p56lck SH2 domain binding motifs from bead binding screening of peptide libraries containing phosphotyrosine surrogates

Phosphorylation reactions are key mediators in a variety of biochemical signal processes. Research into the selective inhibition of protein tyrosine kinases to generate anticancer agents has made O-phosphotyrosyl analogues important pharmacological tools. The simple procedures reported here involving the formation of iterative peptide libraries together with the development of a selective and sensitive bead-binding assay have made it possible to rapidly screen peptides incorporating O-phosphotyrosyl surrogates (including O-phospho-2,3,5,6-tetrafluorotyrosine, 4-(phosphono)hydroxymethyl-phenylalanine and 4-(phosphono)fluoromethyl-phenylalanine) for their potential to inhibit the protein tyrosine kinase p56^lck. These procedures can be easily adapted to combinatorial peptide libraries.     

p56lck SH2 domain binding motifs from bead binding screening of peptide libraries containing phosphotyrosine surrogates

Summary Phosphorylation reactions are key meditors in a variety of biochemical signal processes. Research into the selective inhibition of protein tyrosine kinases to generate anticancer agents has madeO-phosphotyrosyl analogues important pharmacological tools. The simple procedures reported here involving the formation of interative peptide libraries together with the development of a selective and sensitive bead-binding assay have made it possible to rapidly screen peptides incorporatingO-phosphotyrosyl surrogates (includingO-phospho-2,3,5,6-tetrafluorotyrosine, 4-(phosphono)hydroxymethyl-phenylalanine and 4-(phosphono)fluoromethyl-phenylalanine) for their potential to inhibit the protein tyrosine kinase p56^lck. These procedures can be easily adapted to combinatorical peptide libraries.     

Protein tyrosine kinase p56lck-deficiency confers hypersusceptibility to rho-fluorophenylalanine (pFPhe)-induced apoptosis by augmenting mitochondrial apoptotic pathway in human Jurkat T cells

Phenylalanine analog, ρ-fluorophenylalanine (pFPhe)-mediated cytotoxicity and several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, Bid cleavage, degradation of PARP and PLCγ-1, and DNA fragmentation were more significant in p56^lck-deficient Jurkat T cells (JCaM1.6) than in wild-type Jurkat T cells (E6.1). The susceptibility of JCaM1.6 toward apoptogenic activity of pFPhe decreased after acquisition of p56^lck by transfection. The p56^lck kinase activity increased 1.6-fold at 15-30 min after pFPhe treatment. The pan-caspase inhibitor (z-VAD-fmk) completely blocked the pFPhe-mediated apoptotic changes except caspase-9 activation. The caspase-8 inhibitor (z-IETD-fmk), which failed to influence pFPhe-induced caspase-9 activation, completely blocked caspase-8 activation and PLCγ-1 degradation with a marked reduction in caspase-3 activation and PARP degradation, indicating pFPhe-induced caspase-8 activation as a downstream event of mitochondria-dependent activation of caspase-9. These results indicate that the deficiency of p56^lck augments pFPhe-induced mitochondrial cytochrome c release and resultant apoptotic cell death in Jurkat T cells.     

Lipoic acid downmodulates CD4 from human T lymphocytes by dissociation of p56(Lck)

Lipoic acid is an antioxidant that suppresses and treats a model of multiple sclerosis, experimental autoimmune encephalomyelitis. We now demonstrate that treatment of human PBMC and T cell lines with LA downmodulated CD4 expression in a concentration-dependent manner. LA treatment of Con A stimulated PBMC specifically removed CD4 from the T-cell surface, but not CD3. Epitope masking by LA was excluded by using monoclonal antibodies targeting different domains of CD4. Incubation on ice inhibited CD4 removal following LA treatment, suggesting that endocytosis was involved in its downmodulation. LA is in a unique category of compounds that induce CD4 downmodulation by various mechanisms (e.g., gangliosides). We hypothesized that LA might induce dissociation of p56(Lck) from CD4, thus leading to its downmodulation. Immunoblot analyses demonstrated reduced co-precipitation of p56(Lck) from Jurkat T-cells following LA treatment and precipitation of CD4. This unique immunomodulatory effect of LA warrants further investigation.     

Assembly of the CD8α/p56 protein complex in stably expressing rat epithelial cells

We have previously characterized the biogenesis of the human CD8α protein expressed in rat epithelial cells. We now describe the biosynthesis, post-translational maturation and hetero-oligomeric assembly of the human CD8α/p56^lck protein complex in stable transfectants obtained from the same cell line. There were no differences in the myristilation of p56^lck, or in the dimerization, O-glycosylation and transport to the plasma membrane of CD8α, between cells expressing either one or both proteins. In the doubly expressing cells, dimeric forms of CD8α established hetero-oligomeric complexes with p56^lck, as revealed by co-immunoprecipitation assays performed with anti-CD8α antibody. Moreover, p56^lck bound in these hetero-oligomeric complexes was endowed with auto- and hetero-phosphorylating activity. The present study shows that: (1) the newly synthesized p56^lck binds rapidly to CD8α and most of the p56^lck is bound to CD8α at steady state; (2) CD8α/p56^lck protein complexes are formed at internal membranes as well as at the plasma membrane; and (3) about 50% of complexed p56^lck reaches the cell surface.     

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.     

Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.     

The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67(phox) and p47(phox). They translocate to the membrane upon cell stimulation and activate flavocytochrome b(558), the membrane-integrated catalytic core of this enzyme system. The activators p67(phox) and p47(phox) form a ternary complex together with p40(phox), an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif- containing domains: p40(phox) associates with p67(phox) via binding of the p40(phox) PC motif to the p67(phox) PB1 domain, while p47(phox) directly interacts with p67(phox) but not with p40(phox). Here we show that p40(phox) enhances membrane translocation of p67(phox) and p47(phox) in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40(phox) carrying the D289A substitution in PC or a p67(phox) with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40(phox) participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67(phox) and p47(phox) via the PB1-PC interaction with p67(phox).     

p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox

p40(phox) activated phagocyte NADPH oxidase without p47(phox) in a cell-free system consisting of p67(phox), Rac and cytochrome b(558) relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47(phox). Addition of p47(phox) slightly increased the activation, but not additively. p40(phox) improved the efficiency of p67(phox) in the activation. The C-terminus-truncated p67(phox), p40(phox)(D289A), p40(phox)(R58A), or p40(phox)(W207R) showed an impaired activation. A peptide corresponding to the p22(phox) Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40(phox) SH3 domain. Thus, p40(phox) can substitute for p47(phox) in the activation, interacting with p22(phox) and p67(phox) through their specific regions.     

Visualization of the endocytic pathway in the filamentous fungus Aspergillus oryzae using an EGFP-fused plasma membrane protein

Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.     

Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell lysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.     

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of the tumour suppressor p33(ING1b)

The tumour suppressor p33(ING1b) ((ING1b) for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33(ING1b) phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33(ING1b) with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33(ING1b). We found that p33(ING1b) is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33(ING1b). Furthermore, ultraviolet irradiation induces p33(ING1b) phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.     

Bornean orangutans on the brink of protein bankruptcy

Protein is a limiting resource that is essential to the growth, maintenance and reproduction of tropical frugivores, yet few studies have examined how wild animals maintain protein balance. During chronic periods of fruit scarcity, Bornean orangutans (Pongo pygmaeus) often catabolize their own fat reserves despite unusually low metabolic requirements. Such energy deficits suggest a marginal existence, and raise the possibility that orangutans also endure periods of negative protein balance. To test this hypothesis, we conducted the first study of protein cycling in a wild primate. Our five year analysis of urinary metabolites revealed evidence of protein recycling when fruit was scarce. During these periods, orangutans consumed more leaves and bark, proteinaceous but tough foods that yielded a mean daily intake of 1.4 g protein kg(-1) metabolic mass. Such an amount is inadequate for humans and one-tenth the intake of mountain gorillas, but sufficient to avert, perhaps narrowly, a severe protein deficit. Our findings highlight the functional and adaptive value of traits that maximize protein assimilation during periods of ecological exigency.     

Overexpressed chloride intracellular channel protein CLIC4 (p64H1) is an essential component of novel plasma membrane anion channels

Chloride intracellular channel protein CLIC4 is a putative organellar anion channel or channel regulator with an unusual dual cytoplasmic and integral membrane localisation. To investigate its contribution to cellular anion channel activity, the protein was overexpressed in stably transfected HEK-293 cells. Patch-clamp recording revealed CLIC4-associated indanyloxyacetic acid-sensitive (IC(50) approximately 100 microM) plasma membrane currents showing mild outward rectification, and novel low conductance (approximately 1pS) CLIC4-associated anion channels were resolved at the single-channel level. The CLIC4-associated channels were inhibited by anti-CLIC4 antibodies, including a monoclonal antibody directed against a FLAG epitope fused to the C-terminus of CLIC4, but only when these were applied to the cytoplasmic (not the external) face of the membrane. CLIC4 is thus an essential molecular component of novel cellular anion channels and the C-terminus of the integral membrane form of CLIC4 is cytoplasmic.