Regulation of protein turnover versus growth state: ascites hepatoma as a model for studies both in the animal and in vitro

Cell protein turnover states as related to growth phase have been analyzed in a rat ascites hepatoma (Yoshida AH-130), which after transplantation entered a period of exponential growth, followed by a quasi-stationary state. Evaluation of AH-130 cell protein turnover in the animal (slow-turnover protein pool) was combined with rapid assays of proteolytic rates of cells transferred in vitro. Protein accumulation in the exponential phase reflected the balance between sustained synthetic rates and relatively low degradative rates. Cessation of growth resulted from convergent reduction of synthesis (from 3.10 to 1.49%/h) and enhancement of protein breakdown (from 0.61 to 1.43%/h). Endogenous proteolytic rates in vitro were very close to the above degradation rates. As shown by incubation with ammonia or other lysosomal inhibitors, the acidic vacuolar pathway for protein degradation, while totally suppressed in exponential tumor cells, was activated in cells from stationary tumors to such an extent that it fully accounted for the enhanced proteolysis. In contrast, energy metabolism inhibitors were effective on cells in either growth state, the residual ongoing proteolysis being similar in both cells. The possible contribution of cell death to activation of the acidic vacuolar proteolysis in stationary tumors is discussed.     

Regulation of protein turnover versus growth state. III. Growth cessation is associated with activation of autophagy in Yoshida ascites hepatoma AH-340

Yoshida AH-130 ascites hepatoma cells were grown in rats and were examined by quantitative electron microscopy 4 days (exponential growth phase) and 10 days (stationary phase) after intraperitoneal inoculation. No significant differences between growing and growth-inhibited tumors were found in the composition of the cytoplasm, except for a slight increase in the cytoplasm, except for a slight increase in the volume fraction of mitochondria (from 9.6 to 12.1%) and, in particular, a prominent (4.2-fold) increase in the volume fraction of early stage of autophagic vacuoles (from 0.31 x 10(-4) at day 4 to 1.37 x 10(-4) at day 10; P less than 0.001). At the same time, the rate of cell protein degradation was increased twofold, namely from 0.67%/h at day 4 to 1.37%/h at day 10, as measured in vitro after labeling cells with 3H-leucine in vivo. Such elevated proteolytic activity was entirely suppressed by ammonia, which inhibits the lysosomal pathway for protein degradation. The data show that: (i) the regulation of autophagic degradation of cytoplasmic constituents depending on the growth state was maintained in these tumor cells, and (ii) the increase in autophagy measured by morphometric analysis contributed to, yet did not quantitatively explain, the acceleration of protein degradation characterizing the transition from the logarithmic growth phase to the growth-inhibited state.     

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.     

Regulation of mitochondrial membrane protein turnover by thyroid hormone(s)

Effects of thyroidectomy on turnover rate of proteins of rat liver mitochondria, mitochondrial membranes and microsomes were examined. Thyroidectomy resulted in a significant increase in the half-lives of whole mitochondria and inner membrane-matrix, the effect being less pronounced on the half-lives of outer mitochondrial membrane and microsomes.     

Effect of the growth state on protein turnover in two lines of cultured BHK cells

The adherence, phagocytic activity and buoyant density of mouse peritoneal exudate colony forming units (CFU-PE) were investigated. There was a significant enrichment in the proportion of CFU-PE in the adherent cells population, defined as cells adhering to a plastic surface within 30 minutes of incubation. The phagocytic activity of CFU-PE was studied by incubating exudate cells with iron particles for 45 minutes. The cells were then separated into phagocytic and non-phagocytic cell fractions by passing the incubation mixture through a magnetic field. A significant enrichment of CFU-PE was seen in the phagocytic cell fraction. When exudate cells were fractionated in a Ficoll discontinuous density gradient, more than 88% of CFU-PE were recovered at the 16/18% and 18/20% interfaces. It is concluded that CFU-PE are adherent cells, have strong phagocytic activity and have a buoyant density between 1.0562 and 1.0703. When bone marrow cells were studied by these techniques, the committed stem cells for both granulocytes and macrophages (CFU-C) were enriched in both non-adherent cell and non-phagocytic cells populations. In the Ficoll density gradient, CFU-C banded at a heavier density region than CFU-PE.     

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.     

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.     

Intrinsic ADP-ribose transferase activity versus levels of mono(adp-ribose)protein conjugates in proliferating Ehrlich ascites tumor cells.

Transition of proliferating Ehrlich ascites tumor cells (3 days after transplantation) to the non-proliferating status (8--14 days after transplantation) was associated with an increase in total mono (ADP-ribose) protein conjugates. This increase was largely confined to the NH2OH-resistant subfraction. When the amounts of mono-(ADP-ribose) conjugates from 20% trichloroacetic acid precipitates were compared with those from 5% perchloric acid precipitates, no significant differences were seen. This fact excludes histone H1 as a major mono (ADP-ribose) acceptor in vivo in these cells. Transition to the resting state was also associated with a small decrease in NAD levels, and with no significant changes of total ADP-ribose transferase activity. However intrinsic ADP-ribose transferase activity as expressed in permeabilized cells was increased, being correlated with the changes in the level of the NH2OH-resistant mono (ADP-ribose) protein conjugates. This shows that alterations in intrinsic transferase activity may, in general, indicate similar alterations in major subfractions of ADP-ribose conjugates. Intrinsic ADP-ribose transferase activity exhibited an inverse relationship to ornithine decarboxylase activity.     

Time-dependent changes in the denatured state(s) influence the folding mechanism of an all beta-sheet protein

Newt fibroblast growth factor (nFGF-1) is an approximately 15-kDa all beta-sheet protein devoid of disulfide bonds. Urea-induced equilibrium unfolding of nFGF-1, monitored by steady state fluorescence and far-UV circular dichroism spectroscopy, is cooperative with no detectable intermediate(s). Urea-induced unfolding of nFGF-1 is reversible, but the percentage of the protein recovered in the native state depends on the time of incubation of the protein in the denaturant. The yield of the protein in the native state decreases with the increase in time of incubation in the denaturant. The failure of the protein to refold to its native state is not due to trivial chemical reactions that could possibly occur upon prolonged incubation in the denaturant. (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra, limited proteolytic digestion, and fluorescence data suggest that the misfolded state(s) of nFGF-1 has structural features resembling that of the denatured state(s). GroEL, in the presence of ATP, is observed to rescue the protein from being trapped in the misfolded state(s). (1)H-(15)N HSQC data of nFGF-1, acquired in the denatured state(s) (in 8 m urea), suggest that the protein undergoes subtle time-dependent structural changes in the denaturant. To our knowledge, this report for the first time demonstrates that the commitment to adapt unproductive pathways leading to protein misfolding/aggregation occurs in the denatured state ensemble.     

Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.     

The phosphorylation of an acidic protein of the large ribosomal subunit of Krebs II ascites cells.

The ribosomes of Krebs II ascites cells contain an acidic protein, apparently analogous to proteins L7/12 of Escherichia coli. When ascites cells were incubated with [32P]Pi, this protein became labelled, indicating that it is a phosphoprotein.