Bram Laukens
Ghent University
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Publication
Featured researches published by Bram Laukens.
Cell Death & Differentiation | 2011
Nele Vanlangenakker; T Vanden Berghe; Pieter Bogaert; Bram Laukens; Kerry Zobel; Kurt Deshayes; Domagoj Vucic; Simone Fulda; Peter Vandenabeele; Mathieu J.M. Bertrand
Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.
Embo Molecular Medicine | 2011
Nele Festjens; Pieter Bogaert; Anjana Batni; Erica Houthuys; Evelyn Plets; Dieter Vanderschaeghe; Bram Laukens; Bob Asselbergh; Eef Parthoens; Riet De Rycke; Monique A. Willart; Peggy Jacques; Dirk Elewaut; Peter Brouckaert; Bart N. Lambrecht; Kris Huygen; Nico Callewaert
Mycobacterium bovis bacille Calmette‐Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α‐1,2‐oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer‐term survival. Persistence of the SapM‐mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC‐IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
Future Microbiology | 2015
Bram Laukens; Charlotte De Visscher; Nico Callewaert
Yeast has advanced as an alternative for mammalian cell culture for the production of recombinant therapeutic glycoproteins. Engineered yeast strains not only allow to mimic the human N-glycosylation pathway but also specific types of human O-glycosylation. This is of great value for therapeutic protein production and indispensable to determine the structure-function relationships of glycans on recombinant proteins. However, as the technology matures, some limitations have come up that may hamper biomedical applications and must be considered to exploit the full potential of the unprecedented glycan homogeneity obtained on relevant biopharmaceuticals. In this special report, we focus on the recent developments in N- and O-glycosylation engineering in yeasts of industrial importance, to produce recombinant therapeutics with customized glycans.
Methods of Molecular Biology | 2015
Bram Laukens; Charlot De Wachter; Nico Callewaert
Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.
Protein Expression and Purification | 2016
Fabrice Cerini; Hubert Gaertner; Knut R. Madden; Ilya Tolstorukov; Scott Brown; Bram Laukens; Nico Callewaert; Jay C. Harner; Anna Oommen; John T Harms; Anthony R Sump; Robert C Sealock; Dustin J Peterson; Scott K. Johnson; Stephan B Abramson; Michael M. Meagher; Robin E. Offord; Oliver Hartley
In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.
Clinical Chemistry and Laboratory Medicine | 2017
Justin C. Cikomola; Antoine S. Kishabongo; Karl Vandepoele; Marieke De Mulder; Philippe Katchunga; Bram Laukens; Loes Van Schie; Hendrik Grootaert; Nico Callewaert; Marijn M. Speeckaert; Joris R. Delanghe
Abstract Background: Fructosamine 3 kinase (FN3K) is a deglycating enzyme, which may play a key role in reducing diabetes-induced organ damage by removing bound glucose from glycated proteins. We wanted to develop a simple colorimetric method for assaying FN3K activity in human body fluids. Methods: Glycated bovine serum albumin (BSA) was obtained by glycation with a 10% glucose solution at 37 °C. After 72 h, glycated BSA was dialyzed against phosphate buffered saline (0.1 mol/L, pH 7.4). The dialyzed solution (containing ±1000 µmol/L fructosamine) was used as an FN3K substrate. In the assay, 300 µL of substrate was incubated with 50 µL of serum and 100 µL of MgCl2 (0.7 mmol/L)/ATP (3.2 mmol/L). The fructosamine concentration was determined at the start and after incubation (120 min, 25 °C). The decrease in fructosamine concentration over time is a measure for the FN3K activity (1 U corresponding to 1 µmol/min). Concomitantly, the FN3K SNP rs1056534 and the ferroportin SNP rs1156350 were genotyped. Results: Within-assay CV was 6.0%. Reference values for FN3K activity in serum were 14.2±1.6 U/L (n=143). Reference values for FN3K were neither age- nor sex-dependent. The various FN3K SNP rs1056534 genotypes showed no significant differences in serum FN3K activity. In diabetics (n=191), values (14.0±2.2 U/L) were comparable to those of the controls. FN3K activity in erythrocytes was significantly higher (170.3±7.6 U/L). The intra-erythrocytic FN3K activity makes the results prone to hemolysis. FN3K activity depended on the ferroportin Q248H genotypes, with the highest value for the wild type genotype. Neither transferrin saturation nor ferritin were confounders for the FN3K activity. FN3K activity was significantly (p<0.0001) correlated with HbA1c values, although the correlation between FN3K and HbA1c was weak. Conclusions: The simple colorimetric method allows determining FN3K activity in human serum. The assay may be useful for studying the impact of deglycation processes in diabetes mellitus.
Cancer Research | 2012
Barbara Schenk; Bram Laukens; Claudia Jennewein; Nele Vanlangenakker; Silvia Cristofanon; Domagoj Vucic; Irmela Jeremias; Mathieu J.M. Bertrand; Peter Vandenabeele; Simone Fulda
Evasion of apoptosis contributes to treatment resistance, one of the major, yet unresolved obstacles in oncology. Searching for new strategies to bypass apoptosis resistance we investigated the potential of Smac mimetic in acute lymphoblastic leukemia (ALL) cells deficient in FADD or caspase-8. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant leukemia cells for TNFα-induced necroptosis as an alternative mode of cell death. The interaction of Smac mimetic and TNFα occurs is highly synergistic as calculated by combination index (CI Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2278. doi:1538-7445.AM2012-2278
Neoplasia | 2011
Bram Laukens; Claudia Jennewein; Barbara Schenk; Nele Vanlangenakker; Alexander Schier; Silvia Cristofanon; Kerry Zobel; Kurt Deshayes; Domagoj Vucic; Irmela Jeremias; Mathieu J.M. Bertrand; Peter Vandenabeele; Simone Fulda
Plant Biotechnology Journal | 2015
Thomas De Meyer; Bram Laukens; Jonah Nolf; Els Van Lerberge; Riet De Rycke; Ans De Beuckelaer; Sylvie De Buck; Nico Callewaert; Anna Depicker
ACS Synthetic Biology | 2016
Katrien Claes; Kristof Vandewalle; Bram Laukens; Toon Laeremans; Olivier Vosters; Ingrid Langer; Marc Parmentier; Jan Steyaert; Nico N. Callewaert