Publications by authors named "Holger Kramer"

61 Publications

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

Diabetologia 2021 Apr 25;64(4):850-864. Epub 2021 Jan 25.

Section of Cell Biology and Functional Genomics, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital, London, UK.

Aims/hypothesis: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B.

Methods: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry.

Results: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca. Binding partners for both included secretory-granule-localised PTPRN2/phogrin.

Conclusions/interpretation: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans.

Data Availability: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.
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http://dx.doi.org/10.1007/s00125-020-05350-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829492PMC
April 2021

Purified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction.

Mol Cell 2020 12 9;80(6):1039-1054.e6. Epub 2020 Dec 9.

Cell Cycle Group, MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN, UK. Electronic address:

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.
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http://dx.doi.org/10.1016/j.molcel.2020.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758880PMC
December 2020

Clinical features which predict neuronal surface autoantibodies in new-onset focal epilepsy: implications for immunotherapies.

J Neurol Neurosurg Psychiatry 2021 Mar 20;92(3):291-294. Epub 2020 Nov 20.

Oxford Autoimmune Neurology Group, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK

Objective: To generate a score which clinically identifies surface-directed autoantibodies in adults with new-onset focal epilepsy, and evaluate the value of immunotherapy in this clinical setting.

Methods: Prospective clinical and autoantibody evaluations in a cohort of 219 consecutive patients with new-onset focal epilepsy.

Results: 10.5% (23/219) of people with new-onset focal epilepsy had detectable serum autoantibodies to known or novel cell surface antigenic targets. 9/23 with autoantibodies were diagnosed with encephalitis, by contrast to 0/196 without autoantibodies (p<0.0001). Multivariate analysis identified six features which predicted autoantibody positivity (area under the curve=0.83): age ≥54 years, ictal piloerection, lowered self-reported mood, reduced attention, MRI limbic system changes and the absence of conventional epilepsy risk factors. 11/14 (79%) patients with detectable autoantibodies, but without encephalitis, showed excellent long-term outcomes (modified Rankin Score=0) despite no immunotherapy. These outcomes were superior to those of immunotherapy-treated patients with confirmed autoantibody-mediated encephalitis (p<0.05).

Conclusions: Seizure semiology, cognitive and mood phenotypes, alongside inflammatory investigation findings, aid the identification of surface autoantibodies among unselected people with new-onset focal epilepsy. The excellent immunotherapy-independent outcomes of autoantibody-positive patients without encephalitis suggests immunotherapy administration should be guided by clinical features of encephalitis, rather than autoantibody positivity. Our findings suggest that, in this cohort, immunotherapy-responsive seizure syndromes with autoantibodies largely fall under the umbrella of autoimmune encephalitis.
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http://dx.doi.org/10.1136/jnnp-2020-325011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892387PMC
March 2021

Systemic muscle wasting and coordinated tumour response drive tumourigenesis.

Nat Commun 2020 09 16;11(1):4653. Epub 2020 Sep 16.

Medical Research Council London Institute of Medical Sciences, Du Cane Road, London, W12 0NN, UK.

Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. Here, we use a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. We demonstrate coordinate induction of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. We identify Indole-3-propionic acid as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network.
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http://dx.doi.org/10.1038/s41467-020-18502-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495438PMC
September 2020

Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction.

Nat Commun 2020 08 17;11(1):4118. Epub 2020 Aug 17.

Lymphocyte Development Group, MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.
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http://dx.doi.org/10.1038/s41467-020-17823-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431861PMC
August 2020

Caspr2 interacts with type 1 inositol 1,4,5-trisphosphate receptor in the developing cerebellum and regulates Purkinje cell morphology.

J Biol Chem 2020 09 16;295(36):12716-12726. Epub 2020 Jul 16.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

Contactin-associated protein-like 2 (Caspr2) is a neurexin-like protein that has been associated with numerous neurological conditions. However, the specific functional roles that Caspr2 plays in the central nervous system and their underlying mechanisms remain incompletely understood. Here, we report on a functional role for Caspr2 in the developing cerebellum. Using a combination of confocal microscopy, biochemical analyses, and behavioral testing, we show that loss of Caspr2 in the knockout mouse results in impaired Purkinje cell dendritic development, altered intracellular signaling, and motor coordination deficits. We also find that Caspr2 is highly enriched at synaptic specializations in the cerebellum. Using a proteomics approach, we identify type 1 inositol 1,4,5-trisphosphate receptor (IPR1) as a specific synaptic interaction partner of the Caspr2 extracellular domain in the molecular layer of the developing cerebellum. The interaction of the Caspr2 extracellular domain with IPR1 inhibits IPR1-mediated changes in cellular morphology. Together, our work defines a mechanism by which Caspr2 controls the development and function of the cerebellum and advances our understanding of how Caspr2 dysfunction might lead to specific brain disorders.
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http://dx.doi.org/10.1074/jbc.RA120.012655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476715PMC
September 2020

Optical Interrogation of Sympathetic Neuronal Effects on Macroscopic Cardiomyocyte Network Dynamics.

iScience 2020 Jul 1;23(7):101334. Epub 2020 Jul 1.

University of Oxford, Department of Physiology, Anatomy and Genetics, British Heart Foundation Centre of Research Excellence, Parks Road, Oxford OX1 3PT, UK; McGill University, Department of Physiology, McIntyre Medical Sciences Building, Room 1128, 3655 Promenade Sir William Osler, Montréal, QC H3G 1Y6, Canada. Electronic address:

Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. We present approaches to study neuron-cardiomyocyte interactions involving optogenetic selective probing and all-optical electrophysiology to measure activity in an automated fashion. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiomyocyte network dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac behavior, and the applicability of optogenetics in mechanistic in vitro studies. As arrhythmias are emergent behaviors that involve the coordinated activity of millions of cells, we image at macroscopic scales to capture complex dynamics. We show that neurons can both decrease and increase wave stability and re-entrant activity in culture depending on their induced activity-a finding that may help us understand the often conflicting results seen in experimental and clinical studies.
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http://dx.doi.org/10.1016/j.isci.2020.101334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363704PMC
July 2020

Detection of Intravascular Hemolysis in Newborn Infants Using Urinary Carbonic Anhydrase I Immunoreactivity.

J Appl Lab Med 2020 09;5(5):921-934

Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, UK.

Background: Mild hemolysis occurs physiologically in neonates, but more severe forms can lead to life-threatening anemia. Newborns in developing regions are particularly at-risk due to the higher incidence of triggers (protozoan infections, sepsis, certain genetic traits). In advanced healthcare facilities, hemolysis is monitored indirectly using resource-intensive methods that probe downstream ramifications. These approaches could potentially delay critical decisions in early-life care, and are not suitable for point-of-care testing. Rapid and cost-effective testing could be based on detecting red blood cell (RBC)-specific proteins, such as carbonic anhydrase I (CAI), in accessible fluids (e.g., urine).

Methods: Urine was collected from 26 full-term male neonates and analyzed for CAI using immunoassays (ELISA, western blot) and proteomics (mass spectrometry). The cohort included a range of hemolytic states, including admissions with infection, ABO incompatibility, and receiving phototherapy. Data were paired with hemoglobin, serum bilirubin (SBR), and C-reactive protein (CRP) measurements.

Results: Urine from a control cohort (CRP < 20 mg/L, SBR < 125µmol/L) had no detectable CAI, in line with results from healthy adults. CAI excretion was elevated in neonates with raised SBR (>125 µmol/L), including those qualifying for phototherapy. Newborns with low SBR (<125 µmol/L) but elevated CRP (>20 mg/L) produced urine with strong CAI immunoreactivity. Proteomics showed that CAI was the most abundant RBC-specific protein in CAI-immunopositive samples, and did not associate with other RBC-derived peptides, indicating an intravascular hemolytic source followed by CAI-selective excretion.

Conclusions: CAI is a direct biomarker of intravascular hemolysis that can be measured routinely in urine using non-invasive methods under minimal-laboratory conditions.
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http://dx.doi.org/10.1093/jalm/jfaa051DOI Listing
September 2020

Sugar-Induced Obesity and Insulin Resistance Are Uncoupled from Shortened Survival in Drosophila.

Cell Metab 2020 04 19;31(4):710-725.e7. Epub 2020 Mar 19.

MRC London Institute of Medical Sciences, Du Cane Road, London W12 0NN, UK; Institute of Clinical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

High-sugar diets cause thirst, obesity, and metabolic dysregulation, leading to diseases including type 2 diabetes and shortened lifespan. However, the impact of obesity and water imbalance on health and survival is complex and difficult to disentangle. Here, we show that high sugar induces dehydration in adult Drosophila, and water supplementation fully rescues their lifespan. Conversely, the metabolic defects are water-independent, showing uncoupling between sugar-induced obesity and insulin resistance with reduced survival in vivo. High-sugar diets promote accumulation of uric acid, an end-product of purine catabolism, and the formation of renal stones, a process aggravated by dehydration and physiological acidification. Importantly, regulating uric acid production impacts on lifespan in a water-dependent manner. Furthermore, metabolomics analysis in a human cohort reveals that dietary sugar intake strongly predicts circulating purine levels. Our model explains the pathophysiology of high-sugar diets independently of obesity and insulin resistance and highlights purine metabolism as a pro-longevity target.
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http://dx.doi.org/10.1016/j.cmet.2020.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156915PMC
April 2020

TRF1 averts chromatin remodelling, recombination and replication dependent-break induced replication at mouse telomeres.

Elife 2020 Jan 14;9. Epub 2020 Jan 14.

Telomere Replication and Stability group, Medical Research Council - London Institute of Medical Sciences, London, United Kingdom.

Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that mouse telomeres lacking TRF1 undergo protein composition reorganisation associated with the recruitment of DNA damage response and chromatin remodellers. Surprisingly, mTRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for mTRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.
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http://dx.doi.org/10.7554/eLife.49817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986873PMC
January 2020

A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules.

Sci Adv 2019 11 27;5(11):eaay6804. Epub 2019 Nov 27.

Cell Cycle Group, Medical Research Council London Institute of Medical Sciences, Du Cane Road, London W12 0NN, UK.

Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a "permanent bridge" resisting forces over 80 pN and a force-sensitive "reversible bridge." The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. "Permanent" cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.
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http://dx.doi.org/10.1126/sciadv.aay6804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881171PMC
November 2019

Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern.

Nat Commun 2019 10 28;10(1):4910. Epub 2019 Oct 28.

Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
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http://dx.doi.org/10.1038/s41467-019-12711-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817910PMC
October 2019

Single-nucleotide polymorphisms in Orai1 associated with atopic dermatitis inhibit protein turnover, decrease calcium entry and disrupt calcium-dependent gene expression.

Hum Mol Genet 2020 07;29(11):1808-1823

Department of Physiology, Anatomy and Genetics, Parks Road, Oxford, OX1 3PT UK.

Loss-of function mutations in Orai1 Ca2+ channels lead to a form of severe combined immunodeficiency, auto-immunity, muscle hypotonia and defects in dental enamel production and sweat gland function. Two single-nucleotide polymorphisms (SNPs) in Orai1 have been found and localize to the second extracellular loop. These polymorphisms associate with atopic dermatitis but how they affect Ca2+ signalling and cell function is unknown. Here, we find that Orai1-SNPs turnover considerably more slowly than wild type Orai1 and are more abundantly expressed in the plasma membrane. We show a central role for flotillin in the endocytotic recycling of Orai1 channels and that endocytosed wild type Orai1 is trafficked to Rab 7-positive late endosomes for lysosomal degradation. Orai1-SNPs escape the degradation pathway and instead enter Rab 11-positive recycling endosomes, where they are returned to the surface membrane through Arf6-dependent exocytosis. We find that Orai1-SNPs escape late endosomes through endosomal pH regulation of interaction between the channel and flotillin. We identify a pH-sensitive electrostatic interaction between positively charged arginine in extracellular loop 2 (K210) and a negatively charged aspartate (D112) in extracellular loop 1 that helps determine Orai1 turnover. The increase in membrane Orai1-SNP leads to a mis-match in Orai1-STIM stoichiometry, resulting in inhibition of Ca2+ entry and Ca2+-dependent gene expression. Our results identify new strategies for targeting atopic dermatitis.
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http://dx.doi.org/10.1093/hmg/ddz223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372555PMC
July 2020

FACT mediates cohesin function on chromatin.

Nat Struct Mol Biol 2019 10 3;26(10):970-979. Epub 2019 Oct 3.

Cell Cycle Group, MRC London Institute of Medical Sciences (LMS), London, UK.

Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.
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http://dx.doi.org/10.1038/s41594-019-0307-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779571PMC
October 2019

Use of Modified Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Int J Mol Sci 2019 Sep 26;20(19). Epub 2019 Sep 26.

Charité-Universitätsmedizin Berlin, Institute of Clinical Physiology, Hindenburgdamm 30, 12203 Berlin, Germany.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.
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http://dx.doi.org/10.3390/ijms20194774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801472PMC
September 2019

PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection.

Nucleic Acids Res 2019 11;47(20):10706-10727

Cell Cycle and Genome Stability Group, Institute of Functional Biology and Genomics (IBFG), Spanish National Research Council (CSIC). University of Salamanca (USAL), C/ Zacarías González 2, Salamanca 37007, Spain.

The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.
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http://dx.doi.org/10.1093/nar/gkz794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6846210PMC
November 2019

Sex Differences in Intestinal Carbohydrate Metabolism Promote Food Intake and Sperm Maturation.

Cell 2019 08;178(4):901-918.e16

MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized.
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http://dx.doi.org/10.1016/j.cell.2019.07.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700282PMC
August 2019

Small-molecules that covalently react with a human prolyl hydroxylase - towards activity modulation and substrate capture.

Chem Commun (Camb) 2019 Jan;55(8):1020-1023

Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.

We describe covalently binding modulators of the activity of human prolyl hydroxylase domain 2 (PHD2) and studies towards a strategy for photocapture of PHD2 substrates. Reversible active site binding of electrophile bearing compounds enables susbsequent covalent reaction with a lysine residue (K408) in the flexible C-terminal region of PHD2 to give a modified protein that retains catalytic activity.
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http://dx.doi.org/10.1039/c8cc07706aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350621PMC
January 2019

Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin.

Mol Cell 2018 11 1;72(4):739-752.e9. Epub 2018 Nov 1.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany; MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN, UK; Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. Electronic address:

The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside" of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.
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http://dx.doi.org/10.1016/j.molcel.2018.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242706PMC
November 2018

Binding of sulphonylureas to plasma proteins - A KATP channel perspective.

PLoS One 2018 17;13(5):e0197634. Epub 2018 May 17.

Oxford Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic β-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic β-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug's therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197634PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957440PMC
December 2018

Transient and Partial Nuclear Lamina Disruption Promotes Chromosome Movement in Early Meiotic Prophase.

Dev Cell 2018 04;45(2):212-225.e7

Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, 1030 Vienna, Austria. Electronic address:

Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.
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http://dx.doi.org/10.1016/j.devcel.2018.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920155PMC
April 2018

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca Channels and Mast Cell Migration through PAR4 Receptors.

Mol Cell 2018 04;70(2):228-241.e5

Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK. Electronic address:

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca channels and suggest a therapeutic strategy for treating mite-induced asthma.
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http://dx.doi.org/10.1016/j.molcel.2018.03.025DOI Listing
April 2018

Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins.

Cancer Genomics Proteomics 2017 01;14(1):35-51

Department of Pharmaceutical & Biological Chemistry, UCL School of Pharmacy, London, U.K.

Background: Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear.

Materials And Methods: A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes.

Results: A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease.

Conclusion: Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer.
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http://dx.doi.org/10.21873/cgp.20017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267499PMC
January 2017

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein.

Cell Rep 2016 Mar 10;14(11):2695-706. Epub 2016 Mar 10.

Oxford Glycobiology Institute and Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.
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http://dx.doi.org/10.1016/j.celrep.2016.02.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805854PMC
March 2016

The value of in vitro studies in a case of neonatal diabetes with a novel Kir6.2-W68G mutation.

Clin Case Rep 2015 Oct 8;3(10):884-7. Epub 2015 Sep 8.

Department of Paediatrics and Child Health, Cork University Hospital Cork, Ireland.

In infants, especially with novel previously undescribed mutations of the KATP channel causing neonatal diabetes, in vitro studies can be used to both predict the response to sulphonylurea treatment and support a second trial of glibenclamide at higher than standard doses if the expected response is not observed.
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http://dx.doi.org/10.1002/ccr3.370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614663PMC
October 2015

Systemic Administration of Glibenclamide Fails to Achieve Therapeutic Levels in the Brain and Cerebrospinal Fluid of Rodents.

PLoS One 2015 30;10(7):e0134476. Epub 2015 Jul 30.

Department of Physiology, Anatomy and Genetics and OXION, University of Oxford, Oxford, United Kingdom.

Activating mutations in the Kir6.2 (KCNJ11) subunit of the ATP-sensitive potassium channel cause neonatal diabetes (ND). Patients with severe mutations also suffer from neurological complications. Glibenclamide blocks the open KATP channels and is the treatment of choice for ND. However, although glibenclamide successfully restores normoglycaemia, it has a far more limited effect on the neurological problems. To assess the extent to which glibenclamide crosses the blood-brain barrier (BBB) in vivo, we quantified glibenclamide concentrations in plasma, cerebrospinal fluid (CSF), and brain tissue of rats, control mice, and mice expressing a human neonatal diabetes mutation (Kir6.2-V59M) selectively in neurones (nV59M mice). As only small sample volumes can be obtained from rodents, we developed a highly sensitive method of analysis, using liquid chromatography tandem mass spectrometry acquisition with pseudo-selected reaction monitoring, achieving a quantification limit of 10ng/ml (20nM) glibenclamide in a 30μl sample. Glibenclamide was not detectable in the CSF or brain of rats after implantation with subcutaneous glibenclamide pellets, despite high plasma concentrations. Further, one hour after a suprapharmacological glibenclamide dose was administered directly into the lateral ventricle of the brain, the plasma concentration was twice that of the CSF. This suggests the drug is rapidly exported from the CSF. Elacridar, an inhibitor of P-glycoprotein and breast cancer resistance protein (major multidrug resistance transporters at the BBB), did not affect glibenclamide levels in CSF and brain tissue. We also identified a reduced sensitivity to volatile anaesthetics in nV59M mice and showed this was not reversed by systemic delivery of glibenclamide. Our results therefore suggest that little glibenclamide reaches the central nervous system when given systemically, that glibenclamide is rapidly removed across the BBB when given intracranioventricularly, and that any glibenclamide that does enter (and is below our detection limit) is insufficient to influence neuronal function as assessed by anaesthesia sensitivity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134476PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520580PMC
May 2016

Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor.

J Biol Chem 2015 Aug 25;290(32):19726-42. Epub 2015 Jun 25.

From the Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom and

The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia sensing in animals. In humans, they comprise three prolyl hydroxylases (PHD1-3 or EGLN1-3) and factor inhibiting HIF (FIH). FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription. Like the PHDs, FIH is proposed to have a hypoxia-sensing role in cells, enabling responses to changes in cellular O2 availability. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia sensor due to its relatively high sensitivity to changes in O2 concentration and slow reaction with O2. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O2. Consistent with previous reports, we found lower Km(app)(O2) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O2 of the FIH reaction with ankyrin repeat domain (ARD) substrates. FIH has lower Km(app)(O2) values for the tested ARDs than HIF-α substrates, and pre-steady-state O2-initiated reactions were faster with ARDs than with HIF-α substrates. The results correlate with cellular studies showing that FIH is active at lower O2 concentrations than the PHDs and suggest that competition between HIF-α and ARDs for FIH is likely to be biologically relevant, particularly in hypoxic conditions. The overall results are consistent with the proposal that the kinetic properties of individual oxygenases reflect their biological capacity to act as hypoxia sensors.
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http://dx.doi.org/10.1074/jbc.M115.653014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528135PMC
August 2015