Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 μM or forskolin at 10 μM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 μM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 μM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists → changes in the level of calcium ions in cytoplasm → increased protein kinase activities → protein phosphorylation → modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.     

Age-related features of protein synthesis rhythm. Effects of extracellular medium

Cell interactions have been studied in cultures pf hepatocytes from young and old rats. The rhythm of protein synthesis is an index of cell interaction and synchronization in culture, while the amplitude of oscillations characterized cell cooperation in an aggregate rhythm. The mean rhythm amplitude in the culture of hepatocytes from old rats is twice lower than that from young rats. Gangliosides (mixture, bovine brain gangliosides) and ₁-adrenomimetic phenylephrine enhanced synchronization of cultures of the cells from old rats and increased the amplitude of oscillations to the level of young animals. Addition of rat blood serum (10%) to the medium revealed the rhythm of protein synthesis in the culture, asynchronous in the control, i.e., led to their synchronization. In media with young and old rat blood sera, oscillations were intense, with high amplitudes, and low, respectively. Addition of bovine brain gangliosides to a medium with old rat blood serum increased the amplitudes of oscillations to a level of the rhythm stimulated by the young rat serum. Thus, the cells of old animals can fully perceive synchronizing factors and, in the case of their increased concentration, the rhythm of protein synthesis in old animals did not differ from that in young rats. Current data on biochemical mechanisms underlying intercellular cooperation in the formation of population rhythm of protein synthesis have been discussed.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 9–17.Original Russian Text Copyright © 2005 by Brodsky, Nechaeva, Zvezdina, Novikova, Gvazava, Fateeva, Malchenko.     

Protein synthesis rhythm.

The three-dimensional structure of a protein molecule appears to depend on the amino acid sequence of the protein in an as yet incompletely described manner. If the amino acid sequence is replaced by a numerical sequence of values representing a physical or chemical property of amino acids, the resulting numerical sequence is amenable to autocorrelation analysis. Further, if certain geometrical parameters are calculated from the three-dimensional structure of a protein to form a configurational series, pairs of property series and configurational series can be analyzed by cross-correlation techniques. The data base for the analysis was the three-dimensional structures of ten proteins as determined by X-ray crystallography. Such analysis yields the result that the hydrophobicity of an amino acid residue in a protein influences the orientation angle of the amino acid side chain. This result is consistent with the widely current “oil-drop” model of protein structure. Hydrophobicity also appears to influence the backbone dihedral angle φ, but not ψ Such a directional effect cannot be explained by a current model of information transfer in protein helices. The magnitude of the cross correlations does not appear to be satisfactory for construction of a transfer function model for the prediction of general features of protein structure from amino acid sequences.     

Mechanism of action of sparsomycin in protein synthesis.

Before CI isomerizes to C*I, we detect a competitive phase of inhibition (Ki = k5/k4 = 0.05 microM) which eventually, by increasing the concentration of I, becomes linear mixed noncompetitive and involves C*I in place of CI. The equilibration of C and I according to reaction 2 is much slower than the equilibration between C and S in reaction 1 (time-dependent inhibition). The inactivation plots obey reaction 2 and allow us to estimate k6 as equal to 2.2 min-1. The isomerized C*I, free of excess I, can be studied as a mixture with complex C. From the kinetics of the regeneration of C from C*I, in the presence of puromycin, we can estimate k7 to be between 0.22 min-1 and 0.06 min-1. Although the isomerized C*I survives after adsorption on cellulose nitrate filter disks, it does not survive after gel chromatography on a Sepharose CL-4B column but is converted quantitatively to complex C containing D of unchanged reactivity. This result does not support the proposed [Flynn, G. A., & Ash, R. J., (1990) Biochem. Biophys. Res. Commun. 166, 673-680] chemical reaction between D and I toward new products. The isomerized C*I can be obtained not only from the already-made complex C but also de novo from D, R, and M. In the latter case, the reactions which lead to C are represented by the following hypothetical scheme: D + R + M in equilibrium with DRM or C (binding reaction). When C*I is formed de novo, this reaction is coupled to reaction 2 and the ultimate product is a mixture of C and C*I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related features of protein synthesis rhythm in hepatocytes. Effects of extracellular medium

Cell interactions have been studied in cultures pf hepatocytes from young and old rats. The rhythm of protein synthesis is an index of cell interaction and synchronization in culture, while the amplitude of oscillations characterized cell cooperation in an aggregate rhythm. The mean rhythm amplitude in the culture of hepatocytes from old rats is twice lower than that from young rats. Gangliosides (mixture, bovine brain gangliosides) and alpha1-adrenomimetic phenylephrine enhanced synchronization of cultures of the cells from old rats and increased the amplitude of oscillations to the level of young animals. Addition of rat blood serum (10%) to the medium revealed the rhythm of protein synthesis in the culture, asynchronous in the control, i.e., led to their synchronization. In media with young and old rat blood sera, oscillations were intense, with high amplitudes, and low, respectively. Addition of bovine brain gangliosides to a medium with old rat blood serum increased the amplitudes of oscillations to a level of the rhythm stimulated by the young rat serum. Thus, the cells of old animals can fully perceive synchronizing factors and, in the case of their increased concentration, the rhythm of protein synthesis in old animals did not differ from that in young rats. Current data on biochemical mechanisms underlying intercellular cooperation in the formation of population rhythm of protein synthesis have been discussed.     

Cadherin-related protein 24 induces morphological changes and partial cell polarization by facilitating direct cell-cell interactions

Cadherin-related protein 24 (CDHR24) is a potential tumor suppressor located apically as well as laterally in polarized cells. Here, the role of CDHR24 in contributing to cell morphology and polarity is examined. CDHR24 was predominantly localized at the nonattached part of nonpolarizing cells while another apically sorted protein, aminopeptidase N, was equally distributed over the plasma membrane. Furthermore, CDHR24 expression induced cell aggregation capacity, indicating direct cell-cell interaction. The transepithelial resistance, however, was elevated in polarized MDCK cells, but not in nonpolarizing CHO cells. Our data propose a model in which CDHR24 is directly involved in cell and tissue morphogenesis.     

Aftereffect of synchronizers of protein synthesis rhythm in hepatocytes in vitro

The effect of gangliosides and phenylephrine synchronizing the protein synthesis rhythm was preserved in hepatocytes cultured in the normal serum-free medium for one-two days. Hence, the membrane signal triggers intracellular, as was shown by us earlier, calcium-dependent processes, which regulate the kinetics of protein synthesis for a certain time after the signal perception.     

Aftereffect of synchronizers of protein synthesis rhythm in hepatocytes in vitro

The effect of gangliosides and phenylephrine synchronizing the protein synthesis rhythm was preserved in hepatocytes cultured in the normal serum-free medium for one-two days. Hence, the membrane signal triggers intracellular, as was shown by us earlier, calcium-dependent processes, which regulate the kinetics of protein synthesis for a certain time after the signal perception.     

Mechanism of protein synthesis inhibition in CNS during postischaemic reperfusion.

We have used brain (dog, rat) and spinal cord (dog, rabbit) cell-free systems to study early postischaemic inhibition of protein synthesis. Ischaemia alone produced a relatively small decrease in activity of all subcellular systems used. When 15 min of normoxic reperfusion was used, more than 30% decrease (p less than 0.01) in [14C]-leucine incorporation was detected. A translational inhibitor that appeared in the postribosomal supernatant fraction at the early stage of reperfusion reduced translational capacity of an initiating cell-free system. It also phosphorylated the small (38 kDa) subunit of eukaryotic initiation factor 2 (eIF-2) in vitro. Effect of the inhibitor can be reversed by addition of partially purified intact eIF-2 and/or high concentration (2 mmol/l) of GTP. A prevention of postischaemic free oxygen radical formation by the reoxygenation with hypoxaemic blood, containing 37.5 mm Hg O2 at 0-5 min and 56 mm Hg O2 at 6-10 min of recirculation, that was followed by 5 min of normoxic reperfusion, resulted in a significant increase (p less than 0.02) of polypeptide chain synthesis in vitro when compared with normoxic reperfusion.     

Predicting direct protein interactions from affinity purification mass spectrometry data

Background  

Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) in vivo. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest.     

Mechanism of corticotropin action on adrenal protein synthesis. The effects of inhibitors of protein synthesis and calcium chelators

We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E, in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [14C]- or [3H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 micrograms) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 micrograms cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 micrograms per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 micrograms per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 micrograms per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.     

Polarity and hydrophobicity interactions in protein synthesis process

About 30 years ago, experiments found that there are polarity and hydrophobicity (P and H) correlations and affinity between amino acids and their anticodons. Although it is shown that these experimental findings are important for explaining the origins of the genetic code, the great potential of P and H interactions in investigating other bio-functions have not been fully explored. Here, through raising, discussing and answering seven relevant questions hidden in tRNA aminoacylation, the formation of peptide bonds, and the ending of translations, etc., we show our theoretical findings that the P and H correlations and affinity take vital roles in the protein synthesis process. We found the relationship between the 3' end ACCN sequences of tRNA molecules and the activated amino acids and its biological significance, the rRNAs' consensus sequences 5'NCC...TGG3' or 5'TGG...NCC3' which may perform as functional segments of rRNAs to help triggering the reaction of peptide formation, and common nature of releasing factors that the first amino acid residue of releasing factors ERF, RF1 and RF2 are all Methionine, except a few Alanine, which may be necessary for releasing the translated polypeptide and stopping the translating process. In the terms of P and H correlations and affinity, we provide explanations of why only using the poly (G) as mRNA template cannot get the poly (Gly) in experiments deciphering the genetic code, why Gly often appears in beta turns and why translational bypassing might occur when translating 5'GGAUGA on mRNA. Since amino acids and nucleotides are the subunits, respectively, for composing proteins and nucleic acids, these findings will help in further understanding interactions among the bio-macromolecules. These findings are also helpful for investigating rRNAs, further understanding the protein synthesis process and analysing similar bio-problems, and should be proved useful for experimental biologists.     

Self-synchronization of the protein synthesis rhythm in HaCaT cultures of human keratinocytes

In cultures of human keratinocytes HaCaT contained in a serum-free medium on glass, a circahoralian rhythm of protein synthesis was found similar to the one in hepatocytes in vitro. The intensity of the synthesis was determined by the inclusion of ³H-leucine corrected for the pool of free marked leucine. Rhythm was studied in washed 1- or 2-day cultures after the change of the medium. The medium conditioned with keratinocytes HaCaT synchronized the rarefied hepatocyte cultures nonsynchronous in the control. Therefore, the keratinocytes liberate synchronizing factors into the medium. A BAPTA-AM chelator of calcium ions eliminates the protein synthesis rhythm both in dense hepatocyte cultures synchronous in the control and in the HaCaT keratinocyte cultures. The effect of the H7 inhibitor of protein kinases was analogous. Thus, both in keratinocytes and hepatocytes, self-synchronization of fluctuations of the intensity of protein synthesis takes place. The mechanism of self-synchronization is the calcium-depending phosphorylation of cell proteins.     

Demonstration of a hourly rhythm of protein synthesis in human stomach biopsies

The rhythmic changes in the rate of protein synthesis with a period of 30-40 minutes were demonstrated in biopsy specimens of patients with duodenal ulcer. The oscillation amplitude was up to 20-40%. Eight patients of the 22 did not show such a rhythm.     

Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell.