Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.
The solid phase synthesis of the known proteasome inhibitor ZL3VS, using Kenner's safety catch protocol, is described. This methodology has been extended to the synthesis of various novel peptide vinyl sulfone and peptide epoxyketone derivatives. (C) 2000 Elsevier Science Ltd.
Nat Med, 6 (10), pp. 1064. | Citations: 29 (Scopus) | Read more2000. LMP2 expression and proteasome activity in NOD mice.
MOLECULAR BIOLOGY OF THE CELL, 10 pp. 90A-90A. | Citations: 1 (Web of Science Lite)1999. Tight metabolic coupling of the proteasome with downstream oligopeptidases.
Journal of Biological Chemistry, 274 (12), pp. 8344. | Citations: 1 (Scopus)1999. Erratum: T cell recognition of hapten. Anatomy of T cell receptor binding of a H-2K(d)-associated photoreactive peptide derivative (Journal of Biological Chemistry (1999) 274 (3622-3631)
To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.
J Immunol, 161 (12), pp. 6939-6946. | Citations: 56 (Scopus) | Show Abstract1998. Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.
This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.
J Immunol, 161 (2), pp. 553-562. | Citations: 81 (Scopus) | Show Abstract1998. The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement.
Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS(252-260) (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-gamma production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.
FASEB JOURNAL, 12 (5), pp. A936-A936. | Show Abstract1998. The efficiency of antigen recognition by CTL is determined by the frequency of serial TCR engagement.
To investigate the relation between the efficiency of antigen recognition and TCR-ligand binding, we utilized H-2-Kd-restricted CD8+CTL clones that permit assessment of TCR-ligand interactions by TCR photoaffinity labeling. The clones were specific for a derivative of the Plasmodium berghei cirumsporozoite peptide PbCS 252-260 (SYIPSAEKI) containing photoreactive iodo-4-azidosalicylic acid (IASA) in place of PbCS S-252 and 4- azidobenzoic acid (ABA) on PbCS K-259. Selective photoactivation of IASA permitted crosslinking to Kd and photoactivation of ABA to TCR. By assessing antigen recognition (cytotoxicity and IFNγ production) and TCR-ligand binding (TCR photoaffinity labeling) for 12 peptide derivative variants on seven CTL clones, several cases were found, for which the functional response was either ≥ 5-fold lower (antagonists and weak agonists) or higher (strong agonists) than TCR-ligand binding. The efficiency of antigen recognition correlated with the rates of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. Strong agonist exhibited faster TCR-ligand complex dissociation than weak agonists, suggesting that CTL activation depends on the frequency of serial TCR engagement. Consistent with this, we observed that acceleration of TCR-ligand complex dissociation, brought about by blocking of CD8, can increase the efficiency of antigen recognition. On the other hand intercellular TCR-ligand photocrosslinking abrogated CTL function by an active, probably phosphatase mediated, inhibition of T cell activation. Our results indicate that CTL activation is driven by the frequency of serial TCR engagement.
Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR-ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR-ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.
We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.
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