Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling

Microcapillary HPLC electrospray ionization tandem mass spectrometry was used to sequence 15 peptides eluted from HLA-B7. Sequence alignment implicated four peptide positions in specific interactions with the class I molecule, and their importance was confirmed using synthetic peptides. Because no crystal structure for HLA-B7 was available, computer-assisted modeling was used to understand novel aspects of peptide binding specificity and to accurately predict the effect of defined changes in peptide structure. The results demonstrate that mass-spectrometric sequencing coupled with computer-assisted modeling can be used in the absence of a crystal structure to make accurate predictions concerning requirements for peptide binding to class I molecules. These techniques may be valuable to predict or engineer T cell epitopes.     

Liquid chromatography/tandem mass spectrometry of synthesis products associated with the viral protein US11

US11 is a small basic protein composed of 161 amino acid residues, and is among the most abundant viral proteins in cells infected with herpes simplex viruses HSV1 and HSV2. The amino acid sequence [91-121] is considered essential for the binding of this protein with RNA. Automated solid phase synthesis of this fragment resulted in a crude reaction mixture containing the desired sequence as well as a number of unknown side products. On-line liquid chromatography/electrospray mass spectrometry (LC/ES-MS) and LC/ES tandem mass spectrometry (MS/MS) allowed the identification of the separated components and furnished relevant sequencing information. The unusual sequences of the monitored components, which consist of a tandemly repeated three-amino-acid motif with the sequence Arg-X-Pro, where X is an acidic or uncharged polar amino acid residue, yielded product ion spectra lacking substantial sequence information and rich in fragment ions manifesting the neutral losses 17, 42 and 60 u.     

Ig receptor binding protein 1 (alpha4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A

Rapamycin is an immunosuppressant that effectively controls various immune responses; however, its action in the signal transduction of lymphocytes has remained largely unknown. We show here that a phosphoprotein encoded by mouse alpha4 (malpha4) gene transmitting a signal through B-cell antigen receptor (BCR) is associated with the catalytic subunit of protein phosphatase 2A (PP2Ac). The middle region of alph4, consisting of 109 amino acids (94-202), associates directly with PP2Ac, irrespective of any other accessory molecule. Rapamycin treatment disrupts the association of PP2Ac/alpha4 in parallel with the inhibitory effect of lymphoid cell proliferation. The effect of rapamycin was inhibited with an excess amount of FK506 that potentially completes the binding to FKBP. Rapamycin treatment also suppresses the phosphatase activity of cells measured by in vitro phosphatase assay. Introduction of the malpha4 cDNA into Jurkat cells or the increased association of PP2Ac/alpha4 by the culture with low serum concentration confers cells with rapamycin resistance. Moreover, glutathione S-transferase (GST)-alpha4 augments the PP2A activity upon myelin basic protein (MBP) and histone in the in vitro assay. These results suggest that alpha4 acts as a positive regulator of PP2A and as a new target of rapamycin in the activation of lymphocytes.     

Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides

Cell surface proteins SspA and SspB in Streptococcus gordonii and SpaP in Streptococcus mutans are members of the antigen I/II family of polypeptides produced by oral streptococci. These proteins are adhesins and mediate species-specific binding of cells to a variety of host and bacterial receptors. Here we show that antigen I/II polypeptides are involved in the attachment of oral streptococci to collagen and that they also determine the ability of these bacteria to invade human root dentinal tubules. Wild-type S. gordonii DL1 (Challis) cells showed heavy invasion of tubules to a depth of approximately 200 microm, whereas the abilities of cells of isogenic mutant strains OB220 (sspA) and OB219 (sspA sspB) to invade were 50 and >90% reduced, respectively. Likewise, wild-type S. mutans NG8 cells invaded dentinal tubules, whereas cells of isogenic mutant strain 834 (spaP) did not. The invasive abilities of strains OB220 and OB219 were restored by heterologous expression of S. mutans SpaP polypeptide in these strains. The extents of tubule invasion by various wild-type and mutant strains correlated with their levels of adhesion to type I collagen, a major component of dentin. Furthermore, S. gordonii DL1 cells exhibited a growth response to collagen by forming long chains. This was not shown by ssp mutants but was restored by the expression of SpaP in these cells. The production of SspA polypeptide by S. gordonii DL1, but not production of SspB polypeptide by strain OB220 (sspA), was enhanced in the presence of collagen. These results are the first to demonstrate that antigen I/II family polypeptides bind collagen and mediate a morphological growth response of streptococci to collagen. These antigen I/II polypeptide activities are critical for intratubular growth of streptococci and thus for establishment of endodontic infections.     

Platelet hyperactivation in patients with essential thrombocythemia is not associated with vascular endothelial cell damage as judged by the level of plasma thrombomodulin, protein S, PAI-1, t-PA and vWF

The occurrence of thrombotic events remains an important clinical problem in Essential Thrombocythemias (ET). Thus, hemostatic, fibrinolytic and vascular status was investigated in 16 patients (5 males and 11 females) with ET. Among them five presented thromboses in their past history. Platelet hyperactivation, as evidenced by a mean three-fold increase in plasma betathromboglobulin (beta TG), was observed in 13 among 16 patients; surprisingly this activation was present even when the platelet count was normal (in two patients) or subnormal, below 600 x 10(9)/l (in 11 patients). The mean value was 104 +/- 57 IU/ml significantly different from that of normal controls (35 +/- 16.5 IU/ml) (p < 0.001). An artefactual in vitro platelet activation was ruled out by the concomitant measurement of platelet factor 4 (PF4). D-dimers fibrin degradation products (D-Di FDP) were normal in all patients. Vascular endothelial cell function parameters were not markedly modified. The mean value of plasma thrombomodulin (TM) was found slightly but not significantly increased (60.1 +/- 4.9 ng/ml versus 49.1 +/- 10.0 ng/ml in controls). The values of plasma TM correlated neither with that of the platelet count nor with that of plasma beta TG or plasma PF4. The mean values of plasma protein S, von Willebrand factor (vWF), plasminogen activator inhibitor type 1 (PAI-1), tissue plasminogen activator (tPA) were normal and were not correlated neither with that of plasma TM nor with that of plasma beta TG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of T lymphocyte activation by cAMP is associated with down-regulation of two parallel mitogen-activated protein kinase pathways, the extracellular signal-related kinase and c-Jun N-terminal kinase

The induction of T cell proliferation requires signals from the TCR and a co-receptor molecule, such as CD28, that activate parallel and partially cross-reactive signaling pathways. These pathways are disrupted by agonists that utilize adenylate cyclase and cAMP-dependent protein kinase A (PKA). We found that the adenylate cyclase activator, forskolin, inhibits anti-CD3-induced shift in Lck electrophoretic mobility, suggesting an intervention at the TCR-coupled phosphoinositide turnover that precedes the activation of PKC. The shift of Lck following direct PKC activation by 12-O-tetradecanoyl phorbol 13-acetate, which bypasses early receptor-triggered biochemical events, is insensitive to forskolin. Nevertheless, forskolin also inhibits PKC downstream events, such as c-jun expression, which is critical for the activation process of T cells. To further analyze potential cross points between positively and negatively regulating signaling pathways in T cells, we tested the effects of activators of the adenylate cyclase or PKA on two parallel mitogen-activated protein kinase signaling pathways mediated by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase. Using a PKC-specific inhibitor, GF109203X, or PKC-depleted T cells, we found that a large part of the anti-CD3-induced ERK activation is PKC dependent. Both PKC-dependent and -independent activation of ERK were sensitive to inhibition by forskolin or a cell-permeable cAMP analogue, dbcAMP. Furthermore, the effect of 12-O-tetradecanoyl phorbol 13-acetate and ionomycin, which synergized to fully activate c-Jun N-terminal kinase, was also sensitive to inhibition by forskolin. Our results suggest that PKA inhibits T cell activation by interfering with multiple events along the two signaling pathways operating downstream of the TCR and the CD28 co-receptor molecules.     

G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells

UCN-01 (7-hydroxyl-staurosporine) was originally isolated as a Ca2+- and phospholipid-dependent protein kinase C selective inhibitor and now is being developed as an anticancer agent. Results from our and other laboratories have suggested that UCN-01 induces preferential G1-phase accumulation in several human tumor cell lines tested. To elucidate this mechanism, we examined the effects of UCN-01 on several cell cycle-regulatory proteins critical for G1-S-phase transition in p53-mutated human epidermoid carcinoma A431 cells. After 24 h exposure at around 50% growth-inhibitory concentrations (IC50s), 260 and 520 nM, UCN-01 induced the accumulation of pRb (the dephosphorylated retinoblastoma protein form). The protein expression of cyclin A but not cyclin E was markedly reduced and that of cyclin D1 was partially reduced under the same condition. UCN-01 also showed the concentration-dependent inhibitions of the activity of cyclin-dependent kinase 2 (CDK2) using histone H1 and pRb as substrates in vitro (IC50, 530 and 640 nM, respectively). In addition, CDK2 activities of the cells pretreated with UCN-01 for 24 h at 260 and 520 nM were markedly inhibited, giving IC50s of far less than 260 nM. When the same cell lysates were analyzed by Western blotting for CDK2, the lower band (e.g., active and phosphorylated CDK2) was remarkably reduced, in accordance with the reduced activity. Furthermore, UCN-01 induced the expression of the CDK inhibitor p21 protein and its complex formation with CDK2 after 24 h exposure at 260 and 520 nM, whereas the expression level was very low or undetectable in untreated or DNA-damaged cells. The increase of p21 mRNA levels was also induced under the same condition. UCN-01 further increased luciferase activities in A431 cells transiently transfected with p21 promoter-luciferase reporter plasmid after 24 h exposure at 260 and 520 nM. UCN-01 also increased the expression of the CDK inhibitor p27 protein after 24 h exposure at 260 and 520 nM. These results suggest that G1-phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitors p21 and p27.