John E. Kay

Polyamine synthesis during lymphocyte activation. Induction of ornithine decarboxylase and S-adenosyl methionine decarboxylase.

Abstract Lymphocyte stimulation by phytohaemagglutinin (PHA) is accompanied by marked increases in the activities of ornithine decarboxylase and S -adenosyl methionine decarboxylase, two key enzymes for the synthesis of polyamines. Both enzymes increase in a biphasic manner, with the rises in S -adenosyl methionine decarboxylase preceding the increases in ornithine decarboxylase. The initial rises precede the initiation of DNA synthesis, and seem to correlate with the increased rate of ribosomal RNA synthesis. Selective inhibition of ribosomal RNA synthesis inhibits the increases in the activity of both enzymes, especially ornithine decarboxylase, more than the increase in the overall rate of protein synthesis. Both enzymes are metabolically unstable and have half-lives of less than 1 h, although the half-life of ornithine decarboxylase depends on the amino acid concentration in the culture medium. While effects of PHA on the stability of the enzymes have not been ruled out, at least part of the PHA-dependent increases in activity are due to increased synthesis or activation of the enzymes. The synthesis of S -adenosyl-methionine decarboxylase declines rapidly after inhibition of RNA synthesis, but ornithine decarboxylase activity declines at about the same rate as protein synthesis as a whole. The activities of both enzymes also increase during lymphocyte stimulation by concanavalin A, lentil extract and staphylococcal filtrate.

Chlamydia trachomatis Mip‐like protein has peptidylprolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection

The Mip‐like protein of Chlamydia trachomatis has sequence similarity with both the Mip protein of Legionella pneumophila, a virulence factor necessary for optimal intracellular infection, and FK506‐binding proteins (FKBPs) of both prokaryotic and eukaryotic origin. FKBPs contain a site for peptidyl‐prolyl cis/trans isomerase activity, which is blocked upon binding of the drugs, FK506 or rapamycin. In this paper we report that the recombinant chlamydial Mip‐like protein exhibits a peptidyl‐prolyl cis/trans isomerase activity which is inhibited by either rapamycin or FK506. To assess the role of the Mip‐like protein in chlamydial infection, rapamycin or FK506 (25μM), were used in either treatment of chlamydial organisms prior to inoculation, or were present at different intervals through the infection. Pretreatment of organisms alone reduced infectivity for McCoy cells by 30%, with inhibition rising to 80% on more prolonged exposure from 0 to 8h and 8 to 16 h post‐inoculation and declining thereafter. When drug was present during the developmental cycle at intervals from 0 to 24h post‐inoculation abnormal chlamydiae were induced in residual inclusions. The results suggest that inhibition of the isomerase of the Mip‐like protein interferes with one or more early events in the infective process that determine productive intracellular infection.

Control of protein synthesis during the activation of lymphocytes by phytohaemagglutinin

Abstract During the activation of lymphocytes by phytohaemagglutinin the rate of synthesis of proteins increases markedly. This is accompanied by a large increase in the rate of synthesis of ribosomal RNA but the increase in the total numbers of ribosomes is less dramatic. Calculation of the average rate of protein synthesis per ribosome in lymphocyte cultures at different stages of activation showed that this rate increased considerably during activation, especially in the period preceeding the initiation of DNA synthesis. The percentage of lymphocyte ribosomes active in protein synthesis has been determined by their resistance to dissociation to subunits at high ionic strength. More than 70 % of the ribosomes of resting lymphocytes were inactive at any instant as judged by this criterion. This proportion decreases to about 30 % during the pre-DNA synthetic stage of lymphocyte stimulation, and remains low throughout the S period. Activation of inactive ribosomes accounts for much of the increase in the average rate of protein synthesis per ribosome seen during the early period of activation, but at later stages there must also be an increase in the average rate of protein synthesis per active ribosome. Ribosomes synthesized before and after addition of phytohaemagglutinin to lymphocytes are equally active. Investigation of the properties of the inactive ribosomes of resting lymphocytes has shown that, unlike those of stimulated cells, they do not become attached to polyribosomes when protein synthesis is partially inhibited by cycloheximide. They are, however, capable of attaching to the synthetic messenger RNA poly(U).

Effect of inhibition of spermidine formation on protein and nucleic acid synthesis during lymphocyte activation

In many types of mammalian cells the activities of the enzymes involved in polyamine synthesis increase very markedly with the growth rate, and the polyamine content correlates well with the RNA content [l] . While a variety of effects of polyamines on protein and nucleic acid synthesis in mammalian cells, and cell-free systems derived from them, have been reported [ 1, 21, their biological function is still far from clear. Perhaps the most favoured suggestion is that they may be involved in the control of ribosomal RNA synthesis [3, 41, which is itself intimately connected with the growth rate. Methyl glyoxal bis (guanylhydrazone) (MGBG) is a potent inhibitor of the putrescine-dependent S-adenosyl methionine decarboxylase isolated from yeast and animal cells [5], and thus should prevent the synthesis of spermidine from putrescine [6]. Injection of this drug causes a depression of the growth of leukaemic cells in mice that is prevented by simultaneous injection of spermidine [7] . We have used this inhibitor to study the requirement for spermidine synthesis during the activation of cultured human lymphotyces by phytohaemagglutinin (PHA). Human lymphocytes do not normally synthesize DNA or divide in culture, but addition of PHA to the culture medium induces them to initiate DNA synthesis after a lag of about 30 hr [8] . A number of metabolic changes occur prior to the initiation of DNA synthesis, including increases in the rates of RNA and protein synthesis. The increase in the rate of synthesis of ribosomal RNA is especially marked [9]. Prominent amongst these early changes are marked rises in the

Identification and comparative analysis of the peptidyl-prolyl cis/trans isomerase repertoires of H. sapiens, D. melanogaster, C. elegans, S. cerevisiae and Sz. pombe

The peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins comprises three member families that are found throughout nature and are present in all the major compartments of the cell. Their numbers appear to be linked to the number of genes in their respective genomes, although we have found the human repertoire to be smaller than expected due to a reduced cyclophilin repertoire. We show here that whilst the members of the cyclophilin family (which are predominantly found in the nucleus and cytoplasm) and the parvulin family (which are predominantly nuclear) are largely conserved between different repertoires, the FKBPs (which are predominantly found in the cytoplasm and endoplasmic reticulum) are not. It therefore appears that the cyclophilins and parvulins have evolved to perform conserved functions, while the FKBPs have evolved to fill ever-changing niches within the constantly evolving organisms. Many orthologous subgroups within the different PPIase families appear to have evolved from a distinct common ancestor, whereas others, such as the mitochondrial cyclophilins, appear to have evolved independently of one another. We have also identified a novel parvulin within Drosophila melanogaster that is unique to the fruit fly, indicating a recent evolutionary emergence. Interestingly, the fission yeast repertoire, which contains no unique cyclophilins and parvulins, shares no PPIases solely with the budding yeast but it does share a majority with the higher eukaryotes in this study, unlike the budding yeast. It therefore appears that, in comparison with Schizosaccharomyces pombe, Saccharomyces cerevisiae is a poor representation of the higher eukaryotes for the study of PPIases.

Cyclolinopeptide A (CLA) mediates its immunosuppressive activity through cyclophilin-dependent calcineurin inactivation

The immunosuppressive cyclic nonapeptide cyclolinopeptide A inhibits calcium‐dependent, but not calcium‐independent, activation of T lymphocytes comparably to the actions of cyclosporin A and FK506. The concentration required for complete inhibition, however, is 10 times higher than that of cyclosporin A. In addition, we demonstrate that calcineurin, a phosphatase which plays an important role in T lymphocyte signalling, is inhibited in vitro by cyclolinopeptide A by a mechanism dependent on the peptidyl‐prolyl cis‐trans isomerase (PPIase) cyclophilin A but not FKBP12. Direct binding of cyclolinopeptide A to cyclophilin A was confirmed using tryptophan fluorescence studies and PPIase assays. These results represent a third example of the production of a natural product that neutralises calcineurin by a mechanism dependent on the primary binding to a PPIase.

Ornithine decarboxylase and ribosomal RNA synthesis during the stimulation of lymphocytes by phytohaemagglutinin

Omithine decarboxylase (ODC) catalyses the first step in the biosynthesis of polyarnines, the conversion of omithine to putrescine [l] . Its activity rises dramatically at an early stage in the conversion of many types of cells from a non-growing to a growing state [2-41 and it has also been reported to have an unusually short half life [5] . The consequent rapid fluctuations in enzyme activity in response to growth stimulation suggest that this enzyme might be involved in cell growth regulation mechanisms. Prominent among the sequence of metabolic changes invariably associated with the change from the non-growing to the growing state is an early increase in the synthesis of ribosomal RNA (rRNA), and there are some suggestions that ODC activity may be associated with this [6,7] . The effect of growth stimulation on rRNA synthesis has been studied in most detail during the activation of cultured human lymphocytes by phytohaemagglutinin (PHA). Separate effects of the growth stimulant on the synthesis of ribosomal precursor RNA and on the rate and efficiency of the maturation of the precursor rRNA have been identified [8-lo] . We have found that ODC activity increases greatly when lymphocytes are cultured with PHA. This increase correlates with, and is dependent upon, the major increase in the net synthesis of rRNA.

The control of protein synthesis during the stimulation of lymphocytes by phytohaemagglutinin: III. Poly(U) translation and the rate of polypeptide chain elongation

1. Cell-free systems from phytohaemagglutinin-stimulated pig lymphocytes are much more active in both endogenous protein synthesis and the translation of poly(U) than those from unstimulated lymphocytes. 2. Addition of tRNA stimulates the translation of poly(U) and greatly reduces the difference between systems from stimulated and unstimulated lymphocytes. Endogenous protein synthesis is not increased by added tRNA. 3. Systems from stimulated lymphocytes have an increased capacity to form aminoacyl-tRNA with several different amino acids. This reaction is limited by the amount of tRNA present in the cell-free system. 4. The rate of polypeptide elongation is not affected by lymphocyte stimulation. The increased rate of protein synthesis must therefore be due to an increase in the frequency of initiation of the synthesis of protein molecules.

Mechanisms of T lymphocyte activation

The proliferation of T lymphocytes in response to an antigenic stimulus requires the successive ligation of a range of T lymphocyte receptors by MHC-presented antigen, molecules expressed on the surfaces of accessory cells and lymphokines. A range of intracellular signalling systems are involved, with the possibility of alternative activation pathways utilising different intracellular signalling systems, though protein kinase C activation appears to play a key role. Appreciation of the complexity of the response may allow more selective clinical modulation of T lymphocyte-dependent immunological responses.

The human FK506-binding proteins: characterization of human FKBP19

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas.