Peptide length variants p2Ca and QL9 present distinct conformations to L(d)-specific T cells

Recent advances have provided insights into how the TCR interacts with MHC/peptide complexes and a rationale to predict optimal epitopes for MHC binding and T cell recognition. For example, peptides of nine residues are predicted to be optimal for binding to H2-L(d), although 8 mer epitopes have also been identified. It has been predicted that 8 mer and 9 mer length variant peptides bound to L(d) present identical epitopes to T cells. However, in contrast to this prediction, we demonstrate here that the 8 mer peptide p2Ca and its 9 mer length variant QL9, extended by an N-terminal glutamine, assume distinct conformations when bound to L(d). We generated self-L(d)-restricted CTL clones specific for p2Ca that recognize L(d)/QL9 poorly if at all. This result is in sharp contrast to what has been observed with L(d)-alloreactive T cells that possess a much higher affinity for L(d)/QL9 than for L(d)/p2Ca. Alanine substitutions of the N-terminal residues of the QL9 peptide rescue detection by these self-L(d)/p2Ca-specific T cells, but decrease recognition by the L(d)-alloreactive 2C T cell clone. In addition, 2C T cell recognition of the p2Ca peptide is affected by different alanine substitutions compared with 2C T cell recognition of the QL9 peptide. These data clearly demonstrate that the p2Ca and QL9 peptides assume distinct conformations when bound to L(d) and, furthermore, demonstrate that there is flexibility in peptide binding within the MHC class I cleft.     

Discovery and initial structure-activity relationships of trisubstituted ureas as thrombin receptor (PAR-1) antagonists

Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.     

The role of the complement cascade in ischemia/reperfusion injury: implications for neuroprotection

BACKGROUND: The complement cascade plays a deleterious role in multiple models of ischemia/reperfusion (I/R) injury, including stroke. Investigation of the complement cascade may provide a critical approach to identifying neuroprotective strategies that can be effective at clinically relevant time points in cerebral ischemia. This review of the literature describes the deleterious effects of complement activation in systemic I/R models and previous attempts at therapeutic complement inhibition, with a focus on the potential role of complement inhibition in ischemic neuroprotection. Translation of these concepts into ischemic stroke models and exploration of related neuroprotective strategies are also reviewed. SUMMARY OF REVIEW: We performed a MEDLINE search to identify any studies published between 1966 and 2001 dealing with complement activation in the setting of I/R injury. We also searched for studies demonstrating up-regulation of any complement components within the central nervous system during inflammation and/or ischemia. CONCLUSIONS: The temporal and mechanistic overlap of the complement cascade with other biochemical events occurring in cerebral I/R injury is quite complex and is only beginning to be understood. However, there is compelling evidence that complement is quite active in the setting of acute stroke, suggesting that anticomplement strategies should be further investigated through genetic analysis, nonhuman primate models, and clinical investigations.     

Reovirus binding to cell surface sialic acid potentiates virus-induced apoptosis

Reovirus induces apoptosis in cultured cells and in vivo. Genetic studies indicate that the efficiency with which reovirus strains induce apoptosis is determined by the viral S1 gene, which encodes attachment protein sigma1. However, the biochemical properties of sigma1 that influence apoptosis induction are unknown. To determine whether the capacity of sigma1 to bind cell surface sialic acid determines the magnitude of the apoptotic response, we used isogenic reovirus mutants that differ in the capacity to engage sialic acid. We found that T3SA+, a virus capable of binding sialic acid, induces high levels of apoptosis in both HeLa cells and L cells. In contrast, non-sialic-acid-binding strain T3SA- induces little or no apoptosis in these cell types. Differences in the capacity of T3SA- and T3SA+ to induce apoptosis are not due to differences in viral protein synthesis or production of viral progeny. Removal of cell surface sialic acid with neuraminidase abolishes the capacity of T3SA+ to induce apoptosis. Similarly, incubation of T3SA+ with sialyllactose, a trisaccharide comprised of lactose and sialic acid, blocks apoptosis. These findings demonstrate that reovirus binding to cell surface sialic acid is a critical requirement for the efficient induction of apoptosis and suggest that virus receptor utilization plays an important role in regulating cell death.     

Differential protein phosphorylation in 3T3-L1 adipocytes in response to insulin versus platelet-derived growth factor. No evidence for a phosphatidylinositide 3-kinase-independent pathway in insulin signaling

Insulin regulates glucose metabolism in adipocytes via a phosphatidylinositide 3-kinase (PI3K)-dependent pathway that appears to involve protein phosphorylation. However, the generation of phosphoinositides is not sufficient for insulin action, and it has been suggested that insulin regulation of glucose metabolism may involve both PI3K-dependent and -independent pathways, the latter being insulin specific. To test this hypothesis, we have designed a phosphoprotein screen to study insulin-specific phosphoproteins that may be either downstream or in parallel to PI3K. Nineteen insulin-regulated phosphospots were detected in the cytosol and high speed pellet fractions, only six of which were significantly regulated by platelet-derived growth factor. Importantly, almost all (92%) of the insulin-specific phosphoproteins identified using this approach were sensitive to the PI3K inhibitor wortmannin. Thus, we obtained no evidence for an insulin-specific, PI3K-independent signaling pathway. A large proportion (62%) of the insulin-specific phosphoproteins were enriched in the same high speed pellet fraction to which PI3K was recruited in response to insulin. Thus, our data suggest that insulin specifically stimulates the phosphorylation of a novel subset of downstream targets and this may in part be because of the unique localization of PI3K in response to insulin in adipocytes.     

Colon cancer cells adhesion and spreading on autocrine laminin-10 is mediated by multiple integrin receptors and modulated by EGF receptor stimulation

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.     

Amyloid beta -peptide-binding alcohol dehydrogenase is a component of the cellular response to nutritional stress

Amyloid beta-peptide-binding alcohol dehydrogenase (ABAD) is a member of the family of short chain dehydrogenase/reductases whose distinctive properties include the capacity to bind amyloid beta-peptide and enzymatic activity toward a broad array of substrates including n-isopropanol and beta-estradiol. In view of the wide substrate specificity of ABAD and its high activity on l-beta-hydroxyacyl-CoA derivatives, we asked whether it might also catalyze the oxidation of the ketone body d-3-hydroxybutyrate. This was indeed the case, and oxidation proceeded with K(m) of approximately 4.5 mm and V(max) of approximately 4 nmol/min/mg protein. When placed in medium with d-beta-hydroxybutyrate as the principal energy substrate, COS cells stably transfected to overexpress wild-type ABAD (COS/wtABAD) better maintained 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, cellular energy charge, and morphologic phenotype compared with COS/vector cells. Using a severe model of metabolic perturbation, transgenic mice with targeted neuronal expression of ABAD subjected to transient middle cerebral artery occlusion showed strokes of smaller volume and lower neurologic deficit scores in parallel with increased brain ATP and decreased lactate, compared with nontransgenic controls. These data suggest that ABAD contributes to the protective response to metabolic stress, especially in the setting of ischemia.     

Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.     

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.