The effects of amino acids on protein degradation and translocation of non-histone proteins to the nucleus in lymphocytes

Tryptophan, phenylalanine and leucine have two parallel effects in cultured lymphocytes, they inhibit cellular proteolysis and increase the translocation of non-histone proteins to the nucleus. The latter is associated with an increased cellular binding of [3H]actinomycin D, indicating an altered structure of chromatin. The amino acids also inhibit the cellular uptake of [3H]chloroquine, suggesting that inhibited protein degradation is lysosomal. Several amine catabolites of tryptophan and phenylalanine, some of which are known to play a role as biogenic amines, have similar actions, and can explain, at least in part, the effects of their parent amino acids. Fractionation of the nuclear 3H-labeled non-histone proteins according to pH 2.5-6.5 shows that such proteins with a high rate of degradation in untreated cells correspond to the 3H-labeled non-histone proteins with a high rate of translocation in tryptophan treated cells. These data suggest that the degradation and the translocation of the non-histone proteins are linked and that the increased translocation of the non-histone proteins to the nucleus may be the consequence of inhibited lysosomal degradation of these proteins by the amino acids.     

The effects of lysosomotropic amines on protein degradation, migration of nonhistone proteins to the nucleus, and cathepsin D in lymphocytes

Various lysosomotropic amines have two parallel effects in human lymphocytes: they inhibit the degradation of cellular proteins and increase the migration of nonhistone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H] actinomycin D, indicating an altered structure of chromatin. The agents inhibit the degradation of short- and long-lived proteins equally. Fractionation of the [3H] NHP of the nucleus according to pH 2.5-6.5 shows that [3H] NHP with a high rate of degradation in untreated cells correspond to [3H] NHP with a high rate of migration in cells treated with the agents. Eserine, amantadine, nicotine, atropine, benzylamine, and propranolol inhibit cathepsin D in concentrations causing proteolytic inhibition in cell cultures or in concentrations believed to be attained in lysosomes. The agents strongly inhibit the cellular accumulation of [3H] chloroquine. The data support the proposal that the migration of NHP from the cytoplasm to the nucleus is the direct consequence of inhibited degradation of these proteins in lysosomes by the amines.     

Endocytosed non-histone proteins translocate in part to the nucleus in lymphocytes

[3H]Non-histone proteins ([3H]NHP), dissolved in the culture medium, are endocytosed by lymphocytes and equilibrate rapidly between the cytoplasm and the nucleus. During incubation, the proteins are gradually degraded in the lysosomes. The lysosomotropic agents conA, NaF, eserine and atropine have two parallel effects on resting lymphocytes, after they have endocytosed [3H]NHP: inhibition of degradation and increased translocation of [3H]NHP from the cytoplasm to the nucleus. This indicates that lysosomal degradation and translocation of [3H]NHP to the nucleus are linked and suggests that this translocation may be the result of inhibited lysosomal degradation of the [3H]NHP. The behaviour of endocytosed [3H]NHP appears similar to that of endogenous [3H]NHP in cells prelabeled with [3H]leucine, when subjected to the same lysosomotropic agents, reported previously (Polet, H, Exp cell res 148 (1983) 345). This observation may provide a model to study the mechanism(s) controlling nucleo-cytoplasmic traffic of NHP.     

Effects of fibroblastic growth factor on protein degradation, the migration of non-histone proteins to the nucleus and DNA synthesis in diploid fibroblasts

Stimulation of resting WI38 cells, prelabeled with [3H]leucine, with fibroblastic growth factor (FGF) or serum, caused increased nuclear translocation of [3H]non-histone proteins [( 3H]NHP) and DNA synthesis, and a parallel decrease of proteolysis. [3H]NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5 showed that [3H]NHP fractions with high degradation rates in resting cells corresponded to the [3H]NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H]NHP are linked. FGF inhibited cellular uptake of [3H]chloroquine, suggesting that FGF inhibits NHP degradation via lysosomes. The lysosomotropic amine eserine had similar effects as FGF. It is proposed that FGF induces NHP migration to the nucleus by inhibiting their lysosomal degradation. FGF also caused migration of [3H]histones, however, the mechanism is not clear.     

Studies on the degradation of HeLa non-histone proteins

The hydrolysis of HeLa non-histone nuclear proteins over 24 h has been monitored in dilute alkali at 4, 15 and 25°C using the standard ninhydrin estimation, dansylation and various electrophoresis techniques. Under conditions (up to 0.2 N NaOH, 4°C) that do not release a significant quantity of ninhydrin-positive material or new N-terminal end group considerable breakdown was observed by two-dimensional electrophoresis analysis. The number of stained spots decreased from approx. 140 to 25–30. No internal protease activity could be found.Labelling studies (¹⁴C-labelled amino acids) showed that much of the hydrolysed material was extracted from the gel during normal staining and destaining procedures. Peptides could be extracted from alkali-hydrolysed non-histone protein with acid/ethanol and could be further separated by thin-layer chromatography on silica gel G. Short-term labelling of HeLa cells (¹⁴C-labelled amino acids for up to 60 min) revealed that these peptides probably have a high rate of turnover. [¹⁴C] Glucosamine studies also indicated the presence of considerable carbohydrate material in the low molecular weight products of this alkaline hydrolysis. Various standard proteins and histones were unaffected by hydrolysis in up to 0.2 N NaOH (4°C, 24 h) as judged by gel electrophoresis.Seven different phosphate-splitting enzymes and an esterase had no effect on the non-histone protein electrophoresis patterns but a preparation of phospholipase C which had no protease activity towards eight standard proteins did produce considerable breakdown in HeLa non-histone proteins similar to that produced by 0.2 N NaOH at 4°C.     

Effects of serum and conditioned medium on protein degradation, migration of nonhistone proteins to the nucleus, and DNA synthesis in transformed cells

Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [3H] NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5, showed that [3H] NHP fractions with high degradation rates in resting cells corresponded to the [3H] NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H] NHP are linked. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear.     

Comparison between transport and degradation of leucine and glutamine by peripheral human lymphocytes exposed to concanavalin A

Transport and pathways of leucine and glutamine degradation were evaluated in resting human peripheral lymphocytes and compared with the changes induced by concanavalin A (ConA). Cells were incubated with [1-14C]leucine (0.15 mM), [U-14C]leucine (0.15 mM), or [U-14C]glutamine (0.4 mM) after culture with or without 2, 5, 7, or 10 micrograms/ml ConA for 2, 18, or 24 hours, respectively. Initial rates of transport of leucine and glutamine were augmented 2.7-fold and threefold by the mitogen. Leucine transamination, irreversible oxidation, and catabolism beyond isovaleryl-CoA were increased by 90%, 20%, and 60%, respectively. Glutamine utilization increased threefold; accumulation of glutamate, aspartate, and ammonia increased by 700%, 50%, and 100%, respectively, and 14CO2 production by about 400% in response to ConA. The results indicate that ConA stimulates to about the same extent transport of leucine and glutamine into lymphocytes. Glutamine is mainly channeled into catabolic pathways, while leucine remains largely preserved. It is suggested that these metabolic changes provide more leucine for incorporation into protein and more N- and C-atoms required for the synthesis of macromolecules and energy from glutamine.     

Co-localization of non-histone protein BA with U-snRNPs to the same regions of the cell nucleus

Using indirect immunofluorescence, nuclear non-histone protein BA was localized in a normal rat liver cell line. Protein BA antibodies immunostained nuclear structures producing a speckled immunofluorescent staining pattern. Nuclear structures stained with protein BA antibodies were sensitive to DNase I digestion, but not to RNase. The speckled pattern of nuclear fluorescence observed with protein BA antibodies was similar to that reported earlier for Sm antibodies, which react with U-snRNPs. Using double-label indirect immunofluorescence, the Sm antigen was shown to be concentrated in the same regions of the nucleus which contain protein BA. Immunoblot analysis of total nuclear proteins with the two antibodies demonstrated that protein BA and the major Sm antigen have similar molecular weights, but are antigenically distinct. In addition, they differ in their extractabilities from the cell nucleus.     

The production of migration inhibitory factor (MIF) in rabbit lymphocytes by other than immunological mechanism

The ability of certain substances to activate lymphocytes in terms of the production of biologically active substances was studied. These substances were tested by following their migration inhibitory activity. The capacity of concanavalin A and commercial preparations of phytohaemagglutinin (PHA-M and PHA-P) to induce formation of MIF was confirmed. In addition, similar activities were found even in antirabbit antilymphocyte serum (ALS) and erythrogenic toxin (ET). The production of MIF was usually found in material obtained from rabbits treated with complete Freund adjuvant (CFA). On the other hand, material obtained from non-treated rabbits, was found to be inactive with the exception of thymus from young rabbits. The results support the significance of an increasing pharmacological potency of cells in the course of sensitization with CFA. The evidence concerning the release of MIF by ET was extended by similar finding in rabbits made tolerant to the pyrogenic activity of this particular toxin. Lymphocytes obtained from rabbits that were unable to respond to ET by fever, could not liberate MIF (at the same time) upon incubation with ET, even though these lymphocytes produced MIF normally after incubation with PPD tuberculin or concanavalin A. The importance of these results is discussed in terms of the immunologically nonspecific activation of lymphocytes that can mimic immunologically specific events.     

The effect of phenformin and other adenosine triphosphate (ATP)-lowering agents on insulin binding to IM-9 human cultured lymphocytes

In the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM-9 human cultured lymphocytes. After a 24-hr preincubation, phenformin induced a twofold increase in specific 125I-insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between 125I-insulin binding and energy metabolism in IM-9 cells was investigated. The phenformin-induced increase in insulin binding to IM-9 cells was related to a time- and dose-dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2-deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM-9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a lowering of ATP levels.     

The effects of ionophore A23187 and concanavalin A on the membrane potential of human peripheral blood lymphocytes and rat thymocytes

Effects of the Ca2+-ionophore A23187 and concanavalin A on the membrane potential of human lymphocytes and rat thymocytes have been studied using the fluorescent potential probe diS-C3-(5). At concentrations of 10(-8) to 10(-6) M A23187 changes the membrane potential, inducing both hyper- and depolarization. Depending on concentrations of A23187 and the external Ca2+, and on the type of lymphocytes, one of these effects predominates. The hyperpolarization induced by A23187 is caused by activation of Ca2+-dependent K+ channels. It is blocked by quinine and high concentrations of extracellular K+. The dependence of Ca2+-activated K+ transport on extracellular Ca2+ and its sensitivity to calmodulin antagonists is different for human lymphocytes and for thymocytes. As distinct from lymphocytes, in thymocytes calmodulin is not involved in activation of Ca2+-dependent K+ transport. The depolarization induced in lymphocytes by A23187 is caused by an increase in Na+ permeability of the lymphocyte plasma membrane: it is eliminated in a low-Na+ medium. At mitogenic concentrations concanavalin A does not change the membrane potential of the lymphocytes. The results obtained permit elucidation of the relationship between two early events in lymphocyte activation, namely the increase in intracellular Ca2+ concentration and the increase in lymphocyte plasma membrane permeabilities to monovalent cations.     

Interaction of pregnancy proteins with phytohemagglutinin P and concanavalin A (con A)

It has been established that trophoblast-specific beta-glycoprotein and pregnancy-associated alpha 2-glycoprotein specifically react with non-fixed PHA and Con A. Affinity to the former protein is significantly higher than to the latter one. alpha-Fetoprotein has a low affinity to Con A alone. Affinity to this lectin is in an agreement with the content of carbohydrates contained by pregnancy proteins. A considerable part of human serum proteins bind with Con A; receptors for PHA possess only some serum proteins. As the above-mentioned lectins are often used for stimulation of lymphocyte blast transformation, in is recommended that the constituent parts of the culture medium should be preliminarily tested to specify more accurately their affinity to PHA and Con A.