probe allowed in-gel visualization and selective pull-down of the desired bromodomains using both recombinant and endogenous proteins.</b>

Hendy J, Colonese AC, Franz I, Fernandes R, Fischer R, Orton D, Lucquin A, Spindler L, Anvari J, Stroud E et al. 2018. Ancient proteins from ceramic vessels at Çatalhöyük West reveal the hidden cuisine of early farmers. Nat Commun, 9 (1), pp. 4064. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

The analysis of lipids (fats, oils and waxes) absorbed within archaeological pottery has revolutionized the study of past diets and culinary practices. However, this technique can lack taxonomic and tissue specificity and is often unable to disentangle signatures resulting from the mixing of different food products. Here, we extract ancient proteins from ceramic vessels from the West Mound of the key early farming site of Çatalhöyük in Anatolia, revealing that this community processed mixes of cereals, pulses, dairy and meat products, and that particular vessels may have been reserved for specialized foods (e.g., cow milk and milk whey). Moreover, we demonstrate that dietary proteins can persist on archaeological artefacts for at least 8000 years, and that this approach can reveal past culinary practices with more taxonomic and tissue-specific clarity than has been possible with previous biomolecular techniques.

Mavrommati I, Faedda R, Galasso G, Li J, Burdova K, Fischer R, Kessler BM, Carrero ZI, Guardavaccaro D, Pagano M, D'Angiolella V. 2018. β-TrCP- and Casein Kinase II-Mediated Degradation of Cyclin F Controls Timely Mitotic Progression. Cell Rep, 24 (13), pp. 3404-3412. | Show Abstract | Read more

Orderly progressions of events in the cell division cycle are necessary to ensure the replication of DNA and cell division. Checkpoint systems allow the accurate execution of each cell-cycle phase. The precise regulation of the levels of cyclin proteins is fundamental to coordinate cell division with checkpoints, avoiding genome instability. Cyclin F has important functions in regulating the cell cycle during the G2 checkpoint; however, the mechanisms underlying the regulation of cyclin F are poorly understood. Here, we observe that cyclin F is regulated by proteolysis through β-TrCP. β-TrCP recognizes cyclin F through a non-canonical degron site (TSGXXS) after its phosphorylation by casein kinase II. The degradation of cyclin F mediated by β-TrCP occurs at the G2/M transition. This event is required to promote mitotic progression and favors the activation of a transcriptional program required for mitosis.

Moussa EM, Huang H, Thézénas ML, Fischer R, Ramaprasad A, Sisay-Joof F, Jallow M, Pain A, Kwiatkowski D, Kessler BM, Casals-Pascual C. 2018. Proteomic profiling of the plasma of Gambian children with cerebral malaria. Malar J, 17 (1), pp. 337. | Show Abstract | Read more

BACKGROUND: Cerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection. A number of pathological findings have been correlated with pediatric CM including sequestration, platelet accumulation, petechial haemorrhage and retinopathy. However, the molecular mechanisms leading to death in CM are not yet fully understood. METHODS: A shotgun plasma proteomic study was conducted using samples form 52 Gambian children with CM admitted to hospital. Based on clinical outcome, children were assigned to two groups: reversible and fatal CM. Label-free liquid chromatography-tandem mass spectrometry was used to identify and compare plasma proteins that were differentially regulated in children who recovered from CM and those who died. Candidate biomarkers were validated using enzyme immunoassays. RESULTS: The plasma proteomic signature of children with CM identified 266 proteins differentially regulated in children with fatal CM. Proteins from the coagulation cascade were consistently decreased in fatal CM, whereas the plasma proteomic signature associated with fatal CM underscored the importance of endothelial activation, tissue damage, inflammation, haemolysis and glucose metabolism. The concentration of circulating proteasomes or PSMB9 in plasma was not significantly different in fatal CM when compared with survivors. Plasma PSMB9 concentration was higher in patients who presented with seizures and was significantly correlated with the number of seizures observed in patients with CM during admission. CONCLUSIONS: The results indicate that increased tissue damage and hypercoagulability may play an important role in fatal CM. The diagnostic value of this molecular signature to identify children at high risk of dying to optimize patient referral practices should be validated prospectively.

Bailey J, Davis S, Shaw A, Diotallevi M, Fischer R, Benson MA, Zhu H, Brown J, Bhattacharya S, Kessler BM et al. 2018. Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation. Sci Rep, 8 (1), pp. 14310. | Show Abstract | Read more

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics "biotin-switch" approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.

Vazquez-Rodriguez S, Wright M, Rogers C, Cribbs A, Velupillai S, Philpott M, Lee H, Dunford J, Huber K, Robers M et al. 2018. Design, Synthesis and Characterization of Covalent KDM5 Inhibitors | Show Abstract | Read more

Histone lysine demethylase (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

Zhang T, Wei G, Millard CJ, Fischer R, Konietzny R, Kessler BM, Schwabe JWR, Brockdorff N. 2018. A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes. Nat Commun, 9 (1), pp. 3798. | Show Abstract | Read more

Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, here we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/β, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3 containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.

Motnenko A, Liang C-C, Yang D, Lopez-Martinez D, Yoshikawa Y, Zhan B, Ward KE, Tian J, Haas W, Spingardi P et al. 2018. Identification of UHRF2 as a novel DNA interstrand crosslink sensor protein. PLoS Genet, 14 (10), pp. e1007643. | Show Abstract | Read more

The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.

Berridge G, Menassa DA, Moloney T, Waters PJ, Welding I, Thomsen S, Zuberi S, Fischer R, Aricescu AR, Pike M et al. 2018. Glutamate receptor δ2 serum antibodies in pediatric opsoclonus myoclonus ataxia syndrome. Neurology, 91 (8), pp. e714-e723. | Citations: 3 (Scopus) | Show Abstract | Read more

OBJECTIVE: To identify neuronal surface antibodies in opsoclonus myoclonus ataxia syndrome (OMAS) using contemporary antigen discovery methodology. METHODS: OMAS patient serum immunoglobulin G immunohistochemistry using age-equivalent rat cerebellar tissue was followed by immunoprecipitation, gel electrophoresis, and mass spectrometry. Data are available via ProteomeXchange (identifier PXD009578). This generated a list of potential neuronal surface cerebellar autoantigens. Live cell-based assays were used to confirm membrane-surface antigens and adsorb antigen-specific immunoglobulin Gs. The serologic results were compared to the clinical data. RESULTS: Four of the 6 OMAS sera tested bound rat cerebellar sections. Two of these sera with similar immunoreactivities were used in immunoprecipitation experiments using cerebellum from postnatal rat pups (P18). Mass spectrometry identified 12 cell-surface proteins, of which glutamate receptor δ2 (GluD2), a predominately cerebellar-expressed protein, was found at a 3-fold-higher concentration than the other 11 proteins. Antibodies to GluD2 were identified in 14/16 (87%) OMAS samples, compared with 5/139 (5%) pediatric and 1/38 (2.6%) adult serum controls (p < 0.0001), and in 2/4 sera from patients with neuroblastoma without neurologic features. Adsorption of positive OMAS sera against GluD2-transfected cells substantially reduced but did not eliminate reactivity toward cerebellar sections. CONCLUSION: Autoantibodies to GluD2 are common in patients with OMAS, bind to surface determinants, and are potentially pathogenic.

Ghezraoui H, Oliveira C, Becker JR, Bilham K, Moralli D, Anzilotti C, Fischer R, Deobagkar-Lele M, Sanchiz-Calvo M, Fueyo-Marcos E et al. 2018. 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ. Nature, 560 (7716), pp. 122-127. | Citations: 14 (Scopus) | Show Abstract | Read more

53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5-7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.

Hendy J, Warinner C, Bouwman A, Collins MJ, Fiddyment S, Fischer R, Hagan R, Hofman CA, Holst M, Chaves E et al. 2018. Proteomic evidence of dietary sources in ancient dental calculus. Proc Biol Sci, 285 (1883), pp. 20180977-20180977. | Citations: 2 (Web of Science Lite) | Show Abstract | Read more

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein β-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation.

Li L, Friedrichsen HJ, Andrews S, Picaud S, Volpon L, Ngeow K, Berridge G, Fischer R, Borden KLB, Filippakopoulos P, Goding CR. 2018. A TFEB nuclear export signal integrates amino acid supply and glucose availability. Nat Commun, 9 (1), pp. 2685. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3β phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3β is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.

Markolovic S, Zhuang Q, Wilkins SE, Eaton CD, Abboud MI, Katz MJ, McNeil HE, Leśniak RK, Hall C, Struwe WB et al. 2018. Publisher Correction: The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases. Nat Chem Biol, 14 (10), pp. 988. | Show Abstract | Read more

In the version of this article initially published, authors Sarah E. Wilkins, Charlotte D. Eaton, Martine I. Abboud and Maximiliano J. Katz were incorrectly included in the equal contributions footnote in the affiliations list. Footnote number seven linking to the equal contributions statement should be present only for Suzana Markolovic and Qinqin Zhuang, and the statement should read "These authors contributed equally: Suzana Markolovic, Qinqin Zhuang." The error has been corrected in the HTML and PDF versions of the article.

Markolovic S, Zhuang Q, Wilkins SE, Eaton CD, Abboud MI, Katz MJ, McNeil HE, Leśniak RK, Hall C, Struwe WB et al. 2018. The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases. Nat Chem Biol, 14 (7), pp. 688-695. | Citations: 4 (Web of Science Lite) | Show Abstract | Read more

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(II)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.

Psallidas I, Kanellakis NI, Gerry S, Thézénas ML, Charles PD, Samsonova A, Schiller HB, Fischer R, Asciak R, Hallifax RJ et al. 2018. Development and validation of response markers to predict survival and pleurodesis success in patients with malignant pleural effusion (PROMISE): a multicohort analysis. Lancet Oncol, 19 (7), pp. 930-939. | Citations: 5 (Scopus) | Show Abstract | Read more

BACKGROUND: The prevalence of malignant pleural effusion is increasing worldwide, but prognostic biomarkers to plan treatment and to understand the underlying mechanisms of disease progression remain unidentified. The PROMISE study was designed with the objectives to discover, validate, and prospectively assess biomarkers of survival and pleurodesis response in malignant pleural effusion and build a score that predicts survival. METHODS: In this multicohort study, we used five separate and independent datasets from randomised controlled trials to investigate potential biomarkers of survival and pleurodesis. Mass spectrometry-based discovery was used to investigate pleural fluid samples for differential protein expression in patients from the discovery group with different survival and pleurodesis outcomes. Clinical, radiological, and biological variables were entered into least absolute shrinkage and selection operator regression to build a model that predicts 3-month mortality. We evaluated the model using internal and external validation. FINDINGS: 17 biomarker candidates of survival and seven of pleurodesis were identified in the discovery dataset. Three independent datasets (n=502) were used for biomarker validation. All pleurodesis biomarkers failed, and gelsolin, macrophage migration inhibitory factor, versican, and tissue inhibitor of metalloproteinases 1 (TIMP1) emerged as accurate predictors of survival. Eight variables (haemoglobin, C-reactive protein, white blood cell count, Eastern Cooperative Oncology Group performance status, cancer type, pleural fluid TIMP1 concentrations, and previous chemotherapy or radiotherapy) were validated and used to develop a survival score. Internal validation with bootstrap resampling and external validation with 162 patients from two independent datasets showed good discrimination (C statistic values of 0·78 [95% CI 0·72-0·83] for internal validation and 0·89 [0·84-0·93] for external validation of the clinical PROMISE score). INTERPRETATION: To our knowledge, the PROMISE score is the first prospectively validated prognostic model for malignant pleural effusion that combines biological and clinical parameters to accurately estimate 3-month mortality. It is a robust, clinically relevant prognostic score that can be applied immediately, provide important information on patient prognosis, and guide the selection of appropriate management strategies. FUNDING: European Respiratory Society, Medical Research Funding-University of Oxford, Slater & Gordon Research Fund, and Oxfordshire Health Services Research Committee Research Grants.

Huang H, van Dullemen LFA, Akhtar MZ, Faro M-LL, Yu Z, Valli A, Dona A, Thézénas M-L, Charles PD, Fischer R et al. 2018. Proteo-metabolomics reveals compensation between ischemic and non-injured contralateral kidneys after reperfusion. Sci Rep, 8 (1), pp. 8539. | Show Abstract | Read more

Ischaemia and reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI), which contributes to high morbidity and mortality rates in a wide range of injuries as well as the development of chronic kidney disease. The cellular and molecular responses of the kidney to IRI are complex and not fully understood. Here, we used an integrated proteomic and metabolomic approach to investigate the effects of IRI on protein abundance and metabolite levels. Rat kidneys were subjected to 45 min of warm ischaemia followed by 4 h and 24 h reperfusion, with contralateral and separate healthy kidneys serving as controls. Kidney tissue proteomics after IRI revealed elevated proteins belonging to the acute phase response, coagulation and complement pathways, and fatty acid (FA) signalling. Metabolic changes were already evident after 4 h reperfusion and showed increased level of glycolysis, lipids and FAs, whilst mitochondrial function and ATP production was impaired after 24 h. This deficit was partially compensated for by the contralateral kidney. Such a metabolic balance counteracts for the developing energy deficit due to reduced mitochondrial function in the injured kidney.

Ternette N, Olde Nordkamp MJM, Müller J, Anderson AP, Nicastri A, Hill AVS, Kessler BM, Li D. 2018. Immunopeptidomic Profiling of HLA-A2-Positive Triple Negative Breast Cancer Identifies Potential Immunotherapy Target Antigens. Proteomics, 18 (12), pp. e1700465. | Citations: 3 (European Pubmed Central) | Show Abstract | Read more

The recent development in immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cells in the treatment of cancer has not only demonstrated the potency of utilizing T-cell reactivity for cancer therapy, but has also highlighted the need for developing new approaches to discover targets suitable for such novel therapeutics. Here we analyzed the immunopeptidomes of six HLA-A2-positive triple negative breast cancer (TNBC) samples by nano-ultra performance liquid chromatography tandem mass spectrometry (nUPLC-MS2 ). Immunopeptidomic profiling identified a total of 19 675 peptides from tumor and adjacent normal tissue and 130 of the peptides were found to have higher abundance in tumor than in normal tissues. To determine potential therapeutic target proteins, we calculated the average tumor-associated cohort coverage (aTaCC) that represents the percentage coverage of each protein in this cohort by peptides that had higher tumoral abundance. Cofilin-1 (CFL-1), interleukin-32 (IL-32), proliferating cell nuclear antigen (PCNA), syntenin-1 (SDCBP), and ribophorin-2 (RPN-2) were found to have the highest aTaCC scores. We propose that these antigens could be evaluated further for their potential as targets in breast cancer immunotherapy and the small cohort immunopeptidomics analysis technique could be used in a wide spectrum of target discovery. Data are available via ProteomeXchange with identifier PXD009738.

Dimitrova E, Kondo T, Feldmann A, Nakayama M, Koseki Y, Konietzny R, Kessler BM, Koseki H, Klose RJ. 2018. FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment. Elife, 7 | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment.

Hinrichs A, Kessler B, Kurome M, Blutke A, Kemter E, Bernau M, Scholz AM, Rathkolb B, Renner S, Bultmann S et al. 2018. Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver. Mol Metab, 11 pp. 113-128. | Show Abstract | Read more

OBJECTIVE: Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized. METHODS: CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue. RESULTS: GHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2. CONCLUSION: GHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials.

Partridge T, Nicastri A, Kliszczak AE, Yindom L-M, Kessler BM, Ternette N, Borrow P. 2018. Discrimination Between Human Leukocyte Antigen Class I-Bound and Co-Purified HIV-Derived Peptides in Immunopeptidomics Workflows. Front Immunol, 9 (APR), pp. 912. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

Elucidation of novel peptides presented by human leukocyte antigen (HLA) class I alleles by immunopeptidomics constitutes a powerful approach that can inform the rational design of CD8+ T cell inducing vaccines to control infection with pathogens such as human immunodeficiency virus type 1 (HIV-1) or to combat tumors. Recent advances in the sensitivity of liquid chromatography tandem mass spectrometry instrumentation have facilitated the discovery of thousands of natural HLA-restricted peptides in a single measurement. However, the extent of contamination of class I-bound peptides identified using HLA immunoprecipitation (IP)-based immunopeptidomics approaches with peptides from other sources has not previously been evaluated in depth. Here, we investigated the specificity of the IP-based immunopeptidomics methodology using HLA class I- or II-deficient cell lines and membrane protein-specific antibody IPs. We demonstrate that the 721.221 B lymphoblastoid cell line, widely regarded to be HLA class Ia-deficient, actually expresses and presents peptides on HLA-C*01:02. Using this cell line and the C8166 (HLA class I- and II-expressing) cell line, we show that some HLA class II-bound peptides were co-purified non-specifically during HLA class I and membrane protein IPs. Furthermore, IPs of "irrelevant" membrane proteins from HIV-1-infected HLA class I- and/or II-expressing cells revealed that unusually long HIV-1-derived peptides previously reported by us and other immunopeptidomics studies as potentially novel CD8+ T cell epitopes were non-specifically co-isolated, and so constitute a source of contamination in HLA class I IPs. For example, a 16-mer (FLGKIWPSYKGRPGNF), which was detected in all samples studied represents the full p1 segment of the abundant intracellular or virion-associated proteolytically-processed HIV-1 Gag protein. This result is of importance, as these long co-purified HIV-1 Gag peptides may not elicit CD8+ T cell responses when incorporated into candidate vaccines. These results have wider implications for HLA epitope discovery from abundant or membrane-associated antigens by immunopeptidomics in the context of infectious diseases, cancer, and autoimmunity.

Dimitrova E, Kondo T, Feldmann A, Nakayama M, Koseki Y, Konietzny R, Kessler B, Koseki H, Klose R. 2018. FBXL19 recruits CDK-Mediator to CpG islands of developmental genes to prime them for activation during lineage commitment | Show Abstract | Read more

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment.

Aaboud M, Aad G, Abbott B, Abdinov O, Abeloos B, Abidi SH, Abouzeid OS, Abraham NL, Abramowicz H, Abreu H et al. 2018. Combination of inclusive and differential tt¯ charge asymmetry measurements using ATLAS and CMS data at √s = 7 and 8 TeV Journal of High Energy Physics, 2018 (4), | Citations: 2 (Scopus) | Show Abstract | Read more

© CERN, for the benefit of the ATLAS-CMS Collaboration. This paper presents combinations of inclusive and differential measurements of the charge asymmetry (A C ) in top quark pair (tt) events with a lepton+jets signature by the ATLAS and CMS Collaborations, using data from LHC proton-proton collisions at centreof- mass energies of 7 and 8TeV. The data correspond to integrated luminosities of about 5 and 20 fb –1 for each experiment, respectively. The resulting combined LHC measurements of the inclusive charge asymmetry are A CLHC7 = 0:005±0:007 (stat) ±0:006 (syst) at 7TeV and A CLHC7 = 0:0055 ± 0:0023 (stat) ± 0:0025 (syst) at 8TeV. These values, as well as the combination of A C measurements as a function of the invariant mass of the [formula presented] system at 8TeV, are consistent with the respective standard model predictions.

Sepil I, Hopkins B, Dean R, Thezenas M-L, Charles P, Konietzny R, Fischer R, Kessler B, Wigby S. 2018. Quantitative proteomics identification of seminal fluid proteins in male Drosophila melanogaster | Show Abstract | Read more

Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and particularly in insects, modifying female physiology and behaviour. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance due to dilution in the female derived sample or rapid processing in females. Here we present a new and complementary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster male reproductive tissue, where Sfps are unprocessed, and highly abundant, and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analysed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data was consistent with 124 previously reported Sfps. We found 8 high confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 31 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.

Rohm M, Savic D, Ball V, Curtis MK, Bonham S, Fischer R, Legrave N, MacRae JI, Tyler DJ, Ashcroft FM. 2018. Cardiac Dysfunction and Metabolic Inflexibility in a Mouse Model of Diabetes Without Dyslipidemia. Diabetes, 67 (6), pp. 1057-1067. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

Diabetes is a well-established risk factor for heart disease, leading to impaired cardiac function and a metabolic switch toward fatty acid usage. In this study, we investigated if hyperglycemia/hypoinsulinemia in the absence of dyslipidemia is sufficient to drive these changes and if they can be reversed by restoring euglycemia. Using the βV59M mouse model, in which diabetes can be rapidly induced and reversed, we show that stroke volume and cardiac output were reduced within 2 weeks of diabetes induction. Flux through pyruvate dehydrogenase was decreased, as measured in vivo by hyperpolarized [1-13C]pyruvate MRS. Metabolomics showed accumulation of pyruvate, lactate, alanine, tricarboxyclic acid cycle metabolites, and branched-chain amino acids. Myristic and palmitoleic acid were decreased. Proteomics revealed proteins involved in fatty acid metabolism were increased, whereas those involved in glucose metabolism decreased. Western blotting showed enhanced pyruvate dehydrogenase kinase 4 (PDK4) and uncoupling protein 3 (UCP3) expression. Elevated PDK4 and UCP3 and reduced pyruvate usage were present 24 h after diabetes induction. The observed effects were independent of dyslipidemia, as mice showed no evidence of elevated serum triglycerides or lipid accumulation in peripheral organs (including the heart). The effects of diabetes were reversible, as glibenclamide therapy restored euglycemia, cardiac metabolism and function, and PDK4/UCP3 levels.

Mills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, Jedrychowski MP, Costa ASH, Higgins M, Hams E et al. 2018. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature, 556 (7699), pp. 113-117. | Citations: 71 (Web of Science Lite) | Show Abstract | Read more

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Fye HKS, Mrosso P, Bruce L, Thézénas M-L, Davis S, Fischer R, Rwegasira GL, Makani J, Kessler BM. 2018. A robust mass spectrometry method for rapid profiling of erythrocyte ghost membrane proteomes. Clin Proteomics, 15 (1), pp. 14. | Citations: 1 (Web of Science Lite) | Show Abstract | Read more

Background: Red blood cell (RBC) physiology is directly linked to many human disorders associated with low tissue oxygen levels or anemia including chronic obstructive pulmonary disease, congenital heart disease, sleep apnea and sickle cell anemia. Parasites such as Plasmodium spp. and phylum Apicomplexa directly target RBCs, and surface molecules within the RBC membrane are critical for pathogen interactions. Proteomics of RBC membrane 'ghost' fractions has therefore been of considerable interest, but protocols described to date are either suboptimal or too extensive to be applicable to a larger set of clinical cohorts. Methods: Here, we describe an optimised erythrocyte isolation protocol from blood, tested for various storage conditions and explored using different fractionation conditions for isolating ghost RBC membranes. Liquid chromatography mass spectrometry (LC-MS) analysis on a Q-Exactive Orbitrap instrument was used to profile proteins isolated from the comparative conditions. Data analysis was run on the MASCOT and MaxQuant platforms to assess their scope and diversity. Results: The results obtained demonstrate a robust method for membrane enrichment enabling consistent MS based characterisation of > 900 RBC membrane proteins in single LC-MS/MS analyses. Non-detergent based membrane solubilisation methods using the tissue and supernatant fractions of isolated ghost membranes are shown to offer effective haemoglobin removal as well as diverse recovery including erythrocyte membrane proteins of high and low abundance. Conclusions: The methods described in this manuscript propose a medium to high throughput framework for membrane proteome profiling by LC-MS of potential applicability to larger clinical cohorts in a variety of disease contexts.

Nithichanon A, Rinchai D, Buddhisa S, Saenmuang P, Kewcharoenwong C, Kessler B, Khaenam P, Chetchotisakd P, Maillere B, Robinson J et al. 2018. Immune Control of Burkholderia pseudomallei--Common, High-Frequency T-Cell Responses to a Broad Repertoire of Immunoprevalent Epitopes. Front Immunol, 9 pp. 484. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

Burkholderia pseudomallei (Bp) is an environmental bacterial pathogen that causes potentially lethal sepsis in susceptible individuals and is considered a Category B, Tier-1 biothreat agent. As such, it is crucial to gain an improved understanding of protective immunity and potential vaccine candidates. The nature of immune correlates dictating why most exposed individuals in endemic regions undergo asymptomatic seroconversion while others succumb to life-threatening sepsis is largely uncharted. Bp seroreactive, immunogenic proteins have previously been identified by antigen microarray. We here set out to conduct an analysis of T-cell recognition of the Bp immunome using serodominant antigens represented in the original antigen microarray, examining immune correlates of disease in healthy seropositive individuals and those with acute disease or in convalescence. By screening a library of 739 overlapping peptides representing the sequences of 20 different Bp antigens, we aimed to define immune correlates of protection at the level of immunoprevalent T-cell epitopes. Responses to a large number of epitopes were common in healthy seropositive individuals: we found remarkably broad responsiveness to Bp epitopes, with 235 of 739 peptides recognized by ≥80% of all tested donors. The cumulative response to Bp epitopes in healthy, seropositive, donors from this endemic region were of the order of thousands of spot forming cells per million cells, making Bp recognition a significant component of the T-cell repertoire. Noteworthy among our findings, analysis revealed 10 highly immunoprevalent T-cell epitopes, able to induce Bp-specific IFNγ responses that were high in responding T-cell frequency within the repertoire, and also common across individuals with different human leukocyte antigen types. Acute melioidosis patients showed poor T-cell responses to the immunoprevalent epitopes, but acquired responsiveness following recovery from infection. Our findings suggest that a large repertoire of CD4 T cells, high in frequency and with broad coverage of antigens and epitopes, is important in controlling Bp infection. This offers an attractive potential strategy for subunit or epitope-based vaccines.

Eaton JRO, Alenazi Y, Singh K, Davies G, Geis-Asteggiante L, Kessler B, Robinson CV, Kawamura A, Bhattacharya S. 2018. The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins. J Biol Chem, 293 (16), pp. 6134-6146. | Citations: 2 (Web of Science Lite) | Show Abstract | Read more

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm The CC chemokine-binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties.

Rieblinger B, Fischer K, Kind A, Saller BS, Baars W, Schuster M, Wolf-van Buerck L, Schäffler A, Flisikowska T, Kurome M et al. 2018. Strong xenoprotective function by single-copy transgenes placed sequentially at a permissive locus. Xenotransplantation, 25 (2), pp. e12382. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

BACKGROUND: Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals. METHODS: We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry. Resistance to complement-mediated cell lysis and caspase 3/7 activation were determined in vitro. RESULTS: The ROSA26 locus was retargeted efficiently, and animals were generated by nuclear transfer. RNA and protein analyses revealed abundant expression in all organs analyzed, including pancreatic beta cells. Transgenic porcine kidney fibroblasts were almost completely protected against complement-mediated lysis and showed reduced caspase 3/7 activation. CONCLUSION: Step-by-step placement enables highly expressed single-copy xenoprotective transgenes to be grouped at porcine ROSA26.

Fung E, Richter C, Yang H-B, Schäffer I, Fischer R, Kessler BM, Bassermann F, D'Angiolella V. 2018. FBXL13 directs the proteolysis of CEP192 to regulate centrosome homeostasis and cell migration. EMBO Rep, 19 (3), pp. e44799-e44799. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

Aberrant centrosome organisation with ensuing alterations of microtubule nucleation capacity enables tumour cells to proliferate and invade despite increased genomic instability. CEP192 is a key factor in the initiation process of centrosome duplication and in the control of centrosome microtubule nucleation. However, regulatory means of CEP192 have remained unknown. Here, we report that FBXL13, a binding determinant of SCF (SKP1-CUL1-F-box)-family E3 ubiquitin ligases, is enriched at centrosomes and interacts with the centrosomal proteins Centrin-2, Centrin-3, CEP152 and CEP192. Among these, CEP192 is specifically targeted for proteasomal degradation by FBXL13. Accordingly, induced FBXL13 expression downregulates centrosomal γ-tubulin and disrupts centrosomal microtubule arrays. In addition, depletion of FBXL13 induces high levels of CEP192 and γ-tubulin at the centrosomes with the consequence of defects in cell motility. Together, we characterise FBXL13 as a novel regulator of microtubule nucleation activity and highlight a role in promoting cell motility with potential tumour-promoting implications.

Kanellopoulos AH, Koenig J, Huang H, Pyrski M, Millet Q, Lolignier S, Morohashi T, Gossage SJ, Jay M, Linley JE et al. 2018. Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice. EMBO J, 37 (3), pp. 427-445. | Citations: 7 (Scopus) | Show Abstract | Read more

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope-tagged NaV1.7 mouse that showed normal pain behaviours to identify channel-interacting proteins. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing-response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane-trafficking protein synaptotagmin-2 (Syt2), L-type amino acid transporter 1 (Lat1) and transmembrane P24-trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co-immunoprecipitation (Co-IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein-regulated inducer of neurite outgrowth (Gprin1), an opioid receptor-binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope-tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel.

Thompson AG, Gray E, Thézénas M-L, Charles PD, Evetts S, Hu MT, Talbot K, Fischer R, Kessler BM, Turner MR. 2018. Cerebrospinal fluid macrophage biomarkers in amyotrophic lateral sclerosis. Ann Neurol, 83 (2), pp. 258-268. | Citations: 17 (Scopus) | Show Abstract | Read more

OBJECTIVE: The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is a heterogeneous clinical syndrome involving multiple molecular pathways. The development of biomarkers for use in therapeutic trials is a priority. We sought to use a high-throughput proteomic method to identify novel biomarkers in individual cerebrospinal fluid (CSF) samples. METHODS: Liquid chromatography/tandem mass spectrometry with label-free quantification was used to identify CSF proteins using samples from a well-characterized longitudinal cohort comprising patients with ALS (n = 43), the upper motor neuron variant, primary lateral sclerosis (PLS; n = 6), and cross-sectional healthy (n = 20) and disease controls (Parkinsons' disease, n = 20; ALS mimic disorders, n = 12). RESULTS: Three macrophage-derived chitinases showed increased abundance in ALS: chitotriosidase (CHIT1), chitinase-3-like protein 1 (CHI3L1), and chitinase-3-like protein 2 (CHI3L2). Elevated CHI3L1 was common to ALS and PLS, whereas CHIT1 and CHI3L2 levels differed. Chitinase levels correlated with disease progression rate (CHIT1, r = 0.56, p < 0.001; CHI3L1, r = 0.31; p = 0.028; CHI3L2, r = 0.29, p = 0.044). CHIT1, CHI3L1, and CHI3L2 levels correlated with phosphorylated neurofilament heavy chain (pNFH; r = 0.62, p < 0.001; r = 0.49, p < 0.001; r = 0.41, p < 0.001). CHI3L1 levels, but not CHIT1 or CHI3L2, increased over time in those with low initial levels (gradient = 0.005 log abundance units/month, p = 0.001). High CHIT1 was associated with shortened survival (hazard ratio [HR] 2.84; p = 0.009). Inclusion of pNFH in survival models left only an association of pNFH and survival (HR 1.26; p = 0.019). INTERPRETATION: Neuroinflammatory mechanisms have been consistently implicated through various experimental paradigms. These results support a key role for macrophage activity in ALS pathogenesis, offering novel target engagement and pharmacodynamic biomarkers for neuroinflammation-focused ALS therapy. Ann Neurol 2018;83:258-268.

Moussa E, Huang H, Ahras M, Lall A, Thezenas ML, Fischer R, Kessler BM, Pain A, Billker O, Casals-Pascual C. 2018. Proteomic profiling of the brain of mice with experimental cerebral malaria. J Proteomics, 180 pp. 61-69. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

Cerebral malaria (CM) is a severe neurological complication of malaria infection in both adults and children. In pursuit of effective treatment of CM, clinical studies, postmortem analysis and animal models have been employed to understand the pathology and identify effective interventions. In this study, a shotgun proteomics analysis was conducted to profile the proteomic signature of the brain tissue of mice with experimental cerebral malaria (ECM) in order to further understand the underlying pathology. To identify CM-associated response, proteomic signatures of the brains of C57/Bl6N mice infected with P. berghei ANKA that developed neurological syndrome were compared to those of mice infected with P. berghei NK65 that developed equally high parasite burdens without neurological signs, and to those of non-infected mice. The results show that the CM-associated response in mice that developed neurological signs comprise mainly acute-phase reaction and coagulation cascade activation, and indicate the leakage of plasma proteins into the brain parenchyma. SIGNIFICANCE: Cerebral malaria (CM) remains a major cause of death in children. The majority of these deaths occur in sub-Saharan Africa. Even with adequate access to treatment, mortality remains high and neurological sequelae can be found in up to 20% of survivors. No adjuvant treatment to date has been shown to reduce mortality and the pathophysiology of CM is largely unknown. Experimental cerebral malaria (ECM) is a well-established model that may contribute to identify and test druggable targets. In this study we have identified the disruption of the blood-brain barrier following inflammatory and vascular injury as a mechanism of disease. In this study we report a number of proteins that could be validated as potential biomarkers of ECM. Further studies, will be required to validate the clinical relevance of these biomarkers in human CM.

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