Publications by authors named "Boris Rodenko"

27 Publications

  • Page 1 of 1

Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex.

PLoS Pathog 2020 10 27;16(10):e1008784. Epub 2020 Oct 27.

York Biomedical Research Institute and Department of Biology, University of York, United Kingdom.

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647121PMC
October 2020

Essential roles for deubiquitination in Leishmania life cycle progression.

PLoS Pathog 2020 06 16;16(6):e1008455. Epub 2020 Jun 16.

York Biomedical Research Institute and Department of Biology, University of York, United Kingdom.

The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319358PMC
June 2020

Proteasome Activation by Small Molecules.

Cell Chem Biol 2017 Jun 25;24(6):725-736.e7. Epub 2017 May 25.

Division of Cell Biology II, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands; Department of Chemical Immunology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands. Electronic address:

Drugs that increase 26S proteasome activity have potential therapeutic applications in the treatment of neurodegenerative diseases. A chemical genetics screen of over 2,750 compounds using a proteasome activity probe as a readout in a high-throughput live-cell fluorescence-activated cell sorting-based assay revealed more than ten compounds that increase proteasome activity, with the p38 MAPK inhibitor PD169316 being one of the most potent ones. Genetic and chemical inhibition of either p38 MAPK, its upstream regulators, ASK1 and MKK6, and downstream target, MK2, enhance proteasome activity. Chemical activation of the 26S proteasome increases PROTAC-mediated and ubiquitin-dependent protein degradation and decreases the levels of both overexpressed and endogenous α-synuclein, without affecting the overall protein turnover. In addition, survival of cells overexpressing toxic α-synuclein assemblies is increased in the presence of p38 MAPK inhibitors. These findings highlight the potential of activation of 26S proteasome activity and that this can be achieved through multiple mechanisms by distinct molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2017.05.010DOI Listing
June 2017

Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

PLoS One 2016 22;11(6):e0156462. Epub 2016 Jun 22.

Centre for Infectious Disease Control (Cib), National Institute for Public Health and the Environment, Bilthoven, the Netherlands.

T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917206PMC
July 2017

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

J Immunol 2015 Nov 23;195(9):4085-95. Epub 2015 Sep 23.

Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands;

Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1402455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642839PMC
November 2015

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage.

J Immunol 2015 Nov 23;195(9):4075-84. Epub 2015 Sep 23.

Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands;

The proteasome is able to create spliced Ags, in which two distant parts of a protein are excised and ligated together to form a novel peptide, for presentation by MHC class I molecules. These noncontiguous epitopes are generated via a transpeptidation reaction catalyzed by the proteasomal active sites. Transpeptidation reactions in the proteasome follow explicit rules and occur particularly efficiently when the C-terminal ligation partner contains a lysine or arginine residue at the site of ligation. Lysine contains two amino groups that theoretically may both participate in ligation reactions, implying that potentially not only peptide but also isopeptide linkages could be formed. Using nuclear magnetic resonance spectroscopy, we demonstrate in the present study that the proteasome can use the ε-amino group of an N-terminal lysine residue in transpeptidation reactions to create a novel type of posttranslationally modified epitopes. We show that the overall efficiency of ε ligation is only 10-fold lower as compared with α ligation, suggesting that the proteasome can produce sufficient isopeptide Ag to evoke a T cell response. Additionally, we show that isopeptides are more stable toward further proteasomal processing than are normal peptides, and we demonstrate that isopeptides can bind to HLA-A2.1 and HLA-A3 with high affinity. These properties likely increase the fraction of ε-ligated peptides presented on the cell surface for CD8(+) T cell surveillance. Finally, we show that isopeptide Ags are immunogenic in vivo. We postulate that ε ligation is a genuine posttranslational modification, suggesting that the proteasome can create a novel type of Ag that is likely to play a role in immunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1402454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642838PMC
November 2015

Targeting the parasite's DNA with methyltriazenyl purine analogs is a safe, selective, and efficacious antitrypanosomal strategy.

Antimicrob Agents Chemother 2015 Nov 17;59(11):6708-16. Epub 2015 Aug 17.

College of Medical, Veterinary and Life Sciences, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom

The human and veterinary disease complex known as African trypanosomiasis continues to inflict significant global morbidity, mortality, and economic hardship. Drug resistance and toxic side effects of old drugs call for novel and unorthodox strategies for new and safe treatment options. We designed methyltriazenyl purine prodrugs to be rapidly and selectively internalized by the parasite, after which they disintegrate into a nontoxic and naturally occurring purine nucleobase, a simple triazene-stabilizing group, and the active toxin: a methyldiazonium cation capable of damaging DNA by alkylation. We identified 2-(3-acetyl-3-methyltriazen-1-yl)-6-hydroxypurine (compound 1) as a new lead compound, which showed submicromolar potency against Trypanosoma brucei, with a selectivity index of >500, and it demonstrated a curative effect in animal models of acute trypanosomiasis. We investigated the mechanism of action of this lead compound and showed that this molecule has significantly higher affinity for parasites over mammalian nucleobase transporters, and it does not show cross-resistance with current first-line drugs. Once selectively accumulated inside the parasite, the prodrug releases a DNA-damaging methyldiazonium cation. We propose that ensuing futile cycles of attempted mismatch repair then lead to G2/M phase arrest and eventually cell death, as evidenced by the reduced efficacy of this purine analog against a mismatch repair-deficient (MSH2(-/-)) trypanosome cell line. The observed absence of genotoxicity, hepatotoxicity, and cytotoxicity against mammalian cells revitalizes the idea of pursuing parasite-selective DNA alkylators as a safe chemotherapeutic option for the treatment of human and animal trypanosomiasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00596-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604408PMC
November 2015

Altered peptide ligands revisited: vaccine design through chemically modified HLA-A2-restricted T cell epitopes.

J Immunol 2014 Nov 13;193(10):4803-13. Epub 2014 Oct 13.

Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands;

Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1400800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226423PMC
November 2014

Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting breast cancer metastasis.

Proc Natl Acad Sci U S A 2013 Mar 12;110(13):5139-44. Epub 2013 Mar 12.

Department of Molecular Oncology, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands.

Metastasis confronts clinicians with two major challenges: estimating the patient's risk of metastasis and identifying therapeutic targets. Because they are key signal integrators connecting cellular processes to clinical outcome, we aimed to identify transcriptional nodes regulating cancer cell metastasis. Using rodent xenograft models that we previously developed, we identified the transcription factor Fos-related antigen-1 (Fra-1) as a key coordinator of metastasis. Because Fra-1 often is overexpressed in human metastatic breast cancers and has been shown to control their invasive potential in vitro, we aimed to assess the implication and prognostic significance of the Fra-1-dependent genetic program in breast cancer metastasis and to identify potential Fra-1-dependent therapeutic targets. In several in vivo assays in mice, we demonstrate that stable RNAi depletion of Fra-1 from human breast cancer cells strongly suppresses their ability to metastasize. These results support a clinically important role for Fra-1 and the genetic program it controls. We show that a Fra-1-dependent gene-expression signature accurately predicts recurrence of breast cancer. Furthermore, a synthetic lethal drug screen revealed that antagonists of the adenosine receptor A2B (ADORA2B) are preferentially toxic to breast tumor cells expressing Fra-1. Both RNAi silencing and pharmacologic blockade of ADORA2B inhibited filopodia formation and invasive activity of breast cancer cells and correspondingly reduced tumor outgrowth in the lungs. These data show that Fra-1 activity is causally involved in and is a prognostic indicator of breast cancer metastasis. They suggest that Fra-1 activity predicts responsiveness to inhibition of pharmacologically tractable targets, such as ADORA2B, which may be used for clinical interference of metastatic breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1222085110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612632PMC
March 2013

Development of a hypersensitive periodate-cleavable amino acid that is methionine- and disulfide-compatible and its application in MHC exchange reagents for T cell characterisation.

Chembiochem 2013 Jan;14(1):123-31

Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.

Incorporation of cleavable linkers into peptides and proteins is of particular value in the study of biological processes. Here we describe the synthesis of a cleavable linker that is hypersensitive to oxidative cleavage as the result of the periodate reactivity of a vicinal amino alcohol moiety. Two strategies directed towards the synthesis of a building block suitable for solid-phase peptide synthesis were developed: a chemoenzymatic route, involving L-threonine aldolase, and an enantioselective chemical route; these led to α,γ-diamino-β-hydroxybutanoic acids in diastereoisomerically mixed and enantiopure forms, respectively. Incorporation of the 1,2-amino alcohol linker into the backbone of a peptide generated a conditional peptide that was rapidly cleaved at very low concentrations of sodium periodate. This cleavable peptide ligand was applied in the generation of MHC exchange reagents for the detection of antigen-specific T cells in peripheral blood cells. The extremely low concentration of periodate required to trigger MHC peptide exchange allowed the co-oxidation of methionine and disulfide residues to be avoided. Conditional MHC reagents hypersensitive to periodate can now be applied without limitations when UV irradiation is undesired or less practical.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201200540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561698PMC
January 2013

Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes.

Chembiochem 2012 Oct 25;13(15):2251-8. Epub 2012 Sep 25.

Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201200497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3487179PMC
October 2012

Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis.

J Antimicrob Chemother 2012 Apr 17;67(4):933-43. Epub 2012 Jan 17.

Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK.

Objectives: To evaluate existing protocols, based on Alamar blue (resazurin), for the routine determination of drug susceptibility in trichomonads, develop new ones and validate these by screening small antiprotozoal libraries.

Methods: The resazurin-based assay was evaluated by determining fluorescence development in Trichomonas media with various cell densities after various intervals and in the presence of metronidazole. Similar investigations were performed with the alternative fluorophores propidium iodide (PI) and resorufin. The optimized protocols were used to screen for new antitrichomonal compounds.

Results: Anaerobic cultures of Trichomonas vaginalis rapidly reduced blue resazurin to red, fluorescent resorufin. However, the ascorbic acid in the culture medium produced similar effects, even in the absence of cells, causing high background fluorescence and variability. Moreover, T. vaginalis rapidly metabolized resorufin to the non-fluorescent and colourless metabolite dihydroresorufin, making the fluorescent signal transient. In contrast, resorufin proved to be an excellent viability probe for Trichomonas due to its chemical stability in media and rapid metabolism by the parasite. We also show that staining with PI after cell permeabilization similarly constitutes a reliable measurement of trophozoite numbers. Using the PI and resorufin assays we determined reproducible EC(50) values and identified potent antitrichomonal compounds from a limited screen of phosphodiesterase inhibitors and phosphonium salts.

Conclusions: The resorufin- and PI-based assays are suitable for routine and high-throughput drug screening, whereas resazurin-based assays are not. These assays constitute a major advance in the current protocols as demonstrated by a successful screen for new antitrichomonal lead compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkr572DOI Listing
April 2012

Synthesis of marine-derived 3-alkylpyridinium alkaloids with potent antiprotozoal activity.

ACS Med Chem Lett 2011 Dec 5;2(12):901-6. Epub 2011 Oct 5.

College of Medical, Veterinary and Life Sciences, Institute of Infection, Immunity and Inflammation, University of Glasgow , United Kingdom.

Given the pressing need for new antiprotozoal drugs without cross-resistance with current (failing) chemotherapy, we have explored 3-tridecylpyridinium alkaloids (3TPAs), derivatives of viscosamine, as antiparasitic agents. We have developed a simple synthetic route toward viscosamine and related cyclic and linear monomers and oligomers. Evaluation for cytotoxicity on the protozoan parasites Trypanosoma brucei, Leishmania spp., and Plasmodium falciparum revealed several 3TPAs with antiprotozoal activity in the nanomolar range. Their promising selectivity index in vitro prompted us to study the dynamics of cytotoxicity on trypanosomes in more detail. Parasites were killed relatively slowly at therapeutically safe concentrations, in a process that did not target the cell cycle. Clearance of T. brucei cultures was observed at drug concentrations of 1-10 μM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ml200160kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4018069PMC
December 2011

Fluorescence-based proteasome activity profiling.

Methods Mol Biol 2012 ;803:183-204

Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-61779-364-6_13DOI Listing
March 2012

Generation of peptide MHC class I monomers and multimers through ligand exchange.

Curr Protoc Immunol 2009 Nov;Chapter 18:Unit 18.16

The Netherlands Cancer Institute, Amsterdam, The Netherlands.

The recognition of defined antigen-MHC complexes by antigen-specific T cells forms the molecular basis of T cell immunity. It has been shown that fluorescently labeled recombinant MHC tetramers can be utilized to detect antigen-specific T cells by flow cytometry. Since this first description, MHC tetramers and other types of MHC multimers have become a core tool to monitor the development of disease- and therapy-induced antigen-specific T cell responses both in humans and in animal model systems. This unit describes a set of protocols that transform classical MHC multimer technology into a high-throughput platform, allowing one to produce large collections of MHC class I molecules charged with different peptides. This technology is based on the development of conditional MHC ligands that can be triggered to self-destruct while in the MHC-bound state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/0471142735.im1816s87DOI Listing
November 2009

Class I major histocompatibility complexes loaded by a periodate trigger.

J Am Chem Soc 2009 Sep;131(34):12305-13

Division of Cell Biology II, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide-MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide-MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide-MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja9037565DOI Listing
September 2009

UV-induced ligand exchange in MHC class I protein crystals.

J Am Chem Soc 2009 Sep;131(34):12298-304

Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

High-throughput structure determination of protein-ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, "conditional", peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This "in crystallo" exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC beta2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja9037559DOI Listing
September 2009

High-throughput T-cell epitope discovery through MHC peptide exchange.

Methods Mol Biol 2009 ;524:383-405

Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Recombinant major histocompatibility complex (MHC) class I molecules complexed with pathogen-specific or other disease-associated antigens have become essential reagents for the analysis of adaptive T-cell responses. However, conventional techniques for the production of recombinant peptide-MHC (pMHC) complexes are highly involved and thereby limit the use of pMHC complexes in terms of antigen diversity. To make pMHC-based techniques suitable for high-throughput analyses we developed an MHC peptide exchange technology based on the use of conditional MHC ligands. This technology enables the parallel production of thousands of different pMHC complexes within hours, allowing the development of high-throughput MHC-based assay systems to identify MHC ligands and cytotoxic T-cell responses. These high-throughput assays should prove valuable for the screening of entire disease-associated proteomes, including pathogen-encoded proteomes, tumor-associated antigens, and autoimmune antigens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-59745-450-6_28DOI Listing
July 2009

Transpeptidation and reverse proteolysis and their consequences for immunity.

Int J Biochem Cell Biol 2009 Jan 4;41(1):66-71. Epub 2008 Sep 4.

Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Reverse proteolysis and transpeptidation lead to the generation of polypeptide sequences that cannot be inferred directly from genome sequences as they are post-translational phenomena. These phenomena have so far received little attention although the physiological consequences may reach far. The protease-mediated synthesis of several immunodominant MHC class I antigens was recently reported, underscoring its importance to immunity. Reverse proteolytic and transpeptidation mechanisms as well as conditions that favor successful protease-catalyzed synthetic events are discussed here.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biocel.2008.08.036DOI Listing
January 2009

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7.

Proc Natl Acad Sci U S A 2008 Mar 28;105(10):3825-30. Epub 2008 Feb 28.

Divisions of Immunology and Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0709717105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268811PMC
March 2008

2,N6-disubstituted adenosine analogs with antitrypanosomal and antimalarial activities.

Antimicrob Agents Chemother 2007 Nov 13;51(11):3796-802. Epub 2007 Aug 13.

Van't Hoff Institute for Molecular Sciences, Universiteit van Amsterdam, The Netherlands.

A library of 2,N(6)-disubstituted adenosine analogs was synthesized and the analogs were tested for their antiprotozoal activities. It was found that 2-methoxy and 2-histamino and N(6)-m-iodobenzyl substitutions generally produced analogs with low levels of antiprotozoal activity. The best antiplasmodial activity was achieved with large aromatic substitutions, such as N(6)-2,2-diphenylethyl and naphthylmethyl, which could indicate a mechanism of action through aromatic stacking with heme in the digestive vacuole of Plasmodium spp. The activities against Trypanosoma cruzi trypomastigotes and Leishmania donovani amastigotes were generally low; but several analogs, particularly those with cyclopentylamino substitutions, displayed potent activities against Trypanosoma brucei rhodesiense and T. b. brucei bloodstream forms in vitro. The most active were 2-cyclopentylamino-N(6)-cyclopentyladenosine (compound NA42) and 2-cyclopentylamino-N(6)-cyclopentyladenine (compound NA134), with the nucleobase an order of magnitude more potent than the nucleoside, at 26 +/- 4 nM. It was determined that the mode of action of these purines was trypanostatic, with the compounds becoming trypanocidal only at much higher concentrations. Those 2,N(6)-disubstituted purines tested for their effects on purine transport in T. b. brucei displayed at best a moderate affinity for the transporters. It is highly probable that the large hydrophobic substitutions, which bestow high calculated octanol-water coefficient values on the analogs, allow them to diffuse across the membrane. Consistent with this view, the analogs were as effective against a T. b. brucei strain lacking the P2 nucleoside transporter as they were against the parental strain. As the analogs were not toxic to human cell lines, the purine analogs are likely to act on a trypanosome-specific target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00425-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151466PMC
November 2007

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange.

Nat Protoc 2006 ;1(3):1120-32

Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nprot.2006.121DOI Listing
June 2007

Molecular interactions underlying the unusually high adenosine affinity of a novel Trypanosoma brucei nucleoside transporter.

Mol Pharmacol 2007 Mar 21;71(3):921-9. Epub 2006 Dec 21.

Institute of Biomedical and Life Sciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, United Kingdom.

Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (Km = 0.068 +/- 0.013 microM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3'-hydroxy, 5'-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (DeltaG0 = 12 kJ/mol), explaining the different Km values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/mol.106.031559DOI Listing
March 2007

Design and use of conditional MHC class I ligands.

Nat Med 2006 Feb 5;12(2):246-51. Epub 2006 Feb 5.

Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. 'Empty MHC molecules' generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a high-throughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm1360DOI Listing
February 2006

Biosynthesis and uptake of thiamine (vitamin B1) in bloodstream form Trypanosoma brucei brucei and interference of the vitamin with melarsen oxide activity.

Int J Parasitol 2006 Feb 21;36(2):229-36. Epub 2005 Nov 21.

Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland.

Bloodstream forms of Trypanosoma brucei brucei were cultivated in the presence and absence of thiamine (vitamin B1) and pyridoxine (vitamin B6). The vitamins do not change growth behaviour, indicating that Trypanosoma brucei is prototrophic for the two vitamins even though in silico no bona-fide thiamine-biosynthetic genes could be identified in the T. brucei genome. Intracellularly, thiamine is mainly present in its diphosphate form. We were unable to detect significant uptake of [3H]thiamine and structural thiamine analogues such as pyrithiamine, oxithiamine and amprolium were not toxic for the bloodstream forms of T. brucei, indicating that the organism does not have an efficient uptake system for thiamine and its analogues. We have previously shown that, in the fission yeast Saccharomyces pombe, the toxicity of melarsen oxide, the pharmacologically active derivative of the frontline sleeping sickness drug melarsoprol, is abolished by thiamine and the drug is taken up by a thiamine-regulated membrane protein which is responsible for the utilization of thiamine. We show here that thiamine also has weak effects on melarsen oxide-induced growth inhibition and lysis in T. brucei. These effects were consistent with a low affinity of thiamine for the P2 adenosine transporter that is responsible for uptake of melaminophenyl arsenicals in African trypanosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpara.2005.10.003DOI Listing
February 2006

Solid phase synthesis and antiprotozoal evaluation of di- and trisubstituted 5'-carboxamidoadenosine analogues.

Bioorg Med Chem 2006 Mar 24;14(5):1618-29. Epub 2005 Oct 24.

Van't Hoff Institute for Molecular Sciences, Universiteit van Amsterdam, The Netherlands.

The rapid increase of resistance to drugs commonly used in the treatment of tropical diseases such as malaria and African sleeping sickness calls for the prompt development of new safe and efficacious drugs. The pathogenic protozoan parasites lack the capability of synthesising purines de novo and they take up preformed purines from their host through various transmembrane transporters. Adenosine derivatives constitute a class of potential therapeutics due to their selective internalisation by these transporters. Automated solid-phase synthesis can speed up the process of lead finding and we pursued the solid-phase synthesis of di- and trisubstituted 5'-carboxamidoadenosine derivatives by using a safety-catch approach. While efforts with Kenner's sulfonamide linker remained fruitless, successful application of the hydrazide safety-catch linker allowed the construction of two representative combinatorial libraries. Their antiprotozoal evaluation identified two compounds with promising activity: N(6)-benzyl-5'-N-phenylcarboxamidoadenosine with an IC(50) value of 0.91 microM against Trypanosoma brucei rhodesiense and N(6)-diphenylethyl-5'-phenylcarboxamidoadenosine with an IC(50) value of 1.8 microM against chloroquine resistant Plasmodium falciparum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2005.10.011DOI Listing
March 2006

The mechanism of selective purine C-nitration revealed: NMR studies demonstrate formation and radical rearrangement of an N7-nitramine intermediate.

J Am Chem Soc 2005 Apr;127(16):5957-63

Van't Hoff Institute for Molecular Sciences, Universiteit van Amsterdam, Nieuwe Achtergracht 129, NL-1018 WS Amsterdam, The Netherlands.

Modified purine derivatives are of great importance in biomedical sciences, and substitution reactions on the purine skeleton are intensively studied. In our laboratory, an efficient and selective purine C2-nitration reaction was developed using a mixture of tetrabutylammonium nitrate and trifluoroacetic anhydride. The resulting 2-nitro moiety appeared to be a versatile handle to introduce a variety of pharmacophores onto the purine skeleton. Since the mechanism of this selective purine C2-nitration reaction has remained unclear, we now present an extensive NMR study leading to its elucidation, using N9-Boc-protected 6-chloropurine as a model compound. Direct electrophilic aromatic nitration of the highly electron-deficient C2 position was excluded, and we demonstrate that this reaction occurs in a three-step process. Electrophilic attack by trifluoroacetyl nitrate on the purine N7 position results in a nitrammonium species that is trapped by a trifluoroacetate anion furnishing N7-nitramine intermediate 11. This intermediate was characterized at -50 degrees C by (1)H, (13)C, (15)N, and (19)F NMR. At T > -40 degrees C, the N7-nitramine intermediate undergoes a nitramine rearrangement, which generates a C2-nitro species that immediately eliminates TFA to give 2-nitro-6-chloro-9-Boc purine 10. The involvement of radicals during the nitramine rearrangement was unequivocally established by (15)N-CIDNP. Moreover, the emission signal observed for the rearranged product, 2-nitropurine 10, showed that it is primarily formed in an intermolecular process. A quantitative radical trapping experiment finally disclosed that 65-70% of the nitramine rearrangement takes place intermolecularly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja042590xDOI Listing
April 2005