77 research outputs found
The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing
MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8+ T cell stimulation in vitro similar to the wtgp100209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8+ T cell response also towards N-terminally extended versions of the minimal epitope
Proteinase-activated receptor 2 (PAR2) in hepatic stellate cells – evidence for a role in hepatocellular carcinoma growth in vivo
Background Previous studies have established that proteinase-activated
receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma
(HCC) cells, suggesting a role in HCC progression. Here, we assessed the
impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate
cells (HSCs) and Hep3B cells as model. Methods PAR2 expression and function in
LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron
microscopy, and [Ca2+]i measurements, respectively. The impact of
LX-2-expressed PAR2 on tumour growth in vivo was monitored using HCC
xenotransplantation experiments in SCID mice, in which HCC-like tumours were
induced by coinjection of LX-2 cells and Hep3B cells. To characterise the
effects of PAR2 activation in LX-2 cells, various signalling pathways were
analysed by immunoblotting and proteome profiler arrays. Results Following
verification of functional PAR2 expression in LX-2 cells, in vivo studies
showed that these cells promoted tumour growth and angiogenesis of HCC
xenografts in mice. These effects were significantly reduced when F2RL1
(encoding PAR2) was downregulated by RNA interference (RNAi). In vitro studies
confirmed these results demonstrating RNAi mediated inhibition of PAR2
attenuated Smad2/3 activation in response to TGF-β1 stimulation in LX-2 cells
and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B
cells. Furthermore, PAR2 stimulation with trypsin or a PAR2-selective
activating peptide (PAR2-AP) led to activation of different intracellular
signalling pathways, an increased secretion of pro-angiogenic and pro-mitotic
factors and proteinases, and an enhanced migration rate across a collagen-
coated membrane barrier. Silencing F2RL1 by RNAi or pharmacological inhibition
of Src, hepatocyte growth factor receptor (Met), platelet-derived growth
factor receptor (PDGFR), p42/p44 mitogen activated protein kinase (MAPK) or
matrix-metalloproteinases (MMPs) blocked PAR2-AP-induced migration. Conclusion
PAR2 in HSCs plays a crucial role in promoting HCC growth presumably by
mediating migration and secretion of pro-angiogenic and pro-mitotic factors.
Therefore, PAR2 in stromal HSCs may have relevance as a therapeutic target of
HCC
Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes
Proteasome-catalyzed peptide splicing represents an additional catalytic
activity of proteasomes contributing to the pool of MHC-class I-presented
epitopes. We here biochemically and functionally characterized a new melanoma
gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42
antigenic peptide is generated in vitro and in cellulo by a not yet described
proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced
in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-
specific CD8+ T cell response. Importantly, we demonstrate that different
gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells
with efficacies comparable to non-spliced canonical tumor epitopes and that
gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in
peripheral blood of half of the melanoma patients tested. Our data suggest
that both transpeptidation and condensation reactions contribute to the
frequent generation of spliced epitopes also in vivo and that their immune
relevance may be comparable to non-spliced epitopes
Systemic RNAi in C. elegans Requires the Putative Transmembrane Protein SID-1
. 23. Emission spectra were analyzed with a Fluorolog 2 spectrophotometer controlled by Datamax 2.2 ( Jobin Yvon Spex) and the Grams 3.04 II software package (Galactic Industries, Salem, NH ). Both excitation and emission slits were set at 2 nm and voltage at 950 V. 24. Recombinant S-tagged Ran (2 M) preloaded with either GTP or GDP was incubated with 4 M purified YRC in phosphate-buffered saline on ice for 30 min. The Ran-GTP reaction was split into two aliquots, and one sample was treated with 100 ng of recombinant Ran-GAP. S-tagged importin  (2 M) was incubated with recombinant importin ␣ or YIC (each at 2 M final concentration) in either the presence or absence of Ran preloaded with GTP (4 M). Proteins bound to Ran or importin  were retrieved on protein-S-agarose beads, eluted with sample buffer, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAG
CERT1 mutations perturb human development by disrupting sphingolipid homeostasis
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome
The 20S Proteasome Splicing Activity Discovered by SpliceMet
The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected
The proapoptotic influenza A virus protein PB1-F2 forms a nonselective ion channel
Background: PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization.
Methodology/Principal Findings: Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1)( IAV(PR8)), from A/Brevig Mission/1/1918( H1N1) (IAV(SF2)) or the H5N1 consensus sequence (IAV(BF2)) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns.
Conclusion/Significance: The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development
CERT1 mutations perturb human development by disrupting sphingolipid homeostasis
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.This work was supported by the National Institute of Neurological Disorders and Stroke (NINDS), NIH (R01NS109858, to VAG); the Paul A. Marks Scholar Program at the Columbia University Vagelos College of Physicians and Surgeons (to VAG); a TIGER grant from the TAUB Institute at the Columbia Vagelos College of Physicians and Scientists (to VAG); the Swiss National Science Foundation (SNF 31003A-179371, to TH); the European Joint Program on Rare Diseases (EJP RD+SNF 32ER30-187505, to TH); the Swiss Cancer League (KFS-4999-02-2020, to GD); the EPFL institutional fund (to GD); the Kristian Gerhard Jebsen Foundation (to GD); the Swiss National Science Foundation (SNSF) (310030_184926, to GD); the Swiss Foundation for Research on Muscle Disease (FSRMM, to MAL); the Natural Science and Engineering Research Council of Canada (Discovery Grant 2020-04241, to JEB); the Italian Ministry of Health Young Investigator Grant (GR-2011-02347754, to EL); the Fondazione Istituto di Ricerca Pediatrica – Città della Speranza (18-04, to EL); the Wroclaw Medical University (SUB.E160.21.004, to RS); the National Science Centre, Poland (2017/27/B/NZ5/0222, to RS); Telethon Undiagnosed Diseases Program (TUDP) (GSP15001); the Temple Street Foundation/Children’s Health Foundation Ireland (RPAC 19-02, to IK); the Deutsche Forschungsgemeinschaft (DFG) (PO2366/2–1, to BP); the Instituto de Salud Carlos III, Spain (to ELM, EBS, and BMD); the National Natural Science Foundation of China (81871079 and 81730036, to HG and KX); and the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH (R01 DK115574, to SSC).The DEFIDIAG study is funded by grants from the French Ministry of Health in the framewok of the national French initiative for genomic medicine. The funders were not involved in the study design, data acquisition, analysis, or writing of the manuscript. Funding for the DECIPHER project was provided by Wellcome. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between Wellcome and the Department of Health, and the Wellcome Sanger Institute (grant number WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of Wellcome or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South REC, and GEN/284/12, granted by the Republic of Ireland REC). The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network.S
CERT1 mutations perturb human development by disrupting sphingolipid homeostasis
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome
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