Publications 2019


Demarchi B, Presslee S, Gutiérrez-Zugasti I, González-Morales M, Marín-Arroyo AB, Straus LG, Fischer R. 2019. Birds of prey and humans in prehistoric Europe: A view from El Mirón Cave, Cantabria (Spain) Journal of Archaeological Science: Reports, 24 pp. 244-252. | Show Abstract | Read more

© 2019 Elsevier Ltd Bird eggs can become part of the archaeological record either accidentally or as a result of human activities but, in both instances, they can reveal important aspects of the environment at the site, the ways in which people chose to exploit it, and even the existence of subtle ecological balances between humans and other animals. This is the case for El Mirόn, one of the most important cave sites in Cantabrian Spain, with occupation levels spanning around 40,000 years, from the late Middle Palaeolithic to the Bronze Age. This mountainous area in Cantabria was an ideal environment for hunting medium-sized game and, as such, supported both human and non-human predators, including birds of prey. Here we use a combination of peptide mass fingerprinting (by MALDI-MS) and protein sequencing (by LC-MS/MS) in order to taxonomically identify ninety-five fragments of eggshells recovered from nineteen archaeological layers. We firmly identify these as diurnal birds of prey (Accipitridae) and suggest that the species might have been bearded vulture, based on previous taphonomic studies that highlighted its presence at the cave. The implication is that both species of diurnal predators, humans and birds, inhabited the cave and used the surrounding environment during different periods of the year.

Sapmaz A, Berlin I, Bos E, Wijdeven RH, Janssen H, Konietzny R, Akkermans JJ, Erson-Bensan AE, Koning RI, Kessler BM et al. 2019. USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7. Nat Commun, 10 (1), pp. 1454. | Show Abstract | Read more

The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7-the logistical centerpiece of LE biology-as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system.

Valli A, Morotti M, Zois CE, Albers PK, Soga T, Feldinger K, Fischer R, Frejno M, McIntyre A, Bridges E et al. 2019. Adaptation to HIF1α Deletion in Hypoxic Cancer Cells by Upregulation of GLUT14 and Creatine Metabolism. Mol Cancer Res, pp. molcanres.0315.2018-molcanres.0315.2018. | Show Abstract | Read more

Hypoxia-inducible factor 1α is a key regulator of the hypoxia response in normal and cancer tissues. It is well recognized to regulate glycolysis and is a target for therapy. However, how tumor cells adapt to grow in the absence of HIF1α is poorly understood and an important concept to understand for developing targeted therapies is the flexibility of the metabolic response to hypoxia via alternative pathways. We analyzed pathways that allow cells to survive hypoxic stress in the absence of HIF1α, using the HCT116 colon cancer cell line with deleted HIF1α versus control. Spheroids were used to provide a 3D model of metabolic gradients. We conducted a metabolomic, transcriptomic, and proteomic analysis and integrated the results. These showed surprisingly that in three-dimensional growth, a key regulatory step of glycolysis is Aldolase A rather than phosphofructokinase. Furthermore, glucose uptake could be maintained in hypoxia through upregulation of GLUT14, not previously recognized in this role. Finally, there was a marked adaptation and change of phosphocreatine energy pathways, which made the cells susceptible to inhibition of creatine metabolism in hypoxic conditions. Overall, our studies show a complex adaptation to hypoxia that can bypass HIF1α, but it is targetable and it provides new insight into the key metabolic pathways involved in cancer growth.Implications: Under hypoxia and HIF1 blockade, cancer cells adapt their energy metabolism via upregulation of the GLUT14 glucose transporter and creatine metabolism providing new avenues for drug targeting.

Leimbacher P-A, Jones SE, Shorrocks A-MK, de Marco Zompit M, Day M, Blaauwendraad J, Bundschuh D, Bonham S, Fischer R, Fink D et al. 2019. MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis. Mol Cell, | Show Abstract | Read more

In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.

Ji C, Kriaucionis S, Kessler BM, Jiang C. 2019. From herbal small RNAs to one medicine. Sci China Life Sci, 62 (3), pp. 285-287. | Read more

Yu Z, Huang H, Zhang H, Kessler BM. 2019. Improved profiling of polyamines using two-dimensional gas chromatography mass spectrometry. Talanta, 199 pp. 184-188. | Show Abstract | Read more

Polyamines are a class of poly-cationic aliphatic amines, playing a role in different cellular processes such as maintaining intracellular pH and membrane potential that are relevant for general cellular physiology and ageing. The development of analytical methods for detection and quantitation of this class of compounds has been challenging due to the basic nature of these species. Both liquid chromatography (LC) and gas chromatography (GC) have been applied for separation, mostly coupled to mass spectrometry (MS) for detection. However, current methodologies suffer from lengthy extraction protocols and limitations in separation and detection levels. Here, we present a simplified and optimised method for straightforward extraction of polyamine metabolites including spermine, spermidine, norspermidine, cadaverine and putrescine from cellular and tissue material. We demonstrate that strong acid-based extraction and chemical derivatisation not only improves isolation, but also recovery. Combined with two-dimensional gas chromatography, this method provides clear separation and femtomole sensitivity for the profiling of polyamines.

Parikh K, Antanaviciute A, Fawkner-Corbett D, Jagielowicz M, Aulicino A, Lagerholm C, Davis S, Kinchen J, Chen HH, Alham NK et al. 2019. Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature, 567 (7746), pp. 49-55. | Show Abstract | Read more

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

Davis S, Scott C, Ansorge O, Fischer R. 2019. Development of a Sensitive, Scalable Method for Spatial, Cell-Type-Resolved Proteomics of the Human Brain. J Proteome Res, 18 (4), pp. 1787-1795. | Show Abstract | Read more

While nearly comprehensive proteome coverage can be achieved from bulk tissue or cultured cells, the data usually lacks spatial resolution. As a result, tissue based proteomics averages protein abundance across multiple cell types and/or localizations. With proteomics platforms lacking sensitivity and throughput to undertake deep single-cell proteome studies in order to resolve spatial or cell type dependent protein expression gradients within tissue, proteome analysis has been combined with sorting techniques to enrich for certain cell populations. However, the spatial resolution and context is lost after cell sorting. Here, we report an optimized method for the proteomic analysis of neurons isolated from post-mortem human brain by laser capture microdissection (LCM). We tested combinations of sample collection methods, lysis buffers and digestion methods to maximize the number of identifications and quantitative performance, identifying 1500 proteins from 60 000 μm2 of 10 μm thick cerebellar molecular layer with excellent reproducibility. To demonstrate the ability of our workflow to resolve cell type specific proteomes within human brain tissue, we isolated sets of individual Betz and Purkinje cells. Both neuronal cell types are involved in motor coordination and were found to express highly specific proteomes to a depth of 2800 to 3600 proteins.

Olie CS, Riaz M, Konietzny R, Charles PD, Pinto-Fernandez A, Kiełbasa SM, Aartsma-Rus A, Goeman JJ, Kessler BM, Raz V. 2019. Deacetylation Inhibition Reverses PABPN1-Dependent Muscle Wasting. iScience, 12 pp. 318-332. | Show Abstract | Read more

Reduced poly(A)-binding protein nuclear 1 (PABPN1) levels cause aging-associated muscle wasting. PABPN1 is a multifunctional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome with the proteome in mouse muscles expressing short hairpin RNA to PABPN1 (shPab). We found greater variations in the proteome than in mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be decreased in shPab versus control proteomes (63%). Acetylome profiling in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via alternative polyadenylation site utilization. Most importantly, SIRT1 deacetylase inhibition by sirtinol increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting. VIDEO ABSTRACT.

Unsworth AJ, Bombik I, Pinto-Fernandez A, McGouran JF, Konietzny R, Zahedi RP, Watson SP, Kessler BM, Pears CJ. 2019. Human Platelet Protein Ubiquitylation and Changes following GPVI Activation. Thromb Haemost, 119 (1), pp. 104-116. | Citations: 1 (Web of Science Lite) | Show Abstract | Read more

Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.

D'Ascenzio M, Pugh KM, Konietzny R, Berridge G, Tallant C, Hashem S, Monteiro O, Thomas JR, Schirle M, Knapp S et al. 2019. An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains. Angew Chem Int Ed Engl, 58 (4), pp. 1007-1012. | Show Abstract | Read more

Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from 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 allowed in-gel visualization and pull-down of the desired bromodomains.

Volkmar N, Thezenas M-L, Louie SM, Juszkiewicz S, Nomura DK, Hegde RS, Kessler BM, Christianson JC. 2019. The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis. J Cell Sci, 132 (2), | Citations: 2 (Scopus) | Show Abstract | Read more

The eukaryotic endoplasmic reticulum (ER) membrane contains essential complexes that oversee protein biogenesis and lipid metabolism, impacting nearly all aspects of cell physiology. The ER membrane protein complex (EMC) is a newly described transmembrane domain (TMD) insertase linked with various phenotypes, but whose clients and cellular responsibilities remain incompletely understood. We report that EMC deficiency limits the cellular boundaries defining cholesterol tolerance, reflected by diminished viability with limiting or excessive extracellular cholesterol. Lipidomic and proteomic analyses revealed defective biogenesis and concomitant loss of the TMD-containing ER-resident enzymes sterol-O-acyltransferase 1 (SOAT1) and squalene synthase (SQS, also known as FDFT1), which serve strategic roles in the adaptation of cells to changes in cholesterol availability. Insertion of the weakly hydrophobic tail-anchor (TA) of SQS into the ER membrane by the EMC ensures sufficient flux through the sterol biosynthetic pathway while biogenesis of polytopic SOAT1 promoted by the EMC provides cells with the ability to store free cholesterol as inert cholesteryl esters. By facilitating insertion of TMDs that permit essential mammalian sterol-regulating enzymes to mature accurately, the EMC is an important biogenic determinant of cellular robustness to fluctuations in cholesterol availability.This article has an associated First Person interview with the first author of the paper.

Vazquez-Rodriguez S, Wright M, Rogers CM, Cribbs AP, Velupillai S, Philpott M, Lee H, Dunford JE, Huber KVM, Robers MB et al. 2019. Design, Synthesis and Characterization of Covalent KDM5 Inhibitors. Angew Chem Int Ed Engl, 58 (2), pp. 515-519. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

Histone lysine demethylases (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.

Sepil I, Hopkins BR, Dean R, Thézénas M-L, Charles PD, Konietzny R, Fischer R, Kessler BM, Wigby S. 2019. Quantitative Proteomics Identification of Seminal Fluid Proteins in Male Drosophila melanogaster. Mol Cell Proteomics, 18 (Suppl 1), pp. S46-S58. | Citations: 2 (Web of Science Lite) | 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 behavior. 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 because of 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 analyzed 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 were consistent with 125 previously reported Sfps. We found nine 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 42 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.

Kaisar M, van Dullemen L, Charles P, Akhtar ZM, Thézénas ML, Huang H, Klooster A, Watkins NA, Kessler B, Ploeg RJ. 2019. Subclinical Changes in Deceased Donor Kidney Proteomes Are Associated With 12-month Allograft Function Posttransplantation-A Preliminary Study. Transplantation, 103 (2), pp. 323-328. | Show Abstract | Read more

BACKGROUND: Cerebral injury during donation after brain death may induce systemic damage affecting long-term kidney function posttransplantation. Conventional evaluation of donor organ quality as a triage for transplantation is of limited utility. METHODS: We compared donor kidneys yielding opposing extremes of the continuum of posttransplantation outcomes by several common kidney biopsy evaluation techniques, including Kidney Donor Profile Index and Remuzzi scoring, and analyzed tissue from a minimal sample cohort using label-free quantitation mass spectrometry. Further assessment of the proteomic results was performed by orthogonal quantitative comparisons of selected key proteins by immunoblotting. RESULTS: We show that common evaluation techniques of kidney biopsies were not predictive for posttransplantation outcomes. In contrast, despite the limited cohort size, the proteomic analysis was able to clearly differentiate between kidneys yielding extreme posttransplantation outcome differences. Pathway analysis of the proteomic data suggested that outcome-related variance in protein abundance associated with profibrotic, apoptosis, and antioxidant proteins. Immunoblotting confirmation further supported this observation. CONCLUSIONS: We present preliminary data indicating that there is scope for existing evaluation approaches to be supplemented by the analysis of proteomic differences. Furthermore, the observed outcome-related variance in a limited cohort was supported by immunoblotting and is consistent with mechanisms previously implicated in the development of injury and cytoprotection in kidney transplantation.

Xavier M-AE, Liu S, Bugge TH, Baguna-Torres J, Mosley M, Hopkins SL, Allen PD, Berridge G, Vendrell I, Fisher R et al. 2019. Tumor imaging using radiolabelled matrix metalloproteinase-activated anthrax proteins. J Nucl Med, | Show Abstract | Read more

Purpose: Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used an 111In-radiolabelled form of LF with the PA/LF system for non-invasive in vivo imaging of MMP activity in tumour tissue by single photon emission computed tomography (SPECT). Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484 and MCF7). Uptake of 111In-radiolabelled PA-L1, 111In-PA-WTK563C or 111In-LFE687A (a catalytically inactive LF mutant) in tumour and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080>MDA-MB-231>B8484>MCF7). PA-L1-mediated delivery of 111In-LFE687A was demonstrated, and corroborated using confocal microscopy with fluorescently labelled LFE687A Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111In-PA-L1 in MDA-MB-231 tumour xenografts (5.7±0.9%ID/g) at 3 h post intravenous administration. 111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumour tissue by MMP-activatable PA-L1 (5.98±0.62%ID/g), but not by furin cleavable PA-WT (1.05±0.21%ID/g), or a non-cleavable PA variant control, PA-U7 (2.74 ± 0.24%ID/g). Conclusion: Taken together, our results indicate that radiolabelled forms of mutated anthrax lethal toxin hold promise for non-invasive imaging of MMP activity in tumour tissue.

Total publications on this page: 16

Total citations for publications on this page: 6