Peptide Diffusion, Protection, and Degradation in Nuclear and Cytoplasmic Compartments before Antigen Presentation by MHC Class I

Reits, Eric; Griekspoor, Alexander; Neijssen, Joost; Groothuis, Tom A.M.; Jalink, Kees; Veelen, Peter A. van; Janssen, Hans; Calafat, Jero; Drijfhout, Jan W.; Neefjes, Jacques

doi:10.1016/s1074-7613(02)00511-3

Cited by 273 publications

(294 citation statements)

References 36 publications

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“…To test this, we cocultured M4E and M4T cells overnight before microinjecting one M4T cell with a mixture of a stable, fluorescently labeled (FL) peptide (FL-DRLDRLDR[C-fluorescein]) and dextran-Texas Red (200 kDa) as an injection marker that is too large to diffuse into neighboring cells through GJs (25,26). Peptides used in the experiment were protected from degradation in cells by adding Damino acids with a protective group at the amino terminus to protect it from cytosolic aminopeptidase activity (25). One hour after microinjection, confocal microscopy analysis showed the diffusion of the FL-peptide into the cytoplasm of the surrounding ECs (Fig.…”

Section: Abrogation Of Ec Lysis By Ctls Following Treatment Of M4t Wimentioning

confidence: 99%

Gap Junction Communication between Autologous Endothelial and Tumor Cells Induce Cross-Recognition and Elimination by Specific CTL

Benlalam

Jalil

Hasmim

et al. 2009

The Journal of Immunology

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International audienceCellular interactions in the tumor stroma play a major role in cancer progression but can also induce tumor rejection. To explore the role of endothelial cells in these interactions, we used an in vitro three-dimensional collagen matrix model containing a cytotoxic T lymphocyte CTL clone (M4.48), autologous tumor cells (M4T), and an endothelial cell (M4E) line that are A derived from the same tumor. We demonstrate in this study that specific killing of the endothelial cells by the CTL clone required the autologous tumor cells and involved Ag cross-presentation. The formation of gap junctions between endothelial and tumor cells is required for antigenic peptide transfer to endothelial cells that are then recognized and eliminated by CTL. Our results indicate that gap junctions facilitate an effective CTL-mediated destruction of endothelial cells from the tumor microenvironment that may contribute to the control of tumor progression

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Section: Abrogation Of Ec Lysis By Ctls Following Treatment Of M4t Wimentioning

confidence: 99%

Gap Junction Communication between Autologous Endothelial and Tumor Cells Induce Cross-Recognition and Elimination by Specific CTL

Benlalam

Jalil

Hasmim

et al. 2009

The Journal of Immunology

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“…Peptide pulsing was performed by incubation of spleen cells with 10 g/ml peptide for 30 min at room temperature. After 5 days of incubation, cytotoxic activity of restimulated cells was measured in a standard 4-h 51 Cr-release assay. EL4 target cells were labeled with 100 Ci of sodium [ 51 Cr]chromate and incubated with or without 1 g/ml peptide for 1 h then extensively washed and used as target cells.…”

Section: Immunization Elispot and 51 Cr-release Assaysmentioning

confidence: 99%

“…T cell responses from immunized animals were analyzed either by ex vivo IFN-␥ ELISPOT (Fig. 3A), or by a standard 51 Cr-release assay as described in Materials and Methods (Fig. 3B).…”

Section: A Novel Cleavable Linker Sequence Ensures Optimal Ag Processmentioning

confidence: 99%

“…3B). The differences in rank in potency between the constructs as evaluated by the ex vivo ELISPOT and the 51 Cr-release assay may reflect the change in frequency of reactive CD8 T cells that occur as a result 4 g) HSP70 or the appropriate controls in 50 l of saline containing 0.1 mM ADP, as described in Materials and Methods. Seven days later, mice were euthanized and the spleens harvested for analysis.…”

Section: A Novel Cleavable Linker Sequence Ensures Optimal Ag Processmentioning

confidence: 99%

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High-Affinity Interactions between Peptides and Heat Shock Protein 70 Augment CD8+ T Lymphocyte Immune Responses

Flechtner

Cohane

Mehta

et al. 2006

The Journal of Immunology

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Exogenously delivered antigenic peptides complexed to heat shock proteins (HSPs) are able to enter the endogenous Ag-processing pathway and prime CD8+ CTL. It was determined previously that a hybrid peptide containing a MHC class I-binding epitope and HSP70-binding sequence Javelin (J0) in complex with HSP70 could induce cytotoxic T cell responses in vivo that were more robust than those induced by the minimal epitope complexed with HSP70. The present study introduces a novel, higher-affinity HSP70-binding sequence (J1) that significantly enhances binding of various antigenic peptides to HSP70. A competition binding assay revealed a dissociation constant that was 15-fold lower for the H2-Kb OVA epitope SIINFEKL-J1 compared with SIINFEKL-J0, indicating a substantially higher affinity for HSP70. Further, modifying the orientation of the hybrid epitope and introducing a cleavable linker sequence between the Javelin and the epitope results in even greater immunogenicity, presumably by greater efficiency of epitope processing. The enhanced immunogenicity associated with Javelin J1 and the cleavable linker is consistently observed with multiple mouse and human epitopes. Thus, by creating a series of epitopes with uniform, high-affinity binding to HSP70, successful multiple epitope immunizations are possible, with equal delivery of each antigenic epitope to the immune system via HSP70. These modified epitopes have the potential for creating successful multivalent vaccines for immunotherapy of both infectious disease and cancer.

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“…Additionally, current ideas on protein degradation concede that in vivo monitoring of overall cellular protein degradation is less possible because it is believed that intermediate peptides being produced by the proteasomes or lysosomes are broken down so quickly into amino acids by peptidases that the sequence of protein degradation is undetectable except for special cases such as with the major histocompatibility complexes. 18,19 The proteolytic degradation of proteins in cells on the proteome-wide level basically is still not understood fully.In this work, we introduce a mass spectrometry (MS)-based method to study proteome-wide protein degradation. Yeast Saccharomyces cerevisiae was used as a model organism due to its simplicity and mechanistic homology to higher eukaryotic cells.…”

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confidence: 99%

Mass Spectrometry Analysis of Proteome-Wide Proteolytic Post-Translational Degradation of Proteins

et al. 2008

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Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their 200 protein substrate origins we obtain evidence for new insights into the proteome-wide protein selective degradation in yeast cells. This evidences shows that the yeast cytoplasm is the largest pool for the degradation of proteins with both biochemical and geometric specificities, while the yeast nucleus seems to be a proteolysis-inert organelle under the condition studied. Yeast V-ATPase subunits appear to be degraded during their disassembly, and yeast mitochondrial proteins functioning as precursors, transport carriers and gates are preferentially degraded. Ubiquitylation may be unnecessary for the proteasomal degradation of yeast cytoplasmic regulatory and enzyme proteins according to our observations. This study shows that the intracellular peptides are informational targets for directly probing the protein degradation-involved molecular mechanisms and cell biology processes.Protein degradation is a post-translational modification necessary for protein quality control and cell survival. In eukaryotic cells degradation of proteins is primarily carried out in either the proteasomes or lysosomes. Proteasomes, 1,2 are responsible for protein quality control by breaking down misfolded proteins, 3 and they have a well-defined geometric proteolysiscatalytic center 4,5 that breaks down the proteins to produce peptides with specific lengths. 6,7 Ubiquitylation 7,8 of proteins is the typical initial step for protein degradation through the proteasomes (note: ubiquitylation also functions for non-proteolytic purposes 10 ). Resultant peptides from proteasomal degradation can then be further cleaved into amino acids by cytosolic aminopeptidases. 11 Lysosomes, on the other hand, are membrane bound organellelike structures containing multiple proteases that degrade protein aggregates and membrane proteins. 3,12 Proteins to-be-degraded are transported to the lysosome through e.g., the ATGs/ signaling/regulating systems 13,14 and the following proteolyses are catalyzed by endoproteinases 15,16 that are matured and activated within the lysosome. The resultant peptides continue to break down into amino acids by the lysosomal aminopeptidases and carboxypeptidases, 16,17 and the products (e.g., amino acids and others such as sugars, cholesterols) are then transported back out into the cytosol by the solute transporters in the Requests for materials should be addressed to Yufeng Shen (E-mail: yufeng.shen@pnl.gov) or Richard D. Smith (E-mail: dick.smith@pnl.gov). NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript lysosomal membrane and can then be recycled as nutrients making this de...

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Peptide Diffusion, Protection, and Degradation in Nuclear and Cytoplasmic Compartments before Antigen Presentation by MHC Class I

Cited by 273 publications

References 36 publications

Gap Junction Communication between Autologous Endothelial and Tumor Cells Induce Cross-Recognition and Elimination by Specific CTL

Gap Junction Communication between Autologous Endothelial and Tumor Cells Induce Cross-Recognition and Elimination by Specific CTL

High-Affinity Interactions between Peptides and Heat Shock Protein 70 Augment CD8+ T Lymphocyte Immune Responses

Mass Spectrometry Analysis of Proteome-Wide Proteolytic Post-Translational Degradation of Proteins

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