Publications 2016

New M, Sheikh S, Bekheet M, Olzscha H, Thezenas M-L, Care MA, Fotheringham S, Tooze RM, Kessler B, La Thangue NB. 2016. TLR Adaptor Protein MYD88 Mediates Sensitivity to HDAC Inhibitors via a Cytokine-Dependent Mechanism. Cancer Res, 76 (23), pp. 6975-6987. | Citations: 5 (Web of Science Lite) | Show Abstract | Read more

Histone deacetylase (HDAC) inhibitors have proven useful therapeutic agents for certain hematologic cancers. However, HDAC inhibition causes diverse cellular outcomes, and identification of cancer-relevant pathways within these outcomes remains unresolved. In this study, we utilized an unbiased loss-of-function screen and identified the Toll-like receptor (TLR) adaptor protein MYD88 as a key regulator of the antiproliferative effects of HDAC inhibition. High expression of MYD88 exhibited increased sensitivity to HDAC inhibitors; conversely, low expression coincided with reduced sensitivity. MYD88-dependent TLR signaling controlled cytokine levels, which then acted via an extracellular mechanism to maintain cell proliferation and sensitize cells to HDAC inhibition. MYD88 activity was directly regulated through lysine acetylation and was deacetylated by HDAC6. MYD88 was a component of a wider acetylation signature in the ABC subgroup of diffuse large B-cell lymphoma, and one of the most frequent mutations in MYD88, L265P, conferred increased cell sensitivity to HDAC inhibitors. Our study defines acetylation of MYD88, which, by regulating TLR-dependent signaling to cytokine genes, influences the antiproliferative effects of HDAC inhibitors. Our results provide a possible explanation for the sensitivity of malignancies of hematologic origin to HDAC inhibitor-based therapy. Cancer Res; 76(23); 6975-87. ©2016 AACR.

Vaz B, Popovic M, Newman JA, Fielden J, Aitkenhead H, Halder S, Singh AN, Vendrell I, Fischer R, Torrecilla I et al. 2016. Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair. Mol Cell, 64 (4), pp. 704-719. | Citations: 39 (Scopus) | Show Abstract | Read more

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

Wright TH, Bower BJ, Chalker JM, Bernardes GJL, Wiewiora R, Ng W-L, Raj R, Faulkner S, Vallée MRJ, Phanumartwiwath A et al. 2016. Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity. Science, 354 (6312), pp. aag1465-aag1465. | Citations: 62 (Web of Science Lite) | Show Abstract | Read more

Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp3)-C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cβ-Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

Keating SM, Heitman JW, Wu S, Deng X, Stacey AR, Zahn RC, de la Rosa M, Finstad SL, Lifson JD, Piatak M et al. 2016. Magnitude and Quality of Cytokine and Chemokine Storm during Acute Infection Distinguish Nonprogressive and Progressive Simian Immunodeficiency Virus Infections of Nonhuman Primates. J Virol, 90 (22), pp. 10339-10350. | Citations: 4 (Web of Science Lite) | Show Abstract | Read more

Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δnef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δnef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression. IMPORTANCE: NHP models of HIV infection constitute a powerful tool with which to study viral pathogenesis in order to gain critical information for a better understanding of HIV infection in humans. Here we studied progressive and nonprogressive simian immunodeficiency virus (SIV) infection models in both natural and nonnatural host NHP species. Regardless of the pathogenicity of the virus infection and regardless of the NHP species studied, the magnitude of viremia, as measured by area under the curve, during the first 4 weeks of infection correlated positively with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between nonprogressive and progressive SIV infection models. The qualitative differences in the early immune response in pathogenic and nonpathogenic infections identified here may be important determinants of the subsequent disease course.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2016. Restoring p53 Function in Human Melanoma Cells by Inhibiting MDM2 and Cyclin B1/CDK1-Phosphorylated Nuclear iASPP. Cancer Cell, 30 (5), pp. 822-823. | Citations: 7 (Web of Science Lite) | Show Abstract | Read more

© 2016 Elsevier Inc. (Cancer Cell 23, 618–633; May 13, 2013) In the original Figure 4G, two of the upper panels (His-iASPP(625-828)-FITC; the two rightmost panels) were inadvertently duplicated in the lower set of panels (His-ASPP2(905-1128)-FITC; third and fourth from left). This was a mistake made by the authors during the assembly of the figure. This error does not affect any of the findings reported in the paper. The corrected Figure 4 is presented below. The authors apologize for any confusion that this error may have caused. [figure presented]

Ding S, Medjahed H, Prévost J, Coutu M, Xiang S-H, Finzi A. 2016. Lineage-Specific Differences between the gp120 Inner Domain Layer 3 of Human Immunodeficiency Virus and That of Simian Immunodeficiency Virus. J Virol, 90 (22), pp. 10065-10073. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

Binding of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological layers (layer 1, layer 2, and layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to layer 1 of HIV-1 gp120, the SIVmac239 gp120 layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved tryptophan at position 375 (Trp 375) in HIV-2/SIVsmm. In this study, we investigated the role of SIV layer 3 in viral entry, cell-to-cell fusion, and CD4 binding. We observed that a network of interactions involving some residues of the β8-α5 region in SIVmac239 layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity, which directly contacts CD4. In summary, our results suggest that layer 3 in SIV has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in layer 3 from HIV-1 and that from SIV. IMPORTANCE: CD4-induced conformational changes in the gp120 inner domain involve rearrangements between three topological layers. While the role of layers 1 to 3 for HIV-1 and layers 1 and 2 for SIV on gp120 transition to the CD4-bound conformation has been reported, the role of SIV layer 3 remains unknown. Here we report that SIV layer 3 has a greater impact on CD4 binding than does layer 3 in HIV-1 gp120. This work defines lineage-specific differences in layer 3 from HIV-1 and SIV.

Rose NR, King HW, Blackledge NP, Fursova NA, Ember KJ, Fischer R, Kessler BM, Klose RJ. 2016. RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes. Elife, 5 | Citations: 12 (Web of Science Lite) | Show Abstract | Read more

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation.

Welker F, Hajdinjak M, Talamo S, Jaouen K, Dannemann M, David F, Julien M, Meyer M, Kelso J, Barnes I et al. 2016. Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne. Proc Natl Acad Sci U S A, 113 (40), pp. 11162-11167. | Citations: 38 (Scopus) | Show Abstract | Read more

In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this "transitional" assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal-Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne.

Demarchi B, Hall S, Roncal-Herrero T, Freeman CL, Woolley J, Crisp MK, Wilson J, Fotakis A, Fischer R, Kessler BM et al. 2016. Protein sequences bound to mineral surfaces persist into deep time. Elife, 5 (September), | Citations: 37 (Scopus) | Show Abstract | Read more

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C).

Kaisar M, van Dullemen LFA, Thézénas M-L, Zeeshan Akhtar M, Huang H, Rendel S, Charles PD, Fischer R, Ploeg RJ, Kessler BM. 2016. Plasma degradome affected by variable storage of human blood. Clin Proteomics, 13 (1), pp. 26. | Citations: 7 (Scopus) | Show Abstract | Read more

BACKGROUND: The successful application of-omics technologies in the discovery of novel biomarkers and targets of therapeutic interventions is facilitated by large collections of well curated clinical samples stored in bio banks. Mining the plasma proteome holds promise to improve our understanding of disease mechanisms and may represent a source of biomarkers. However, a major confounding factor for defining disease-specific proteomic signatures in plasma is the variation in handling and processing of clinical samples leading to protein degradation. To address this, we defined a plasma proteolytic signature (degradome) reflecting pre-analytical variability in blood samples that remained at ambient temperature for different time periods after collection and prior to processing. METHODS: We obtained EDTA blood samples from five healthy volunteers (n = 5), and blood tubes remained at ambient temperature for 30 min, 8, 24 and 48 h prior to centrifugation and isolation of plasma. Naturally occurred peptides derived from plasma samples were compared by label-free quantitative LC-MS/MS. To profile protein degradation, we analysed pooled plasma samples at T = 30 min and 48 h using PROTOMAP analysis. The proteolytic pattern of selected protein candidates was further validated by immunoblotting. RESULTS: A total of 820 plasma proteins were surveyed by PROTOMAP, and for 4 % of these, marked degradation was observed. We show distinct proteolysis patterns for talin-1, coagulation factor XI, complement protein C1r, C3, C4 and thrombospondin, and several proteins including S100A8, A9, annexin A1, profiling-1 and platelet glycoprotein V are enriched after 48 h blood storage at ambient temperature. In particular, thrombospondin protein levels increased after 8 h and proteolytic fragments appeared after 24 h storage time. CONCLUSIONS: The overall impact of blood storage at ambient temperature for variable times on the plasma proteome and degradome is relatively minor, but in some cases can cause a potential bias in identifying and assigning relevant proteomic markers. The observed effects on the plasma proteome and degradome are predominantly triggered by limited leucocyte and platelet cell activation due to blood handling and storage. The baseline plasma degradome signature presented here can help filtering candidate protein markers relevant for clinical biomarker studies.

Elliott PR, Leske D, Hrdinka M, Bagola K, Fiil BK, McLaughlin SH, Wagstaff J, Volkmar N, Christianson JC, Kessler BM et al. 2016. SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling. Mol Cell, 63 (6), pp. 990-1005. | Citations: 40 (Scopus) | Show Abstract | Read more

The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.

Miranda F, Mannion D, Liu S, Zheng Y, Mangala LS, Redondo C, Herrero-Gonzalez S, Xu R, Taylor C, Chedom DF et al. 2016. Salt-Inducible Kinase 2 Couples Ovarian Cancer Cell Metabolism with Survival at the Adipocyte-Rich Metastatic Niche. Cancer Cell, 30 (2), pp. 273-289. | Citations: 32 (Scopus) | Show Abstract | Read more

The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer.

Alexopoulou Z, Lang J, Perrett RM, Elschami M, Hurry MED, Kim HT, Mazaraki D, Szabo A, Kessler BM, Goldberg AL et al. 2016. Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease. Proc Natl Acad Sci U S A, 113 (32), pp. E4688-E4697. | Citations: 26 (Scopus) | Show Abstract | Read more

In Parkinson's disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. An important outstanding question is whether ubiquitination in Lewy bodies is directly relevant to α-synuclein trafficking or turnover and Parkinson's pathogenesis. By comparative analysis in human postmortem brains, we found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared with the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly colocalized with α-synuclein in endosomal membranes and, both in cells and after purification, it deubiquitinated K63-linked chains on α-synuclein. Knockdown of Usp8 in the Drosophila eye reduced α-synuclein levels and α-synuclein-induced eye toxicity. Accordingly, in human cells, Usp8 knockdown increased the lysosomal degradation of α-synuclein. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein-induced locomotor deficits and cell loss. These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism that reduces its lysosomal degradation in dopaminergic neurons and may contribute to α-synuclein accumulation in Lewy body disease.

Kaisar M, van Dullemen LFA, Thézénas M-L, Charles PD, Ploeg RJ, Kessler BM. 2016. Plasma Biomarker Profile Alterations during Variable Blood Storage. Clin Chem, 62 (9), pp. 1272-1274. | Citations: 1 (European Pubmed Central) | Read more

Pinto-Fernandez A, Kessler BM. 2016. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets. Front Genet, 7 (JUL), pp. 133. | Citations: 22 (Scopus) | Show Abstract | Read more

Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

Grijzenhout A, Godwin J, Koseki H, Gdula MR, Szumska D, McGouran JF, Bhattacharya S, Kessler BM, Brockdorff N, Cooper S. 2016. Functional analysis of AEBP2, a PRC2 Polycomb protein, reveals a Trithorax phenotype in embryonic development and in ESCs. Development, 143 (15), pp. 2716-2723. | Citations: 20 (Scopus) | Show Abstract | Read more

The Polycomb repressive complexes PRC1 and PRC2 are key mediators of heritable gene silencing in multicellular organisms. Here, we characterise AEBP2, a known PRC2 co-factor which, in vitro, has been shown to stimulate PRC2 activity. We show that AEBP2 localises specifically to PRC2 target loci, including the inactive X chromosome. Proteomic analysis confirms that AEBP2 associates exclusively with PRC2 complexes. However, analysis of embryos homozygous for a targeted mutation of Aebp2 unexpectedly revealed a Trithorax phenotype, normally linked to antagonism of Polycomb function. Consistent with this, we observe elevated levels of PRC2-mediated histone H3K27 methylation at target loci in Aebp2 mutant embryonic stem cells (ESCs). We further demonstrate that mutant ESCs assemble atypical hybrid PRC2 subcomplexes, potentially accounting for enhancement of Polycomb activity, and suggesting that AEBP2 normally plays a role in defining the mutually exclusive composition of PRC2 subcomplexes.

Mathea S, Abdul Azeez KR, Salah E, Tallant C, Wolfreys F, Konietzny R, Fischer R, Lou HJ, Brennan PE, Schnapp G et al. 2016. Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib. ACS Chem Biol, 11 (6), pp. 1595-1602. | Citations: 6 (Scopus) | Show Abstract | Read more

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here, we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The cocrystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions -1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs.

Raducu M, Fung E, Serres S, Infante P, Barberis A, Fischer R, Bristow C, Thézénas M-L, Finta C, Christianson JC et al. 2016. SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development. EMBO J, 35 (13), pp. 1400-1416. | Citations: 12 (European Pubmed Central) | Show Abstract | Read more

Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.

Akhtar MZ, Huang H, Kaisar M, Lo Faro ML, Rebolledo R, Morten K, Heather LC, Dona A, Leuvenink HG, Fuggle SV et al. 2016. Using an Integrated -Omics Approach to Identify Key Cellular Processes That Are Disturbed in the Kidney After Brain Death. Am J Transplant, 16 (5), pp. 1421-1440. | Citations: 3 (European Pubmed Central) | Show Abstract | Read more

In an era where we are becoming more reliant on vulnerable kidneys for transplantation from older donors, there is an urgent need to understand how brain death leads to kidney dysfunction and, hence, how this can be prevented. Using a rodent model of hemorrhagic stroke and next-generation proteomic and metabolomic technologies, we aimed to delineate which key cellular processes are perturbed in the kidney after brain death. Pathway analysis of the proteomic signature of kidneys from brain-dead donors revealed large-scale changes in mitochondrial proteins that were associated with altered mitochondrial activity and morphological evidence of mitochondrial injury. We identified an increase in a number of glycolytic proteins and lactate production, suggesting a shift toward anaerobic metabolism. Higher amounts of succinate were found in the brain death group, in conjunction with increased markers of oxidative stress. We characterized the responsiveness of hypoxia inducible factors and found this correlated with post-brain death mean arterial pressures. Brain death leads to metabolic disturbances in the kidney and alterations in mitochondrial function and reactive oxygen species generation. This metabolic disturbance and alteration in mitochondrial function may lead to further cellular injury. Conditioning the brain-dead organ donor by altering metabolism could be a novel approach to ameliorate this brain death-induced kidney injury.

Lochmatter C, Fischer R, Charles PD, Yu Z, Powrie F, Kessler BM. 2016. Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes. Sci Rep, 6 (1), pp. 24491. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn's disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation.

Chung VY, Konietzny R, Charles P, Kessler B, Fischer R, Turney BW. 2016. Proteomic changes in response to crystal formation in Drosophila Malpighian tubules. Fly (Austin), 10 (2), pp. 91-100. | Citations: 1 (Web of Science Lite) | Show Abstract | Read more

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.


Rahighi S, Braunstein I, Ternette N, Kessler B, Kawasaki M, Kato R, Matsui T, Weiss TM, Stanhill A, Wakatsuki S. 2016. Selective Binding of AIRAPL Tandem UIMs to Lys48-Linked Tri-Ubiquitin Chains. Structure, 24 (3), pp. 412-422. | Citations: 7 (Web of Science Lite) | Show Abstract | Read more

Lys48-linked ubiquitin chains act as the main targeting signals for protein degradation by the proteasome. Here we report selective binding of AIRAPL, a protein that associates with the proteasome upon exposure to arsenite, to Lys48-linked tri-ubiquitin chains. AIRAPL comprises two ubiquitin-interacting motifs in tandem (tUIMs) that are linked through a flexible inter-UIM region. In the complex crystal structure UIM1 binds the proximal ubiquitin, whereas UIM2 (the double-sided UIM) binds non-symmetrically to the middle and distal ubiquitin moieties on either side of the helix. Specificity of AIRAPL for Lys48-linked ubiquitin chains is determined by UIM2, and the flexible inter-UIM linker increases avidity by placing the two UIMs in an orientation that facilitates binding of the third ubiquitin to UIM1. Unlike middle and proximal ubiquitins, distal ubiquitin binds UIM2 through a novel surface, which leaves the Ile44 hydrophobic patch accessible for binding to the proteasomal ubiquitin receptors.

Ghari F, Quirke A-M, Munro S, Kawalkowska J, Picaud S, McGouran J, Subramanian V, Muth A, Williams R, Kessler B et al. 2016. Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response. Sci Adv, 2 (2), pp. e1501257. | Citations: 26 (Scopus) | Show Abstract | Read more

Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.

Keating SM, Heitman JW, Wu S, Deng X, Stacey AR, Zahn RC, de la Rosa M, Finstad SL, Lifson JD, Piatak M et al. 2016. Magnitude and quality of cytokine and chemokine storm during acute infection distinguish nonprogressive and progressive simian immunodeficiency virus infections of nonhuman primates Journal of Virology, 90 (22), pp. 10339-10350. | Citations: 2 (Scopus) | Show Abstract | Read more

© 2016, American Society for Microbiology. All Rights Reserved. Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δnef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony- stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP- 1β), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δnef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression.

Schwenzer A, Jiang X, Mikuls TR, Payne JB, Sayles HR, Quirke A-M, Kessler BM, Fischer R, Venables PJ, Lundberg K, Midwood KS. 2016. Identification of an immunodominant peptide from citrullinated tenascin-C as a major target for autoantibodies in rheumatoid arthritis. Ann Rheum Dis, 75 (10), pp. 1876-1883. | Citations: 16 (Web of Science Lite) | Show Abstract | Read more

OBJECTIVES: We investigated whether citrullinated tenascin-C (cTNC), an extracellular matrix protein expressed at high levels in the joints of patients with rheumatoid arthritis (RA), is a target for the autoantibodies in RA. METHODS: Citrullinated sites were mapped by mass spectrometry in the fibrinogen-like globe (FBG) domain of tenascin-C treated with peptidylarginine deiminases (PAD) 2 and 4. Antibodies to cyclic peptides containing citrullinated sites were screened in sera from patients with RA by ELISA. Potential cross-reactivity with well-established anticitrullinated protein antibody (ACPA) epitopes was tested by inhibition assays. The autoantibody response to one immunodominant cTNC peptide was then analysed in 101 pre-RA sera (median 7 years before onset) and two large independent RA cohorts. RESULTS: Nine arginine residues within FBG were citrullinated by PAD2 and PAD4. Two immunodominant peptides cTNC1 (VFLRRKNG-cit-ENFYQNW) and cTNC5 (EHSIQFAEMKL-cit-PSNF-cit-NLEG-cit-cit-KR) were identified. Antibodies to both showed limited cross-reactivity with ACPA epitopes from α-enolase, vimentin and fibrinogen, and no reactivity with citrullinated fibrinogen peptides sharing sequence homology with FBG. cTNC5 antibodies were detected in 18% of pre-RA sera, and in 47% of 1985 Swedish patients with RA and 51% of 287 North American patients with RA. The specificity was 98% compared with 160 healthy controls and 330 patients with osteoarthritis. CONCLUSIONS: There are multiple citrullination sites in the FBG domain of tenascin-C. Among these, one epitope is recognised by autoantibodies that are detected years before disease onset, and which may serve as a useful biomarker to identify ACPA-positive patients with high sensitivity and specificity in established disease.

Ying S, Chen Z, Medhurst AL, Neal JA, Bao Z, Mortusewicz O, McGouran J, Song X, Shen H, Hamdy FC et al. 2016. DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair. Cancer Res, 76 (5), pp. 1078-1088. | Citations: 29 (Scopus) | Show Abstract | Read more

A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved.

Ternette N, Yang H, Partridge T, Llano A, Cedeño S, Fischer R, Charles PD, Dudek NL, Mothe B, Crespo M et al. 2016. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells. Eur J Immunol, 46 (1), pp. 60-69. | Citations: 20 (Web of Science Lite) | Show Abstract | Read more

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T-cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4(+) T cells and the C8166 human T-cell line. Importantly, one-third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T-cell responses have previously been reported but for which the precise HLA class I-binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation during HIV-1 infection and provide novel targets for T-cell vaccine development.

Total publications on this page: 28

Total citations for publications on this page: 487